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Yeasts are ubiquitous unicellular fungi widespread in natural environments. Yeast have a broad set of carbon sources (e.g., polyols, alcohols, organic acids and amino acids) that they can metabolize but they prefer sugars. Yeast are capable of metabolizing hexoses (glucose, fructose, galactose or mannose) and disaccharides (maltose or sucrose) as well as compounds with two carbons (ethanol or acetate). The metabolic pathways utilized by yeast are Embden-Meyerhof glycolysis, tricarboxylic acid cycle (TCA), the pentose phosphate pathway, and oxidative phosphorylation.
Exercise \(1\)
• The yeasts involved in food fermentation are identified are [ facultative / obligate ] fermenters and may display either respiratory or a fermentative metabolism or even both in a mixed respiratory-fermentative metabolism
Review Metabolism
Embden-Meyerhof Glycolysis is the pathway utilized by most eukaryotes.
Exercise \(2\)
• What is the final product of this glycolysis pathway in aerobic conditions?
• What is the fate of this molecule as it travels through the TCA Cycle?
• What happens in oxidative phosphorylation?
Ethanol Fermentation Key Steps
Ethanol fermentation reaction occurs in two steps, decarboxylation and then hydride reduction.
1. In the first reaction, the enzyme pyruvate decarboxylase removes a carboxyl group from pyruvate, releasing CO2 gas and acetaldehyde.
2. Pyruvate decarboxylate utilizes the TPP ylide (seen previously in the conversion of pyruvate to acetyl CoA).
Exercise \(3\)
Show the curved arrows for this mechanism.
2. The second reaction, catalyzed by the enzyme alcohol dehydrogenase, regenerates NAD+ by reducing the acetaldehyde to ethanol.
Exercise \(4\)
• How many NADH can be converted to NAD+ using the ED Pathway? ________
Ethanol has the added benefit of being toxic to competing organisms. However, it will also start to kill the yeast that is producing the ethanol. at the accumulation of alcohol will become toxic when it reaches a concentration between 14-18%, thereby killing the yeast cells
• Explain why the percentage of alcohol in wine and beer can only be approximately 16%.
• How would you produce beverages (liquor) with higher concentrations of alcohol?
Pyruvate Branch Point
Ethanol fermentation utilizes the pyruvate from glycolysis to regenerate NAD+. This is an alternative pathway to metabolize glucose. The pathway is operated by Saccharomyces and other yeast fermenters that ultimately produces ethanol and CO2.
Exercise \(5\)
When would you expect that an organism would choose to operate each pathway?
• What molecule might be a regulator?
Aerobic or Anaerobic?
Pasteur Effect
Pasteur observed that yeast produce alcohol only as the product of a “starvation process” once they run out of oxygen. This observation has been shown to be incorrect!
Crabtree Effect
The Crabtree effect is the occurrence of alcoholic fermentation under aerobic conditions. The most common yeasts used in fermentation processes (Saccharomyces genus) will produce alcohol in both a beer wort and in bread dough immediately regardless of aeration. While you might expect the cell would perform aerobic respiration (full conversion of sugar and oxygen to carbon dioxide and water) as long as oxygen is present, while reverting to alcoholic fermentation, when there is no oxygen as it produces less energy.
However, if a Saccharomyces yeast finds itself in a high sugar environment, it will immediately start producing ethanol, shunting sugar into the anaerobic respiration pathway while still running the aerobic process in parallel. This phenomenon is known as the Crabtree effect. People have speculated that yeast use the ability to produce ethanol to kill competing organisms in the high-sugar environment.
Exercise \(6\)
Summarize:
• [ Aerobic glycolysis / Alcoholic Fermentation ] is more efficient and yields higher ATP per glucose.
• S. cerevisiae will undergo [ aerobic glycolysis / fermentation] when there is high sugar concentration and plenty of oxygen.
• S. cerevisiae will undergo [ aerobic glycolysis / fermentation ] when there is low sugar concentration and plenty of oxygen.
• S. cerevisiae will undergo [ aerobic glycolysis / fermentation ] when there is no oxygen.
• S. cerevisiae [ can / cannot ] tolerate alcohol while most competing organisms [ can / cannot ] survive in alcohol. | textbooks/chem/Biological_Chemistry/Fermentation_in_Food_Chemistry/1.10%3A_Yeast_Metabolism.txt |
Yogurt Production
Yogurt has been around for several millennia. The mythological story about the discovery of yogurt suggests that sheepherders stored their milk in bags made of the intestinal gut of the animals. The intestines contain natural enzymes that cause the milk to curdle and sour. This soured milk lasted longer so they continued making it. Today, the FDA defines yogurt as a milk product fermented by two bacterial strains: a lactic acid producing bacteria: Lactobacillus bulgaricus and Streptococcus thermophiles.
Yogurt Production Process:
Exercise \(1\)
• What is the purpose of the heat treatment in step 2? Think about the whey proteins.
• How does this process differ from cheese production? How does that affect the texture?
Biochemistry of Yogurt Fermentation
In step 4, yogurt cultures are added to milk. These bacteria are lactic acid fermenters; they use enzymes to produce energy (ATP) from lactose.
Exercise \(2\)
• Draw the structure of lactic acid.
• From the previous discussion of lactic acid fermentation, draw a quick pathway showing the production of lactic acid.
• What is the biochemical purpose of producing this lactic acid?
• Bacteria creating lactic acid as a side product which results in a ____________ [ acidic / basic ] environment.
• When the pH drops, what changes occur to the casein micelles?
Biochemistry of Yogurt Flavors
Yogurt is often tart. This flavor is often attributed to the presence of lactic acid. However, there are also a number of carbonyl compounds like acetoin, diacetyl and acetaldehyde that also contribute to the tangy yogurt flavor.
Acetaldehyde Production
During yogurt fermentation, acetaldehyde could be produced from lactose metabolism as a result of pyruvate decarboxylation. However, the primary source of acetaldehyde in these bacteria is from the conversion of threonine (amino acid) into acetaldehyde and glycine.
Exercise \(3\)
• Add arrows for the mechanism of acetaldehyde production shown below.
Exercise \(4\)
• Goat's milk is richer in glycine than cow's milk. In turn, the level of acetaldehyde is much lower in goat milk yogurt. Explain.
Many yogurt bacteria lack the enzyme, alcohol dehydrogenase.
• How does the lack of the dehydrogenase enzyme impact the concentration of acetaldehyde?
Diacetyl and Acetoin Production
Both Streptococcus thermophilus and Lactobacillus bulgaricus produce diacetyl which provides a distinctive “buttery” flavor to yogurt (and other fermented milk products). Acetoin is the reduced form of diacetyl and it complements the diacetyl with a mild creamy flavor.
Exercise \(5\)
• Add arrows for the mechanism for the formation of acetolactate.
Exercise \(6\)
Propose a mechanism.
Probiotics
Yogurt cultures in the intestinal tract have been shown to release the enzyme lactase which continues to break down lactose in the dairy product. This makes yogurt edible for people who are lactose-intolerant.
Exercise \(7\)
To give their products a longer shelf life, manufacturers often heat-treat yogurt after fermentation. This kills the live cultures. What will happen to lactase if the yogurt has been heat-treated after fermentation?
Lactobacillus bulgaricus and Streptococcus thermophilus are the two main bacteria used for creating yogurt. However, these strains do not survive the gastrointestinal tract. They are destroyed by the acidity of the stomach and the enzymes of the pancreas. It has become common to add ‘probiotic’ bacterial strains to yogurt such as Lactobacillus acidophilus, Lactobacillus casei, or Bifidobacterium spp. There is evidence that these bacteria will make it to the intestine intact.
Example \(8\)
• Read more about probiotics in foods: Scourboutakos, et. al. , Nutrients, 2017, 9(4), 400; https://doi.org/10.3390/nu9040400 and Bisanz & Reid, Science: Translational Medicine, 2011, 3(106), 1-4 (in Canvas)
• Describe their experiments and finding.
• How are probiotics beneficial?
• What are their concerns about the claims in probiotic foods on the market?
When probiotics are added to foods, the food industry often also adds ingredients known as prebiotics, such as inulin, which will, after digestion, aid in the growth of the probiotics in the colon.
Exercise \(9\)
• Draw the structure of inulin.
• Can humans metabolize inulin?
• Why is it added to the yogurt?
Other Fermented Milk Beverages
Kefir
Kefir is a carbonated fermented milk drink. The microbes involved in the production of kefir are a symbiotic culture of lactic acid bacteria and yeasts embedded in a matrix of proteins, lipids, and polysaccharides, ‘kefir grains’.
Kefir Production Process:
During the first fermentation, lactic acid bacteria are responsible for the conversion of the lactose present in milk into lactic acid, which results in a pH decrease and milk preservation.
Exercise \(10\)
• This step is similar to cheese and yogurt production. What is occurring to casein proteins?
Biochemistry of the Flavors of Kefir
Similar to yogurt, the flavor of kefir is often attributed to diacetyl and acetoin (both of which contribute a "buttery" flavor), acetaldehyde, and related carbonyl products.
Exercise \(11\)
• Draw the structure of diacetyl and acetoin.
Non-lactose fermenting yeast and acetic acid bacteria (AAB) also participate in the process. Propionibacteria further break down some of the lactic acid into propionic acid (these bacteria also carry out the same fermentation in Swiss cheese).
Exercise \(12\)
• Draw the structure of acetic acid and propionic acid. Review the pathways for these products.
Second Fermentation
Other kefir microbial constituents include lactose-fermenting yeasts such as Kluyveromyces marxianus, Kluyveromyces lactis, and Saccharomyces fragilis, as well as strains of yeast that do not metabolize lactose, including Saccharomyces cerevisiae, Torulaspora delbrueckii, and Kazachstania unispora.
The lactose-fermenting yeast break the lactose down into ethanol and carbon dioxide resulting in a carbonated taste. Ethanol concentration is typically low, usually 0.2-0.3%.
Exercise \(13\)
Review:
• Recap the pathway that leads to ethanol and carbon dioxide production.
• Why is this step anaerobic?
Summarize:
• Describe the biochemical difference between kefir and yogurt. Include structures and products
Sources
Zourari, Accolas, & Desmazeaud, Metabolism and Biochemical Characteristics of Yogurt Bacteria, A Review. Le Lait, INRA Editions, 1992, 72 (1), pp.1-34. (Available in Canvas) | textbooks/chem/Biological_Chemistry/Fermentation_in_Food_Chemistry/1.11%3A_Yogurt.txt |
Bread Production
Bread is a staple food in many cultures. The key ingredients are a grain starch, water, and a leavening agent. However, there are some breads without leavening agents (tortillas or naan), but these are flat breads.
Typical Steps in Bread Production:
Leavening Organisms and Fermentation
Saccharomyces cerevisiae, also known as baker’s yeast, is the primary leavening agent in the production of most breads. Yeast cells consume the sugars present in dough and generate carbon dioxide (CO2) and ethanol that are responsible for dough leavening during the fermentation phase and the oven rise.
Review:
Exercise $1$
1. What is the biochemical pathway for the formation of CO2 and ethanol in yeast?
2. Why doesn’t bread contain alcohol?
Fermentable Sugars
After flour, yeast and water are mixed, complex biochemical and biophysical processes begin, catalyzed by the wheat enzymes and by the yeast. These processes go on in the baking phase. The primary starches found in most cereal plants are the polymers amylose and amylopectin.
Review:
Exercise $2$
What are the monosaccharides in these polysaccharides? What are the linkages?
These starches in the flour provide most of the sugar for fermentation, but the starch must be broken down into monosaccharides before it can be fermented by the yeast. Here is an overview of the sugars utilized by the yeast for the fermentation process:
Amylases: Two types of amylases are present in wheat flour: $\alpha$-amylases and $\beta$-amylases.
• $\alpha$-Amylases hydrolyze the $\alpha$-(1,4)-linkages inside the starch chain randomly, thereby generating shorter oligosaccharides.
• $\beta$-Amylases cleave maltose from the non-reducing end of the starch chain.
Yeast Invertase and Maltase
• Invertase hydrolyzes several small oligosaccharides.
• Maltase cleaves maltose into the 2 monosaccharides.
Exercise $3$
• Draw Maltose. It is a disaccharide made of which two monosaccharides?
• Draw Sucrose. Label the monosaccharides that compose this structure.
• Draw Raffinose. Label the monosaccharides that compose this structure.
• Draw a Fructan. Label the monosaccharides that compose this structure.
Sometimes, $\alpha$-amylases are added to dough as part of a flour improver.
• Explain the benefits of additional amylase enzymes to the bread production process.
• Explain the benefits of adding sugar (sucrose) to the bread production process.
Gluten Formation
Amongst the most important components of the flour are proteins, which often make up 10-15% of the flour. These include the classes of proteins called glutenins and gliadins. Gliadins are globular proteins with molecular weights ranging from 30,000 to 80,000 kDa. Gliadins contain intramolecular disulfide bonds.
Glutenins consist of a heterogeneous mixture of linear polymers with a large molecular weight sections and low molecular weight branches (LMW). Disulfide bond cross-link the glutenin subunits.
Exercise $4$
• Define chemical cross-links and physical cross-links in polymers.
In the bread-making process, water is added to flour, where it hydrates the glutenin proteins, causing them to swell and become stretchy and flexible.
• Add water molecules to this hydrated glutenin picture.
• What IMF changes might be occurring to cause this conformational change?
• Addition of water increases the flexibility of the protein strands and decreases the chain entanglement. This hydration [ increases / decreases ] the flexibility of dough. Explain what is happening on a molecular level to the flexibility.
• Starch granules will also begin to associate with these glutenin proteins. Add some of these to the picture indicating the IMFs involved.
Prior to kneading, the two main protein types, gliadin and glutanin, remain separate on a molecular level. However, as the dough is mixed and kneaded several things begin happening:
The protease enzymes from the wheat begin to break the glutenin into smaller pieces.
The glutenin and gliadin begin to form chemical crosslinks between the proteins. A complex network of proteins, gluten, is formed.
Exercise $5$
• Predict whether gluten is [ more / less ] elastic than the individual starting proteins.
Starch granules are trapped in the dough and air is incorporated into the dough during kneading. The dough needs to be elastic enough to relax when it rests and expand and hold CO2 when it rises — while still maintaining its shape.
Exercise $6$
1. If too much gluten forms (over-kneaded), what will happen to the texture of the bread?
2. As the bread rises, where does the CO2 come from?
3. As the bread bakes, the yeast will continue to ferment the sugars causing the dough to [ expand / shrink ].
Eventually, the heat of the baking will kill the yeast.
Effect of Other Ingredients on Gluten Formation
Fat and emulsifiers coat proteins.
Exercise $7$
1. The presence of fats (butter, oil, etc.) will [ increase / decrease ] hydration.
2. The presence of fats (butter, oil, etc.) will [ increase / decrease ] gluten development
Salts (table salt, NaCl, or hard water salts such as Ca+2 or Mg+2 ) can strengthen the gluten network.
Exercise $8$
Suggest how the presence of salts might strengthen gluten.
Cookie: Usually quite crumbly and doesn’t rise very much.
Exercise $9$
What would you need for a cookie dough?
• [ Low or High ] gluten formation
• [ Low or High ] fat content
• [ Low or High ] salt content
Pizza: To pull dough as thin as a pizza without breaking, there must be a very strong gluten network.
Exercise $10$
What would you need for a pizza dough?
• [ Low or High ] gluten formation
• [ Low or High ] water content
• [ Low or High ] salt content
Bread: A network is tight enough to trap the yeast’s CO2 allowing it to rise, but not so tight that it is free to expand.
Exercise $11$
• What would you need for a bread dough?
o [ Low or Medium or High ] gluten formation
o [ Low or Medium or High ] water content
Baking
Flavors and Aromas: Maillard Reactions
Brewer’s Journal, Science/Maillard Reaction
In food chemistry, any heating steps involving the presence of sugars and amino compounds lead to a series of reactions called the Maillard reactions. These Maillard reactions are nonenzymatic ‘browning reactions’ that lead to the formation of a wide range of flavorful compounds which include; malty, toasted, bready and nutty flavors.
There are three stages to the Maillard Reactions:
Stage I: A condensation between the sugar and amine followed by the Amadori rearrangement.
Stage II: Formation of Strecker Aldehydes
Stage III: Formation of heterocyclic nitrogen compounds.
Stage 1:
Exercise $12$
• Add curved arrows for the mechanism of the condensation and subsequent Amadori Rearrangement.
Stage 2:
Tautomerizations can convert the Amadori Product to a dicarbonyl.
Exercise $13$
The dicarbonyl reacts with an amino acid (asparagine in this example) to form an imine.
• Draw the curved arrow multi-step mechanism for the formation of an imine. You can use abbreviations.
In the Strecker degradation, the imine product undergoes a decarboxylation and is hydrolyzed to an aldehyde.
Exercise $14$
• Complete the table with the Strecker aldehyde formed from these amino acids.
Amino Acid Strecker Aldehyde Aroma
Leucine Malty, toasted bread
Isoleucine Fruity, roasted
Valine Green, unripe fruit
Phenylalanine Floral
Methionine Vegetable
Stage 3:
In this stage, the Strecker aldehydes form complicated heterocycles in a variety of molecular families.
furanones
‘sweet, caramel’
pyrroles
‘nutty’
Acylpryidines
‘cracker’
furans
‘meaty, burnt’
thiophenes
‘meaty,roasted’
Alkylpryidines
‘bitter, burnt’
pyranones
‘maple, warm, fruity’
pyrazines
‘roasted, toasted’
oxazoles
‘nutty, sweet’
imidazoles
‘chocolate, bitter, nutty’
The molecules can also form polymers and precipitates. | textbooks/chem/Biological_Chemistry/Fermentation_in_Food_Chemistry/1.12%3A_Bread.txt |
Beer Production
Beer has been produced by humans for 6000 to 8000 years. The key ingredients are a malted barley, water, hops, and yeast.
Typical Steps in Beer Production:
Barley
Barley is a widely adaptable and hardy crop that can be produced in temperate and tropical areas. Barley kernels or grains are the fruit of the barley grass. The endosperm contains many starches as a food reserve for the baby plant. The starch and the embryo are surrounded by the husk, a protective layer around the kernel. While people have made beers from other grains, many people define beer as the fermented alcoholic barley drink. In fact, the German beer purity law, known as the Reinheitsgebot, of 1516 allows for only hops, barley, water and yeast in the production of beer.
Step 1: Malting the Barley
The goal of the first stage of beer making, malting the barley, is to access the fermentable carbohydrates.
Exercise $1$
Review: Yeasts can utilize what sugars? What enzymes are used?
The barley grains are soaked, called steeping. This process triggers metabolism in the grain to start germination for 4-5 days. As the baby plant starts to grow, the enzymes begin to break down the starches and the cell wall.
The cell wall surrounding the starch containing endosperm is primarily made of $\beta$-glucan and pentosan.
Exercise $2$
1. $\beta$-glucan is glucose units joined through a mix of 1-3 and 1-4 $\beta$-linkages. Draw a short chain of $\beta$-glucan.
2. Pentosans are polysaccharides made from pentoses such as xylose and arabinose. Draw these two monosaccharides.
$\beta$-glucan and pentosan are structural polysaccharides that are NOT digestible by humans or yeast enzymes (i.e. fiber).
Exercise $3$
Explain why germination is necessary for this step of the beer making process (or any food product that uses barley).
To stop germination and enzymatic processes, the grain is heated, called kilning.
Exercise $4$
• Why must germination and enzymatic processes be stopped?
• The enzymes will be needed during the mashing process (next step). Why?
Kiln Variations
There are many varieties of kilned malts. These are a few of the popular styles:
• Pale malt: low and slow kilning at around 100 F and 120 F for as long as 24 hours. This yields a pale beer.
• Vienna malt: kilned at a relatively low temperature, though it can be heated as high as 160 F. It is known for its toast or biscuit like flavor and the pleasant orange color.
• Munich malt: kilned at a high temperature, between 195 F and 220 F. It has a sweet, bready flavor and imparts a nice amber color.
• Aromatic malt: kilned at a high temperature, between 195 F and 220 F. It is sweet and gives the beer a malty, almost syrupy flavor and aroma.
Roasting the malts promotes Maillard reactions This leads to the complex flavors promoted during this stage.
Exercise $5$
• List some products formed in these reactions that might be found in these malts.
After kilning, the malt grain is then cleaned, transported, and stored. Most breweries purchase their malts rather than prepare them.
A diastatic malt has enough enzymes (such as amylase) to convert the starch into fermentable sugars in the mashing stage.
Exercise $6$
• Predict which of the four malt types above would have enough diastatic power to convert the starches present?
• If the malt does not have enough diastatic power, then what will the brewer need to add to the mash? There are several approaches. Try to come up with difference solutions.
Step 2: Brewing
Brewing involves multiples steps. Here is an overview.
• Mill: grinding the malt into a flour called grist
• Mash: mixing the grist with heated water to allow water and enzymes to hydrolyze the starch to form the ‘wort’, a sugary liquid
• Wort Separation: filtering the wort from the insoluble husk particles and other grain particles. Traditional practices used the husk as the filter; modern breweries use polypropylene filters
• Boiling: hops are added to the wort and the mixture is boiled.
• Clarification: denatured proteins, tannins, and hop remains are removed
There is some important chemistry occurring in these steps. We will look at some of the enzymes, the hops, and the boiling steps in more detail.
Mill:
In this step, the grains are broken up in a mill. The particle size, grist, can be determined by the spacing on the rotors.
A large grist was traditionally favored because the crushed grain was used for the filtering at the end of the brewing process.
Exercise $7$
• A fine small grain was problematic in the filtering. Why?
Modern brewers use small grist because they use polypropylene filters.
Exercise $8$
• Why is a smaller grist favored?
Mashing: Enzymes
Mashing is the brewer's term for the hot water steeping process which hydrates the barley, activates the malt enzymes, and converts the grain starches into fermentable sugars.
Table $1$. Enzymes involved in the mash
Enzyme Optimum Temperature (F) Optimum pH Role
$\alpha$-amylase 154-162 5.3-5.7
$\beta$-amylase 131-150 5.0-5.5
amylo-$\alpha$-1,6-glucosidase 95-113 5.0-5.8
$\beta$-glucanase 95-113 4.5-5.5
peptidase 113-131 4.6-5.3 Hydrolyses small
proteins in mash
protease 113-131 4.6-5.3
Exercise $9$
• Fill in the missing information on the table.
Typically, hot water is added to help solubilize starches.
Exercise $10$
• What happens to these enzymes if the water is too hot?
• What happens to amount of fermentation if water temperature is cold?
• Based on your reading, define the following temperature methods for mashing:
• Infusion:
• Decoction:
• Programmed Temperature:
• Some breweries add un-malted grain like rice or maize, called adjunct, that has been boiled in a separate cooker. What is the purpose of this step?
• For light beers, brewers add amylo-$\alpha$-1,6-glucosidase. What is the purpose of this step?
Mashing: Hops
Hops are a climbing perennial vine and the cone of the flower is used to add ‘bittering’ and aroma flavors to the beer wort during this phase of beer production. Typically, these cones are milled and pressed into pellets for use by the brewer. Other brewers use extracts of the cones.
The main components that hops adds to the beer are alpha acids (table 2) and resins (table 2).
Table $2$ : Typical Alpha-acids
Humulone Cohumulone Adhumulone
Exercise $11$
• How do these acids vary?
These alpha acids isomerize during the boiling process to produce iso-alpha acids (see below)
Exercise $12$
• The iso-alpha acids are [ more / less ] soluble than humulone.
The iso-alpha acids contribute the bitter flavor to most beers. It was also discovered that these compounds disrupt the proton pumps used by gram-positive bacteria.
During the 1700s, the British Empire controlled India by maintaining a large army in India, they had a large demand for British brewed ales to be shipped to India. Unfortunately, many ales would spoil during the long sea journey. It was noticed that beers that were brewed at temperatures with higher concentrations hops were less likely to spoil – the beginning of the India Pale Ale (IPA) beers.
Exercise $13$
• Explain how those two factors improved the shipping of British ales.
‘Lightstruck beer’ or ‘skunk’ beer is one in which the iso-alpha acids have undergone a photochemical reaction to form MBT.
Exercise $14$
• Propose a possible method for preventing the ‘skunking’ of beer.
Mashing: Hop Resins
Table $3$: Types of Compounds from Hop Resins
Hop Resin Constituent
Soft Resin: Alpha Acids
Beta Acids
Hard Resin: Tannins
Polyphenols
Amino Acids
Proteins & Carbohydrates
Exercise $15$
• Beta acids do not isomerize during the boil. Draw some beta acids.
Tannins are astringent polyphenolic compounds.
Tannic Acid (example of a tannin):
The tannin compounds are widely distributed in many species of plants, where they play a role in preventing predation. The astringent flavor predominates in unripe fruit, red wine or tea.
Exercise $16$
• How might these flavors impact the beer?
Mashing: Aroma Hops
Hops added after boiling is called ‘dry hopping’.
Hop oils (essential oil) are sometimes added after boiling of the wort. These ‘aroma hops’ are volatile non-polar compounds that have strong aromas and flavors. There are between 400 and 1000 different compounds in hop oil including structures such as myrcene, humulene, caryophyllene, $\beta$-pinene, geraniol, linalool, and farnesene.
Exercise $17$
• Draw a few of these isoprene natural products.
• What flavors are associated with these structures?
• How soluble do you think these compounds are in beer? Would you predict that they would be present in high quantity?
• Why are these aroma hops added after the boil?
Boiling the Wort:
Exercise $18$
There are several goals of boiling wort. Explain the importance of each of these steps:
1. Isomerize alpha acids
2. Volatile compounds evaporated especially dimethyl sulfide
3. Kill bacteria and wild yeast
4. Deactivate enzymes from grain
5. Concentrate the wort by evaporation since the water used in mashing and sparging has produced a wort lower in specific gravity (concentration of dissolved sugar)
6. Denature proteins from grain so that they clump together (This is usually done in two steps: 1) hot break: denatured proteins coagulate and float to the surface. The trub (remains of the hops, tannins, and the coagulated proteins) can be removed and 2) cold break: After boiling, the wort is cooled down to fermentation temperature and more proteins will coagulate.
Brewing: Adjuncts
Liquid adjuncts (sugars/syrups) are usually added in the wort boiling stage. They may be sugars extracted from plants rich in fermentable sugars, notably sucrose from cane or beet or corn syrup. Liquid adjuncts are frequently called “wort extenders”.
Exercise $19$
• Why are these added after the mash?
• What are the advantages of a wort extender?
• The use of added sucrose can actually decrease the sugar content in the final beer. Explain.
Brewing: Clarification
This is a filtering process that varies by brewer.
Exercise $20$
• List 3-4 components that are removed from the wort during the clarification process?
• Some brewers argue about whether to remove the cold break and trub. What are the advantages of keeping the trub in the wort for fermentation?
Step 3: Fermentation
There are hundreds of strains of yeast. Many beer yeasts are classified as "top-fermenting" type (Saccharomyces cerevisiae) and or "bottom-fermenting" (Saccharomyces uvarum, formerly known as Saccharomyces carlsbergensis). Today, as a result of recent reclassification, both yeast types are considered to be strains of S. cerevisiae.
Top-fermenting
Ale yeast strains are best used at temperatures ranging from 10 to 25°C. These yeasts rise to the surface during fermentation, creating a very thick, rich yeast head. Fermentation by ale yeasts at these relatively warmer temperatures produces a beer high in esters, regarded as a distinctive characteristic of ale beers. These yeasts are used for brewing ales, porters, stouts, Altbier, Kölsch, and wheat beers.
Bottom-fermenting
Lager yeast strains are best used at temperatures ranging from 7 to 15°C. At these temperatures, lager yeasts grow less rapidly than ale yeasts, and with less surface foam they tend to settle out to the bottom of the fermenter as fermentation nears completion. These yeasts are used in brewing Pilsners, Dortmunders, Märzen, Bocks, and American malt liquors.
Wild Yeast
Beer that is brewed from natural/wild yeast and bacteria are called spontaneous fermented beers. One of the typical yeasts is the Brettanomyces lambicus strain which is used to produce traditional lambic beers. This brewing method has been practiced for decades in the West Flanders region of Belgium. We will visit 3 Fonteinen Brewery in Belgium that specializes in lambic beers.
Exercise $21$
• Review: Explain the Crabtree effect.
• Why is it important to add oxygen to the wort during fermentation?
Fermentation Flavors: Higher alcohols (fusel alcohols)
Longer chain alcohols produced by yeast during fermentation can also contribute to the aroma and flavor of beer. Primarily these alcohols can increase the warming of the mouthfeel.
Fusel alcohols are derived from amino acid catabolism via a pathway that was first described by Ehrlich. Amino acids represent a major source of the assimilable nitrogen in the wort. Amino acids that are taken up by the yeasts and converted to fusel alcohols by the Ehrlich pathway (valine, leucine, isoleucine, methionine and phenylalanine).
The Ehrlich pathway is shown below for phenylalanine.
Exercise $22$
• Draw the structures of the alcohols that would be formed from valine, leucine and isoleucine.
Too much of the higher weight fusel alcohols provides a harsh alcoholic taste (in fact, the word fusel is from the German for bad liquor).
Fusel alcohols can be produced by excessive amounts of yeast or fermentation temperatures above 80°F.
Exercise $23$
• How can you prevent too high of levels of yeast production?
Fermentation Flavors: Ester Production
Many of these esters are derived from alcohols reacting with acetyl coA.
Exercise $24$
• Draw the mechanism for this reaction including the intermediate. Remember that thioesters are activated carbonyl derivatives.
• Draw the products of the fusel alcohols reacting with acetyl coA.
Some of the esters are derived from alcohols reacting with activated thioesters from the fatty acid synthesis pathway.
Exercise $25$
• Draw the products of some of these alcohols reacting with various fatty acid derivatives.
Fermentation Flavors: Ester Flavor Profile
Table $4$: Common Esters from Yeast Metabolism and their Flavor Profiles
Ester Structure Flavor Profile
Ethyl butanoate Papaya, butter, apple,
perfumy
Ethyl hexanoate Apple, fruity, sweet
Ethyl 2-methylbutanoate Sweet, fruity, grape-like
Ethyl acetate Solvent, fruity, sweet
Ethyl octanoate Apple, sweet, fruity
Isoamyl acetate Banana, estery, apple,
solvent
2-phenylethyl acetate Roses, honey, apple,
sweet
Isobutyl acetate Banana, estery, apple,
solvent
Usually, brewers want a balance of esters present in the final product but not too many.
Exercise $26$
• Predict how these factors will impact the amount of ester compound and fusel alcohols present in the final product.
• Fermentation Temperature:
• Fermentation with ‘trub’ (contains lots of yeast nutrients):
• Specific Gravity of wort:
• Yeast ‘pitch’ (concentration of innoculation):
Fermentation Flavors: Ketones
While the presence of esters and fusel alcohols can enhance the flavor and aroma of beers, the presence of ketones is usually considered undesirable.
The most common are the formation of diacetyl and acetoin. Diacetyl is most often described as a buttery flavor. It is desired in small quantities in many ales, but it can be unpleasant in larger quantities and in lagers; it may even take on rancid overtones.
Exercise $27$
• Review: What is the pathway for the production of diacetyl (and the reduced forms)?
Diacetyl can be the result of the normal fermentation process or the result of a bacterial infection. Diacetyl is produced early in the fermentation cycle by the yeast and is gradually metabolized towards the end of the fermentation.
Beer sometimes undergoes a "diacetyl rest", in which its temperature is raised slightly for two or three days after fermentation is complete.
Exercise $28$
• What is the purpose of this rest time?
Beer Types
Beer style is a term used to differentiate and categorize beers by various factors, including appearance, flavor, ingredients, production method, history, or origin. There is no agreed upon method for distinguishing beer styles.
There are some general categories that are used in describing beer styles:
Yeasts: Ales vs Lagers
• Ale: Top-fermenting strains that tend to produce more esters.
• Lager: Bottom-fermenting strains that tend to produce more sulfur compounds.
• Weizen Yeast: Used in German-style wheat beers and is considered an ale yeast.
• Brettanomyces: Wild yeast with flavors like barnyard, tropical fruit, and more.
Malt types:
• It is the main fermentable ingredient and there are a wide variety of roasts. Darker roasts impart more chocolate, coffee, caramel, and toasty flavors.
• Adjuncts can impart different flavors.
Hops:
• Hops provide a range of compounds that influence beer’s aroma, flavor, bitterness, head retention, and astringency.
• Quantity and strain and timing can impact the flavor range.
• Flavor and aroma ranges: citrus, tropical, fruity, floral, herbal, onion-garlic, sweaty, spicy, woody, green, pine, spruce, resinous
Alcohol Content:
• Alcohol content can range from 2% to greater than 14%
• Fusel alcohol can also exist in beer
Carbonation Level:
• Carbonation is a main ingredient in beer. Carbonation can be detected as an aroma (carbonic acid) and it has a mouthfeel and flavor. It also affects the foam.
• Carbonation can be naturally occurring (produced by yeast during fermentation) or added to beer under pressure. N2 can also be added to beer, providing smaller bubbles and a softer mouthfeel than CO2.
Craft Beer.com provides a nice style guide on the different names of beers with information about the yeast strains, hop aroma, IBU (International Bitterness Units), alcohol content, carbonation for hundreds of beer styles.
John Palmer also provides a nice table that places a wide range of beer styles on a chart comparing a number of ales and lagers on malty vs fruity and sweet vs bitter.
• John Palmer, How to Brew, Chapter 19, A Question of Style, Ales vs Lagers
Exercise $29$
Choose your favorite breweries or breweries chosen by your instructor.
• Pick 3-6 beers from these breweries and look some of their categories – ale vs lager, hops, post-brewing conditioning, malt types, etc. | textbooks/chem/Biological_Chemistry/Fermentation_in_Food_Chemistry/1.13%3A_Beer.txt |
Cider Production
Cider is a drink made from apples. In the US, cider can refer to apple juice or the fermented, alcoholic version. This section will focus on the fermented, alcoholic drink.
Typical Steps in Cider Production:
Step 1: Apple Preparation
Choosing Apple Varietals
Apples are the primary material used in cider production; thus, the final cider product quality and style depend heavily upon the quality of the apples used. Apples must be juicy, sweet, and ripened. A full-bodied cider requires the use of several different types of apples to give it a balanced flavor including a mix of sweet and tart apples.
Exercise $1$
There are four main apple varietals. List them and their flavors.
Milling and Pressing
Apples are not peeled as the skin of the apples contains many of the compounds that contribute to the taste of the cider. The apples are ground and then pressed to extract the juice. The primary components of an apple are shown in Table 14.1. The fiber and insoluble carbohydrates are mostly removed in the pressing process.
Table $1$ Raw Apple Components
Constituent Approximate Composition
Water 80%
Carbohydrates (mono-, disaccharides) 5%
Carbohydrates (cellulose, mostly removed in pressing) 5%
Malic Acid 4-6%
Pectin 1%
Polyphenols, tannins --
Vitamins, Minerals 4%
Proteins --
After pressing, the juice can be pasteurized and sold as apple juice or it can be further processed with fermentation to produce the alcoholic beverage.
Components of Cider Apple Juice: Sugars
The primary sugars found in cider apple juice before fermentation are fructose, glucose, and sucrose.
Exercise $2$
• Draw these three structures of fructose, glucose, and sucrose.
• Would most bacteria or yeast be able to ferment these saccharide structures? Or would these sugars need to be hydrolyzed first?
On a commercial scale, there are considerable cost advantages to supplementing the raw apple juice with glucose syrup and water as they are cheaper than apple juice. In fact, many commercial ciders are now made from around 35% juice and 65% glucose syrup.
Exercise $3$
How would this impact the flavor?
Components of Cider Apple Juice: Pectins
Pectin is a polysaccharide made from a mixture of monosaccharides. While many distinct polysaccharides have been identified and characterized within these ‘pectic polysaccharide family’, most contain stretches of linear chains of $\alpha$-(1–4)-linked D-galacturonic acid.
Exercise $4$
Draw a linear chain of linear chains of $\alpha$-(1–4)-linked D-galacturonic acid.
Most wild type yeasts cannot ferment galacturonic acid but failure to remove the pectin can lead to the formation of jelly during concentration. Thus, pectolytic enzymes are sometimes added prior to fermentation. This pectinase treatment often results in a release of higher concentrations of anthocyanins, tannins, and polyphenols from the apple pressings.
Exercise $5$
How could the increased level of tannins and phenols impact the flavor of the final cider?
Remaining pectin polysaccharides cause a haze in finished ciders, so pectinase is also sometimes added after fermentation to clear the cider.
Exercise $6$
How would the presence of alcohol in the finished cider impact the effectiveness of the pectinase?
French cider is often prepared with an initial step, ‘defecation’, in which pectins and other substances are separated from the juice using a gelatin. T hen, the clear juice is fermented slowly.
Exercise $7$
These ciders are fruitier than others. Explain.
Sulfuring
Many cider makers add sulphur dioxide (or potassium metabisulphite) to inhibit the growth of most spoilage yeasts and bacteria, while permitting the desirable fermenting yeasts (such as Saccharomyces cerevisiae or uvarum) to facilitate the conversion to alcohol.
Exercise $8$
• Draw the Lewis structure of sulfur dioxide, $\ce{SO2}$.
• Sulfur dioxide in water is rapidly converted to $\ce{H2SO4}$. Show a mechanism.
Most natural weak acid preservatives (such as vinegar, benzoic acid or sorbic acid) are believed to work by diffusing through bacterial cell membranes. The increased acidity of the cytoplasm disrupts the cell homoeostasis and the cell has to work very hard to pump out protons to restore the pH. Eventually, the cells run out of ATP and die. Sulphite is believed to work in the same way as other weak acid preservatives.
Step 2: Fermentation
Fermentation Yeasts
Apple juice (must) was traditionally fermented with the bacteria and yeast already present on the apples.
The main yeasts found in wild fermentations is Saccharomyces bayanus. But Saccharomyces cerevisiae, Lachancea cidri, Dekkera anomala and Hanseniaspora valbyensis are also present is substantial amounts. Other species are present in small amounts: Candida oleophila, C. sake, C. stellate, C. tropicalis, H. uvarum, Kluyveromyces marxianus, Metschnikowia pulcherrima, Pichia delftensis, P. misumaiensis and P. nakasei.
There are three phases in the cider process based on the dominant yeast species present.
1. The first phase, which they called ‘the fruit yeast’ phase, is dominated by Hanseniaspora uvarum/Kloeckera apiculata yeasts.
2. The second phase, or ‘fermentation phase’ where the alcoholic fermentation occurs with the replacement of non-Saccharomyces yeasts by the strong fermenting Saccharomyces yeasts, such as S. bayanus and S. cerevisiae.
3. The last ‘maturation phase’ is dominated by Brettanomyces/Dekkera yeasts.
Exercise $9$
• Many large industrial cider factories use heavy sulfiting to _______________ and then add purified Saccharomyces wine yeasts.
• Suggest a reason that industrial cider factories choose not to use wild fermentation.
• Craft cider-makers will often still use wild fermentation. Suggest a reason that they choose to use wild fermentation.
• During alcoholic fermentation, sugars are converted mainly into _______ and ________ by yeasts (mainly Saccharomyces sp.). The varietal choice and maturity of the fruits influence the sugar content of the starting must and, thus, the final ethanol level.
Fermentation should take 5 to 10 days, and up to 4 - 6 weeks at cool temperatures. Nearly all the sugar will then have been used by the yeast and the yeast will become dormant.
Exercise $9$
Propose how cider-makers would determine when to stop fermentation.
During alcoholic fermentation, many secondary metabolites are produced by the yeasts. Esters provide mainly fruity and floral notes; higher alcohols provide ‘background flavors’; whereas the phenolic compounds can generate interesting or unpleasant aromatic notes.
Esters are the main volatile compounds in cider. They are characterized by a high presence of ethyl acetate, which alone can represent up to 90% of the total esters.
Exercise $10$
• What are the fusel alcohols and how are they formed?
• Draw ethyl acetate and review the metabolic process for its synthesis from fermenting yeasts.
Dioxanes, key flavor components of cider, are described as a ‘green, cidery’ flavor that results only from alcoholic fermentation of apples (and pears).
These dioxanes are formed from reaction of acetaldehyde or other aldehydes (fermentation byproduct) with diols which are found almost exclusively in apples.
Exercise $11$
Propose a mechanism for this reaction of acetaldehyde and octane-1, 3-diol.
Another dioxane found in cider is formed from acetaldehyde and (R)-5(Z)-octene-1,3-diol.
Exercise $12$
Draw the product found in cider.
Step 3: Post-fermentation Processing
Racking and Fining
Racking is the process of moving the cider from its lees (the sediment formed).
This is usually a filtration or centrifugation process.
Pectinase may be added at this point.
Exercise $13$
What is the purpose of this step?
After racking, the cider maker may choose to do a secondary fermentation. Yeast might be added to ensure a sparkling cider, or a malolactic acid fermentation will be used to improve the flavor. (See next sections).
Aging
Cider was traditionally stored in wooden barrels to age, but this is not essential if chilling and fining have been properly carried out after the fermentation.
The bacteria needed for malolactic acid are often founded in the wood barrels.
Secondary Fermentation: Malolactic Fermentation
Cider fermentation with LAB bacteria convert the sugars and the malic acids into lactate. Malolactic fermentation is primarily completed by Leuconostoc oenos, a heterofermentative organism. This process tends to create a rounder mouthfeel to the final cider. Malic acid is typically associated with the taste of green apples, while lactic acid has a richer taste.
Table $2$
Nutrient Homofermentive LAB Heterofermentive LAB
Glucose Lactate Lactate, ethanol, CO2
Fructose Lactate Lactate, ethanol, CO2
Malate Lactate, CO2 Lactate, ethanol, CO2
Citrate or Pyruvate Acetoin, Diacetyl, CO2 Lactate, acetate, CO2
Exercise $14$
• Review: What is the difference between homofermentive and heterofermentive?
• Malolactic fermentation is favored by [ high / low ] sulphiting during fermentation.
Secondary Fermentation: Malolactic Fermentation Pathway
The malolactic fermentation involves the conversion of malic acid into lactic acid and carbon
dioxide. Some LAB bacteria convert the malic acid in one step; while others utilize these steps
that include intermediates from the TCA cycle.
Exercise $15$
Complete the steps in this biochemical pathway to convert malic acid to lactic acid.
• What is the net NAD+ / NADH change?
• For many bacteria, the goal of fermentation is to regenerate the [ NAD+ / NADH ] utilized in the glycolysis pathway as they do not have the enzymes for oxidative
phosphorylation where this occurs in eukaryotes.
If malolactic fermentation is not fulfilling this function, then there must be some energy gain for the organism in completing this process.
Secondary Fermentation: Malolactic Fermentation Energy and pH
MLF process is shown. This reaction allows cells to regulate their internal pH.
Exercise $16$
What happens to the overall H+ concentration inside the cell?
This reaction allows cells to gain energy by creating a proton gradient across cell membranes. Some bacteria can utilize citrate or malate. The process allows out 1-2 proton atoms to be pumped out of the cell into the periplasm.
Exercise $17$
Suggest a method for pumping out 2 H+ instead of 1 H+ in this process.
The proton gradient created from MLF is coupled to an ATPase which captures the energy in the production of new ATP molecules.
In cider production, this is important to reduce the malic acid content AND the overall raw acidic flavor of the cider.
Exercise $18$
The proton pump will [ increase / decrease ] the acidity of the cider product (outside the cell).
Depending upon the organism, these processes are inhibited with higher alcohol content and below pH of 3-4.
Exercise $19$
If a cider producer wants to inhibit MLF in the cider, the pH of the must can be [ lowered / raised ] to prevent the process.
Sweet Still Cider
Ciders are naturally ‘dry’. The term ‘dry’ means that there is little sweetness from remaining sugars, but more flavor from alcohol, fusel alcohols, esters, etc. Some consumers prefer a sweet still cider.
Exercise $20$
Propose at least two methods for ensuring a sweet cider.
Sparkling Ciders: Gasification and Bottling
To get some bubbles into cider, excess carbon dioxide under pressure can be added and then the cider is bottled or put in a keg which will withstand the pressure.
Sparkling Ciders: Secondary Fermentation for Gasification
Commercial cider-makers will sometimes inoculate with active dry yeast (Saccharomyces cerevisiae) before bottling to obtain a naturally-carbonated beverage.
Exercise $21$
Because there is not much sugar left in the cider at this point, __________ is often added when using a second fermentation.
This can be very successful although the bottom of each bottle will inevitably be a little cloudy when poured, because there will always be some yeast deposit which will be roused up when the pressure is released.
Note: Bottles used for carbonated ciders must be designed to withstand the pressure generated by the gas!
Stabilization
After all fermentation processes are complete, the must is either pasteurized or treated with ascorbic acid or sulfur dioxide.
Exercise $22$
• What is the purpose of this step?
This step also decreases the chance of contamination by Acetobacter.
• What happens if Acetobacter is present?
Sources
1. Kavvadias, et. al. J. Agric. Food Chem. 1999, 47 (12), 5178-5183.
2. Cox and Henick-Kling, Chemiosmotic Energy from Malolactic Fermentation, J. Bacteriol. 1989, 5750-5752. | textbooks/chem/Biological_Chemistry/Fermentation_in_Food_Chemistry/1.14%3A_Cider.txt |
Wine Production
Overview
Wine is defined as the fermented juice of a fruit. Wines have been produced from all kinds of plant materials and fruits. However, the most classic version is made from grapes.
Typical Steps in Wine Production:
Grape Preparation
Grapes
The grape pulp has a high concentration of fermentable sugars while the skin and seeds have a lot of flavorful compounds.
Grapes: Varietals
The grape is the fruit of the vine, Vitis vinifera (wine) and Vitis labrusca (table grapes). There are over 5000 varietals of grapes which all have different flavor and aroma profiles.
A list of varietals (and pronunciations) is available from J. Henderson, Santa Rosa Junior College. The Wine Spectator has an article by J. Laube and J. Molesworth on Varietal Characteristics.
In Europe, wines are usually categorized by their geographic region. In America, Australia, South Africa and New Zealand, wines are usually labelled by their varietal names.
Grapes: Terroir
The grapes will develop a different profiles of flavor chemicals depending on soil, temperature, growing practices, rain, etc. The land and climate are referred to as the ‘terroir’.
Chemical Components of Grapes
Carbohydrates in Grapes
As grapes ripen on the vine, they accumulate sugars through the translocation of sucrose molecules that are produced by photosynthesis from the leaves. During ripening the sucrose molecules are hydrolyzed (separated) by the enzyme invertase into glucose and fructose.
Exercise $1$
• Show the hydrolysis products resulting from invertase action. Label the two sugars formed.
By the time of harvest, between 15 and 25% of the grape will be composed of monosaccharides; the total sugar content and the types will vary by cultivar.
This includes glucose, fructose, and sucrose (fermentable sugars) and a small amount of sugars like the five-carbon arabinose, rhamnose and xylose.
arabinose xylose ribose rhamnose
Exercise $2$
• What type of sugars are these?
• Hexose OR pentose
Sugars like arabinose have little flavor to humans and Saccharaomyces cannot metabolize them so they have little impact in wine unless Brettanomyces (wild yeast) or LAB are present.
Exercise $3$
• What will be the products of fermentation of these sugars by LAB? How will that impact the flavor?
Organic Acids in Grapes: Tartaric, Malic, and Citric Acids
Tartaric and malic make up over 90% of grape juice acid. Tartaric acid is rarely found in other fruits. There are some other organic acids present in small amounts including lactic, ascorbic (vitamin C), fumaric, pyruvic and more.
tartic acid malic acid
The majority of the tartaric acid found in grapes is present as the potassium acid salt.
Exercise $4$
Draw the potassium dipotassium salt of tartaric acid.
In wine tasting, the term “acidity” refers to the fresh, tart and sour attributes of the wine which are evaluated in relation to how well the acidity balances out the sweetness and bitter components of the wine such as tannins.
In the mouth, tartaric acid provides most of the tartness to the flavor of the wine, although citric and malic acids also play a role.
To improve the flavor, the winemaker can add tartaric, malic, citric, or lactic to the grape juice
(must).
Exercise $5$
• How would a winemaker decide whether to add more tartaric acid? What test would they run?
Polyphenols: Overall class of compounds
Polyphenols are a class of molecules characterized by the presence of large multiples of phenol structural units. This is a huge class of molecules found many plants. Grapes have a wide variety of polyphenols, most of which are concentrated in the skin and seeds.
The concentration and types of polyphenols varies between grapes based on cultivar, ‘terroir’ – grape growing region (altitude, geological features, soil type, sunlight exposure), temperature during ripening, and environmental stressors such as heat, drought and light intensity.
There are many sub-categories of polyphenols. Here is a simplified outline.
The flavor and appearance of red wines are determined by the phenolic compounds: anthocyanins (responsible for the red color) and tannins (responsible for the sensation of
astringency).
Non-Flavonoid Polyphenols: Cinnamic Acids, Stilbenes, & Hydroxybenzoates
Hydroxycinnamic Acids Hydroxybenzoic Acids Stilbenes
R1 = R2 = H courmaric acid R1 = R2 = OH gallic acid trans-Resveratrol
R1 = OH R2 = H caffeic acid R1 = R2 = OCH3 syringic acid --
R1 = OCH3 R2 = H ferulic acid R1 = OCH3 R2 = H vanillic acid --
Hydroxycinnamic Acids are mostly found in the grape pulp.
Exercise $6$
• During the processing, would you predict that these structures would be soluble or insoluble in the must (mostly water)?
Hydroxycinnamic acids are often found as esters of tartaric acid or with a sugar. During the processing, these esters are hydrolyzed.
Exercise $7$
• Draw the hydrolysis products formed from caftaric acid and tartaric acid.
Hydroxybenzoates have been identified in both grapes and wines. These structures are the basis of hydrolysable tannins (next section)!
Stilbenes have two aromatic rings connected with an alkene (cis or trans). Resveratrol is one of the most common stilbenes found in grapes and wine. It is usually located in the grape skin.
Exercise $8$
• Draw the cis-resveratrol.
Flavonoid Polyphenols: Anthocyanins, Flavonols, and More!
Flavonoids are a class of compounds with a basic structure containing two aromatic rings bound through a three-carbon chain. Flavonoids are grouped into several classes (shown below). They can have many different substituents on the rings.
Exercise $9$
• Briefly summarize the differences in these four structural types.
flavone flavanone flavane (flavanol if R=OH) anthocyanidin
Flavones, Flavanones, and Flavonols are mostly found in the seeds and skin.
Many of these flavonoids are present in the grapes as the glycosides (the sugar moiety can also vary) but are cleaved in the processing to wine.
Exercise $10$
• How does the sugar moiety impact the solubility?
• Show the hydrolysis products of the common flavonol, catechin o-glycoside.
Anthocyanins are also prevalent in wines and grapes. They are usually glycosylated. They are partially responsible for the color of grapes and wines.
Exercise $11$
• These molecules have a [ positive / negative ] charge.
• How does this impact the solubility in the juice?
• Anthocyanins can change color in different pH. Draw the arrows for the changes occurring at different pH.
• Which form is in grape juice?
• Which in wine? Hint: Think about pH of fermented products.
Flavonoid Polyphenols: Tannins
Tannins are polymeric forms of polyphenols.
Most of the natural tannins present in grapes and wine are the ‘condensed type’, often dimers and trimers of polyphenols (flavonoid or non-flavonoid).
Exercise $12$
• For these two condensed type tannins, draw the monomer from which they are derived.
Hydrolysable tannins are also present in grapes and wine. These are usually a sugar with several polyphenols covalently bound.
Exercise $13$
• For this hydrolysable tannin
• Circle the polyphenol
• Box the sugar.
Complex tannins are long polymeric mixtures of these structures.
Grape Processing
Stemming and Crushing
Harvesting of grapes is usually done in late summer and early fall. Harvesting for most large industrial wineries is mostly mechanical. The stems must be removed first to avoid ‘off-flavors’.
The grades are crushed immediately after picking. The goal of crushing is to release the sugars, acids and some of the polyphenols from the skins. For white wines, the juice is separated from the skins so that the color and tannins are not extracted into the must. For red wines, the juice and skin are both fermented.
The grape skin cell walls are composed of polysaccharides (pectins, hemicellulose and cellulose) that prevent the diffusion of polyphenols into the must.
Exercise $14$
• Explain how these structures prevent diffusion of polyphenols. Discuss IMF.
Excessive crushing can release too many polyphenols.
Exercise $15$
• What will happen to the flavor of the wine if too many polyphenols/tannins are extracted?
Too sweet Too high alcohol Too low alcohol Too astringent Too dry
Maceration
During winemaking, phenolic compounds are extracted into the juice by diffusion. A diffusion period, ‘maceration’, can be done as a cold soak, through heating, enzymes, or a variety of techniques intended to increase polyphenol extraction. Maceration can be before, during, or after fermentation.
Exercise $16$
• The [ more / less ] water soluble compounds will diffuse easily into the juice.
• [ More / Less ] hydroxyl substituents present will increase diffusion into the juice.
• [ Polymer / Monomer ] tannins and proanthocyanins will diffuse easily into the juice.
Maceration enzymes (pectinases and cellulases) are often added during this process.
Exercise $17$
• Explain how these enzymes can increase polyphenolic content in must.
Polyphenols: Oxidation Reactions
Polyphenols are susceptible to oxidation with Fe and O2 in solution or through the action of some yeast enzymes.
This oxidation is called browning because the quinones are a brown, muddy color.
Exercise $18$
• This is undesirable in all wines but is particularly problematic in white wines. Why?
Winemakers will usually add SO2 to correct for the oxidation processes.
Sulfite also prevents ethanol oxidation.
Exercise $19$
• Explain why this is important in wine.
It is important to remember that sulfite has another role; it can slow or prevent growth of spoilage organisms.
Phenolic Changes
This is a chart of some typical reactions that can occur to anthocyanins in the wine-making process including during fermentation including oxidation and condensations with yeast byproducts.
Exercise $20$
• Fill in the boxes with polyphenol products.
• Are these products more or less soluble in wine?
These are just a few of the types of reactions that anthocyanins undergo during maceration and aging.
Polymeric pigment formation increases progressively during maceration and aging ultimately leading to color changes, modification of mouthfeel properties, and, sometimes, precipitation.
An important polymerization is the reaction of an anthocyanin with a flavanol (shown below).
Exercise $21$
• Label the electrophile and the nucleophile in this reaction.
• Use curved arrows to show how this product can reform the aromatic ring.
• This reaction continues on to very long polymers. Draw this as a 4-mer.
These large polymers start to precipitate and form a sediment.
Exercise $22$
• In the beginning, polyphenolic materials increase in the must during maceration. If the maceration times are too long, the number of polyphenolic compounds in the final wine decrease. Explain this observation.
Adjusting Sugar Content in the Must
Sugar content is important as it effects the alcohol level of the final wine as well as the sweetness of the wine.
Degrees Brix’ is a density measurement that represents the sugar concentration in wine.
$\text{1 degree Brix (°B) = (% by weight) = 1 gram of sugar per 100 grams solution (water & sugar combined)}$
Sucrose and/or grape juice can be added to the grape must.
Exercise $23$
• How will this addition impact alcohol content?
• How will this addition impact flavor?
Adjusting pH in the Must
A wine with low acidity will taste "flat" whereas one with too high an acid level will be unpleasantly tart.
Acid content is important for flavor and is important in some of the reactions involved in polyphenolic changes.
Wine-makers will add tartaric, malic, citric, or lactic acids to adjust pH for the tartness of a wine. For most adjustments, tartaric acid is used because it disassociates best (lowers the pH more/gram).
Exercise $24$
• A wine with a low pH will be [ more / less ] microbially stable (less likely to be spoiled).
Fermentation
Alcoholic Fermentation: Pathway
Fermentation of the ‘grape must’ is an alcoholic fermentation by yeasts.
Exercise $25$
• Review: Redraw the chemical process and talk about the benefit to the microorganisms.
• Is this an aerobic or anaerobic process?
• Unlike brewer’s wort, the oxygen is usually not added until after inoculation. This is to prevent oxidation of _________________ in the grape must.
Alcoholic Fermentation: Organisms
Wine-makers can utilize wild fermentation or inoculation with a specific yeast strains of Saccharomyces cerevisiae.
In spontaneous wine fermentation, the fermentation begins with non-Saccharomyces yeasts until the ethanol concentration reaches 3–4%. As the alcohol concentration increases, these yeasts die off, and Saccharomyces dominates the fermentation process.
In inoculated ferments, S. cerevisiae is used to begin the fermentation process and its primary role is to catalyze the rapid, complete and efficient conversion of grape sugars to ethanol.
Exercise $26$
• Review: Explain the Crabtree effect.
Glycerol Production
A good wine will have the components of alcohol, acidity, sweetness, fruitiness and tannin structure complement each other so that
no single flavor overwhelms the others.
Recently, there has been a demand for a ‘richer’ red wine flavor; this has led winemakers to harvest grapes at a later stage to obtain more polyphenols and flavors. However, more mature grapes have increased sugar concentration.
Exercise $27$
• Increased sugar content leads to [ increased / decreased ] alcohol content in the wine.
In an attempt to develop full-bodied wines with lower alcohol content, researchers have been attempting to create strains of S. cerevisiae that produce glycerol instead of ethanol. Glycerol tastes slightly sweet with a slightly ‘oily’ mouthfeel but it does not dramatically change the overall sensory perception of the wine.
Exercise $28$
• Draw the structures of dihydroxyacetone, glycerol-3- phosphate, and glycerol.
• The addition of sulfite can bind to acetaldehyde preventing ethanol production leading to a [ increase / decrease ] of NAD+ .
• The organism then shifts to the glycerol synthesis pathway which will [ increase / decrease ] production of NAD+ .
Secondary Fermentation: Malolactic Fermentation (red wines)
Malic acid is described as a harsher or more aggressive acidic flavor. Wines with high levels of malic acid are submitted to malolactic fermentation (MLF). In general, winemakers use MLF to treat red wines more than whites. There are exceptions; oaked Chardonnay is often put through MLF.
Malolactic Fermentation is described in detail in “Cider”.
Exercise $29$
• Review: Redraw the chemical process and talk about the benefit to the microorganisms.
The bacteria behind this process can be found naturally in the winery, usually in the oak wine barrels used for aging. Alternatively, these bacteria can be introduced by the winemaker.
The bacteria used in MLF are usually Pediococcus (homofermentative), Leuconostoc (heterofermentative, Oenococcus (heterofermentative) or Lactobacillus (either).
Summarize MLF:
Exercise $30$
• You are more likely to find malic acid and [ lower / higher ] acidity in [ red / white ] wines.
• In MLF, bacteria convert the stronger malic acid into the softer __________acid.
• Thus, after MLF, wine has a [ lower / higher ] pH (less acidic), and a different mouthfeel.
• Winemakers (like cider makers) wishing to control or prevent MLF can use ____________ to inhibit the bacteria.
Aging
The wine aging has two phases: 1) ‘maturation’, changes after fermentation and before 2) ‘bottling’. During the aging process, changes in taste and flavor occur.
Traditional maturation involves the storage of wine in barrels for a few months to a few years (or even longer!). During this time, the wine undergoes reactions and absorbs compounds from the wood of the barrels.
Chemical Aging: Oxidations and Polymerizations
The polyphenolic component of the wine continues to undergo oxidations and polymerizations and condensations.
Chemical Aging: Compounds from wooden barrels
The main phenolic compounds extracted from the wood to the wine during barrel ageing are hydrolysable tannins and phenolic acids.
The volatile compounds extracted from wood are mainly furfural compounds, guaiacol, oak or whisky lactone, eugenol, vanillin, and syringaldehyde.
Exercise $31$
• Look up the structures and flavors of these compounds coming from the woods.
• Guaiacol
• Eugenol
• Vanillin
• Syringaldehyde
• Oak Lactone
Processing: Clarification & Filtration of Wine
As a wine ages, phenolic molecules combine to form tannin polymers that fall to the bottom of the bottle.
Unlike beer and cider, filtration is not a common process for wines so many older wines will have sediment. Many winemakers leave the sediments in the wine bottle. Wine drinkers can ‘decant’ the wine before drinking – pour off the wine leaving behind the sediment.
Fining is a technique that is used to remove unwanted juice/wine components that affect flavor and aroma.
Addition of Bentonite for Hazing
Bentonite is a clay made of soft silicate mineral that will absorb positively charged proteins that cause hazing of wines (particularly white wines).
Addition of Proteins for Astringency
Bovine Serine Albumin (BSA) or gelatin or casein are added to bind with excess tannins and precipitate out of the wine.
Exercise $32$
• Draw a picture of how a protein might interact with a polymeric tannin. Show IMF.
Filtration
Filtration is sometimes used to help control both MLF and Acetic Acid bacteria and other spoilage organisms since lees are a food source for the bacteria. LAB can continue the fermentation leading to off-flavors. Membrane filtration can be helpful at this point to remove organisms.
Stabilization
Exercise $33$
• What is another post-fermentation additive that might help with spoilage organisms?
Flavors and Aromas
Composition
The flavor and aroma components, including polyphenols, acids, aldehydes, esters, and fusel alcohols are a very small percentage of the overall beverage.
Exercise $34$
• What types of flavors do these components provide?
• Sugars:
• Ethanol:
• Polyphenols:
• Acids (tartaric, malic, lactic, citric):
Sweetness/Dry
A dry wine has little residual sugars, so it isn't sweet. Sugars are the main source of perceived sweetness in wine, and they come in many forms.
Exercise $35$
• To make a sweet wine, the easiest way is to stop fermentation before it is complete. Name 3-4 possible approaches to stop fermentation.
• Wine-makers will occasionally add sugar or juice after fermentation. There are regulations on this depending on the region. How do these practices impact flavor?
• Pre-fermentation:
• Post-fermentation:
While it seems paradoxical, many people have noticed that wines with higher sugar content last longer even when open to the air.
Exercise $36$
• [ Lower / Higher ] sugar levels would be more likely to support spoilage organisms.
Osmotic pressure seems to play a part: high concentrations of sugar force the water within a microbe to rush outward, and its cell walls collapse.
Exercise $37$
• Osmotic pressure and high levels of alcohol [ inhibit / increase ] microorganism growth.
Sweetness from Aging Processes
In 2017, scientists in Bordeaux discovered a set of molecules called quercotriterpenosides, which are released from oak during aging. These molecules are small but mighty, influencing the taste of wine at even low doses due to their extreme sweetness.
Other oak flavors can evoke sweetness: guaiacol, eugenol, and vanillin.
Exercise $39$
• Draw the structures of these three important phenolic compounds from oak barrels.
Glycerol can also provide a sweet sensation.
• Review: How does glycerol end up in the wine?
Aroma and flavors: Esters and alcohols
During alcoholic fermentation, many secondary metabolites are produced by yeast. Esters provide mainly fruity and floral notes; higher alcohols provide ‘background flavors’; whereas the phenolic compounds can generate interesting or unpleasant aromatic notes.
Esters are the main volatile compounds in cider. They are characterized by a high presence of ethyl acetate, which alone can represent up to 90% of the total esters.
Esters and Fusel Alcohols were covered in the ‘Beer’ Section.
Exercise $40$
• Review: What are the fusel alcohols and how are they formed?
• Review: Draw ethyl acetate and review the metabolic process for its synthesis from fermenting yeasts.
Too many esters or fusel alcohols are considered a fault in wines.
Wine faults: Microbial Byproducts
Spoilage: Acetic acid
Acetic acid is responsible for the sour taste of vinegar. During fermentation, activity by yeast cells naturally produces a small amount of acetic acid.
If the wine is exposed to oxygen, Acetobacter bacteria will convert the ethanol into acetic acid and is considered a fault.
The process for ‘acetification’ (conversion of ethanol to acetic acid by AAB is covered in the ‘Vinegar’ section.
Exercise $41$
• Review: Redraw the chemical process and talk about the benefit to the microorganisms.
Taints: Volatile Phenols
Lactobacilli and contaminant yeasts like Brettanomyces are often present during wine-making.
These organisms are often responsible for ‘taints’, unpleasant chemical flavors.
A common taint is the production of volatile phenols, compounds are derived from the naturally occurring hydroxycinnamic acids in grapes/wine.
Humans can taste volatile phenols at very low concentrations and can have a strong influence on wine aroma. These compounds are described as medicinal, animal, leather and ‘horse sweat’ odors.
Exercise $42$
• Draw the volatile phenols the would be formed from these common polyphenols found in grapes.
Taints: Bitterness taint
Bitterness taint is produced by LAB. The bacteria degrade glycerol, a compound naturally found in wine, to 3-hydroxypropionaldehyde. During aging, this is converted to acrolein which reacts with the anthocyanins and other phenols present within the wine.
Exercise $43$
1. What ‘reagent’ is needed in this pathway?
2. Propose a possible product of the reaction of an acrolein with an anthocyanin. These adducts are bitter.
Taints: Mannitol Taint
Mannitol is often described as an ester flavor with a sweet and irritating aftertaste. This was covered in the Cider section.
Exercise $44$
Draw the pathways for the production of mannitol.
Taints: Diacetyl taint
Diacetyl in wine is produced by lactic acid bacteria. This compound has an intense buttery flavor.
This was covered in the Beer section.
Exercise $45$
Draw the pathways for the production of diacetyl.
Taints: Geranium taint
Potassium sorbate is sometimes added to wine as a preservative against yeast. However, LAB will metabolize the sorbic acid into 2-ethoxyhexa-3,5-diene which provides a flavor reminiscent of geranium leaves.
Exercise $46$
• Fill in the missing biological cofactor.
• Many alternate microbial pathways such as the metabolism of sorbate and fructose and acetoin use the same ‘reagent’. Why are the LAB metabolizing these compounds using this cofactor?
Taints: Mousiness
Mousiness is a wine fault that can occur during MLF. The compounds responsible are lysine derivatives. The taints are not volatile but, when mixed with saliva in the mouth, they provide a flavor of mouse urine.
2-ethyltetrahydropyridine 2-acetlypyrroline
Taints: Ropiness
Certain species of Leuconostoc have been found to produce dextran slime or mucilaginous substances in wine.
Sources
Belda, et. al., Microbial Contribution to Wine Aroma, Molecules 2017, 22(2), 189
Casassa, Flavonoid Phenolics in Red Winemaking In Grapes and Wine, A. M. Jordão, Ed., 2018, InTechOpen.
Chantal Ghanam, Study of the Impact of Oenological Processes on the Phenolic Composition of Wines, Thesis, Université de Toulouse.
Dangles & Fenger, The Chemical Reactivity of Anthocyanins, Molecules, 2018, 23(8), 1970-1993.
Danilewicz, Role of Tartaric and Malic Acids in Wine Oxidation, J. Agric. Food Chem. 2014, 62, 22, 5149-5155.
du Toit & Pretorius, Microbial Spoilage, S. Afr. J. Enol. Vitic. 2000, 21, 74-96.
E.J. Bartowsky, Bacterial Spoilage of Wine, Letters in Applied Microbiology, 2009, 48, 149–156.
Garrido & Borges, Wine and Grape Polyphenols, Food Research International, 2013, 54, 1844–1858
Goold, et. al. Yeast's balancing act between ethanol and glycerol production in low-alcohol wines, Microbial Biotechnology 2017, 10(2), 1-15.
He, et. al., Anthocyanins and Their Variation in Red Wines, Molecules, 2012, 17(2), 1483-1519.
J. Harbertson, A Guide to the Fining of Wine, Washington State University
Li, Guo, & Wang, Mechanisms of Oxidative Browning of Wine, Food Chemistry, 2008, 108, 1-13.
Marchal, et. al. Identification of New Natural Sweet Compounds in Wine, Anal. Chem, 2011, 83 (24), 9629-9637.
Niculescu, Paun, and Ionete, The Evolution of Polyphenols from Must to Wine, In Grapes and Wine, A. M. Jordão, Ed., 2018, InTechOpen. | textbooks/chem/Biological_Chemistry/Fermentation_in_Food_Chemistry/1.15%3A_Wine.txt |
Distilled Alcoholic Beverages
Distilled spirits are all alcoholic beverages in which the concentration of ethanol has been increased above that of the original fermented mixture by a method called distillation. More Information about Distilling: Artisanal Distilling, A Guide for Small Distilleries, Kris Berglund
Distilled Spirits Production Steps:
Raw Materials
Any sugar containing fruit or syrup can be used for fermentation and then distilled to prepare spirits. Similarly, grains and potatoes are fermentable and can be used for whiskey or vodka production. Like wine and cider production, the fruits are harvested and mashed to release enzymes and simple mono- and di-saccharides.
Exercise $1$
Review: Describe the steps and any necessary additives (like pectinases and sulfite)
For grain spirits, the process involves malting of the grain, milling, boiling a mash to release the complex carbohydrates.
Exercise $2$
Review: Describe the steps and any necessary additives (like amylases)
Preparation of the Mash
Fruit Spirits: Minimize Methanol Production
As you remember from the ‘Cider’ unit, many fruits contain a large amount of pectin. Pectin is a polymer of the sugar galacturonic acid.
Exercise $3$
• Draw the monomer unit.
• What type of linkages are used in this polysaccharide? Circle the correct designations.
• $\alpha$ or $\beta$
• 1-2 1-3 1-4 1-5 1-6 2-4
This pectin can form a gel that is undesirable in ciders or fruit beverages, so it is necessary to allow native pectolytic enzymes to hydrolyze this polysaccharide. In addition, some producers add extra pectolytic enzymes. Pectin methylesterase is an enzyme found in cherries, pears, and apples that hydrolyzes the esters that are on the side chains of pectin.
Exercise $3$
Show the product of this reaction.
In cider or wines, the small amounts of methanol formed in this process are not a concern. However, when the wine is distilled the methanol is also concentrated and can have toxic impacts on consumers. One way to limit the formation of methanol is by heating of the mash to a temperature of 80- 85 °C.
Exercise $4$
What will this do to the enzyme?
Fermentation
Fermentation is the same process as seen in the previous discussions of Bread, Beer, Cider, and Wine.
Exercise $5$
• In grains, the amylose is converted to the disaccharide ________________ with the ____________ enzyme presented in the grain.
• Mannose is converted to glucose with the yeast enzyme _________________.
• Sucrose (and other di- and tri-saccharides) can be converted to glucose and fructose with the yeast enzyme ___________________.
• The primary fermenting organism is _________________.
• This alcoholic fermentation is [ aerobic or anaerobic ].
• Define the Crabtree effect.
• During fermentation, glucose and/or fructose are converted to _______________ and __________________.
• Sulfur dioxide is often added to these fermentation mashes to prevent ________________.
• The higher the sugar content of the mash or must or pomace, then the final alcohol content will be [ higher / lower ] .
• Due to the toxicity of ethanol, the maximum alcohol content for a fermentation is:
5% 10% 15% 20% 25% 30% 35% 45% 50% 60% 75%
• Fusel alcohols are yeast fermentation side-products derived from:
• Esters are yeast fermentation side-products formed from
Distillation
Theory
Distillation in the concentration of ethanol content in an alcoholic beverage through boiling. Ethanol boils at a lower temperature ( 78.4 °C or 173.12 °F) than water ( 100 °C or 212 °F). When the fermentation mixture is heated, the ethanol is evaporated in a higher concentration in the steam. This is condensed and collected resulting in a product that is approximately 25- 35% alcohol.
Exercise $6$
If a distillery desires a higher concentration of alcohol, then what will they need to do?
Still: The pot still
The still vessel is filled with mash, wine, or beer up to 50-75 % full and then closed. More viscous mashes are diluted with 20 % water. Pomaces which yield a low alcohol content are mixed preferentially with 20 % coarse spirit.
Exercise $7$
1. As the mash is boiled, the ethanol and water are ________________.
2. As the vapors move through the condenser tube which is cooled by water, the ethanol and water will ___________________.
Most distilleries use copper stills as they produce cleaner and aromatic because copper reacts with the sulfur side-products found in mashes to form non-volatile compounds.
Exercise $8$
What is the problem with sulfur side-products in mash (and then the final product)?
Boiling points of different alcohols present in mashes:
Alcohol Boiling Point (oC)
Ethanol 78
Methanol 65
Small Aldehydes, Esters 30-60
Fusel Alcohols, (amyl alcohol, isoamyl, etc) 115-140
Most distillers will collect three fractions from the distillation process: fore-run (head), middlerun (heart), and after-run (tail).
Exercise $9$
What is the primary component(s) in each fraction?
• Fore-run:
• Middle-run:
• After-run:
Which fraction will be sold as a distilled spirit?
With direct heating of the fermentation product in the pot stills, the highly viscous mashes/fruit pulps can lead to burning.
Exercise $10$
The decomposition products of sugar leads to ___________________. The products formed in this process can lend a bitter or burnt flavor to the final distilled spirits.
Direct heat or not?
Wood fires directly below the pot are problematic due to leads to concerns about burning the mash and possible explosions.
Exercise $11$
Why is distillation prone to fires? Hint: consider flammability of the product.
Some whisky distillers choose to use the wood fired heating because they like the flavors. To keep the mash from burning, they use a ‘rummager’ to continuously stir the mash. The fire also requires careful tending, making sure it’s not burning too hot or too cold. To prevent burning the mash, other distillers have moved to steam, hot water baths around the pot, or electrical heating.
Still: The column still
With column distillation, the mash enters near the top of the still and begins flowing downward. This brings it closer to the heating source, and once it’s heated enough to evaporate, the vapor rises up through a series of partitions known as plates or stripping plates. At each plate along the way, the vapor ends up leaving behind some of the higher boiling compounds. It is important to note that pot stills operate on a batch by batch basis, while column stills may be operated continuously allowing higher throughput.
Exercise $12$
• Draw a picture of column still.
Is scorching a problem with this method?
Aging
Chemistry of Aging in Barrels
The aging process is similar to wines. The aging process allows tannins, terpenes, lignins, polyphenols, and minerals from the wood of the barrel to dissolve into the spirits. Many of the barrels have been charred so there are oxidized lignin and wood sugars also available. As these compounds are dissolved into the spirits, new condensation and oxidation reactions can occur during this process.
Some barrels have been previously used for wines so they will also release flavors from the polyphenols of wines that were absorbed into the wood.
Process
Each of the distilled spirits have a slightly different aging process.
• Apple spirits, cherry spirits, and brandies a short storage in oak barrels (or steel tanks) proved to be best.
• Kirsch (cherry) and plum distillates are aged in closed glass carboys or tanks in a warm environment for aging.
• Single malt scotch and Irish whiskeys must all be aged for a minimum of 3 years.
• Bourbon can only be aged in a new barrel while whiskies are usually aged in barrels that previously contained sherry or other wines.
• Not all distilled spirits are aged. Tequila, rum, vodkas, moonshine, and gins are can be unaged.
You will notice that the more northern the climate in which the distilled spirit is produced, the longer it is aged.
Exercise $13$
• Suggest a reason for the differences in aging times in different climates.
Choosing to Age or Not
Exercise $14$
• A pot distilled spirit will have [ more / less ] flavor and aroma compounds.
• A [ column or pot ] distilled spirit will benefit from aging.
Processing
Blending with Water
Most distillates have greater than 40-45 % alcohol content. In order to be drinkable, they have to be watered down.
Cool Storage
The distilled spirits still contain a variety of flavor and aroma compounds from the original mash, the fermentation process, the Maillard reaction in the still, or from the wood barrels in the aging process. Some of these compounds can cause a cloudy or hazy appearance to the distilled spirits.
Distillers will often cool the spirits to between 0 and -10 °C.
Exercise $15$
• A compound with low solubility will _______________ at these temperatures.
Filtration
After cold storage, the distilled spirits are filtered to remove any precipitates.
Bottling
The bottling of the distilled spirits is straightforward.
Exercise $16$
• Unlike beer, cider, and wine, there is usually not a problem with contamination from microorganisms. Why?
Flavors and Aromas
There are many flavors in distilled spirits. It is highly dependent upon the original raw materials, yeast fermentation process, presence of any microbial contaminants, aging, etc.
However, distillation can intensify flavors that are found in the middle-run, but many other flavors do not get transferred from the pot to the distillate.
It is important to note that the addition of flavorings, sugars or other sweetening products after distillation is forbidden for distilled beverages such as rum, whisky, fruit distillates or wine brandy. The addition of caramel in fruit distillates is not allowed, while whiskey is allowed plain caramel coloring only.
Exercise $17$
• The flavors of the original grains or fruits would be expected to be [ strong / light ].
• The color of most distilled spirits is _________________.
• Distilled spirits are [ sweet / not sweet ].
• The primary components of distilled spirits are water and _____________.
Types of Distilled Spirits
The most common spirits are those derived from grains (whiskey, vodkas), grapes (cognac, brandy), molasses (rum), and agave (tequila).
Whiskey
Whisky is a distilled beverage from cereal grains and matured in barrels. There are different regional variations on this drink. The malt from corn, barley, rye, or wheat is mashed in a process similar to beer. The wort is then directly distilled.
Exercise $18$
• Look up the differences in the grains, malting, distillation, and aging process for these whiskies:
• Scotch Whisky:
• Irish Whiskey:
• American Bourbon:
• Rye Whiskey (Canadian Whiskey):
Brandy
Brandy is a distilled wine beverage.
Exercise $19$
• Describe the process for the production of brandy. Comment on the variations such as Armagnac, Cognac, and Pisco.
Rum
Rum is a distilled beverage from sugar cane.
Exercise $20$
• Describe the process for the production of rum.
Tequila
Tequila is a distilled beverage from agave.
Exercise $21$
• Describe the process for the production of tequila.
Eau de vie
An eau de vie is a clear fruit brandy that is produced by means of fermentation and double distillation. For example, Framboise is a double distilled raspberry brandy. Unlike liqueurs, eae de vie are not sweetened. Although eau de vie is a French term, similar beverages are produced in other countries (e.g. German Schnapps, German Kirschwasser, Turkish rakı, Hungarian pálinka, and Sri Lankan coconut arrack).
Exercise $22$
• The fruit flavor in eau de vie is typically [ strong / light ].
Liqueurs
Liqueurs are drinks made by adding fruit, herbs or nuts to neutral distilled spirits. Usually a distilled beverage like vodka is used as it is mostly alcohol and little flavoring. They are often also heavily sweetened. They are often served with dessert. You might drink it straight, with coffee, used in cocktails, or in cooking.
Typical Liqueurs
Liqueur Flavor
Absinthe Brandy with anise, fennel, wormwood
Amaretto Apricot and almond flavors
Bailey’s Irish Whiskey and chocolate
Benedictine Brandy with 27 herbs and spices
Cherry Brandy Brandy with cherries
Cointreau Distillates from bitter and sweet orange peels
Drambuie Scotch Whisky with herbs and honey
Grand Marnier Cognac blended with bitter orange and sugar
Kahlua Rum with coffee, sugar, vanilla
Malibu Rum with coconut
Sambuca Anis, sugar
Exercise $23$
• The flavors in liqueurs are typically [ strong / light ] compared to a brandy or eau de vie.
• The flavors are added [ before / after ] distillation.
• Unlike spirits, liqueurs have added _____________.
Sources
Coldea, Mudura & Socaciu, Chapter 6: Advances in Distilled Beverages Authenticity and Quality Testing, In Ideas and Applications Toward Sample Preparation for Food and Beverage Analysis, M. Stauffer, Ed., IntechOpen, 2017.
S. Canas, Phenolic Composition and Related Properties of Aged Wine Spirits: Influence of Barrel Characteristics. A Review, Beverages, 2017, 3(4), 55-77.
Schaller, Structure and Reactivity, Purification of Molecular Compounds, PM3: Distillation
N. Spaho, Ch 6: Distillation Techniques in the Fruit Spirits Production, In Distillation – Innovative Applications and Modeling, M. Mendes, Ed., IntechOpen, 2017. | textbooks/chem/Biological_Chemistry/Fermentation_in_Food_Chemistry/1.16%3A_Distilled_Spirits.txt |
Carbohydrates, also known as sugars, are found in all living organisms. They are essential to the very source of life (ex. Ribose sugars in DNA and RNA) or sustaining life itself (e.g., Metabolic conversion of carbohydrates into usable biochemical energy, ATP). Another important role of carbohydrates is structural (ex. Cellulose in plants). General names for carbohydrates include sugars, starches, saccharides, and polysaccharides. The term saccharide is derived from the Latin word "sacchararum" from the sweet taste of sugars. The name "carbohydrate" means a "hydrate of carbon." The name derives from the general formula of carbohydrate is Cx(H2O)y - x and y may or may not be equal and range in value from 3 to 12 or more. For example glucose is: C6(H2O)6 or is more commonly written, C6H12O6.
Thumbnail: Haworth formula of D-glucose.
Carbohydrates Fundamentals
There are a variety of interrelated classification schemes. The most useful classification scheme divides the carbohydrates into groups according to the number of individual simple sugar units. Monosaccharides contain a single unit; disaccharides contain two sugar units; and polysaccharides contain many sugar units as in polymers - most contain glucose as the monosaccharide unit.
Monosaccharides Disaccharides
Carbohydrate Isomers
Glyceraldehyde, the simplest carbohydrate, exhibits properties of a chiral or optical isomer compound. This molecule forms the basis for the designation of the isomers of all of the carbohydrates.
Introduction
Glyceraldehyde can exist in two isomeric forms that are mirror images of each other which are shown below. The absolute configuration is defined by the molecule on the far left as the D-glyceraldehyde. With the aldehyde group in the "up" direction, the the -OH group must project to the right side of the molecule for the D isomer. Chemists have used this configuration of D-glyceraldehyde to determine the optical isomer families of the rest of the carbohydrates. All naturally occurring monosaccharides belong to the D optical family. It is remarkable that the chemistry and enzymes of all living things can tell the difference between the geometry of one optical isomer over the other.
Monosaccharides are assigned to the D-family according to the projection of the -OH group to the right on the chiral carbon that is the farthest from the carbonyl (aldehyde) group. This is on carbon # 5 if the carbonyl carbon is # 1.
Note: For whatever reason, the ball and stick model does not completely match the projections of the -OH groups on carbons # 2 and 4. It is in the way that the flat Fischer model has been defined.
How many chiral carbons can you find? List them. If necessary Review Chiral Compounds to find the definitions.Then check the answer from the drop down menu.
Compare Glucose and Galactose
Examine the structures of glucose and galactose carefully. Which -OH group determines that they both are the D isomer? Then check the answer from the drop down menu.
Isomers have different arrangements of atoms. Which carbon bonding to -OH and -H is different in glucose vs. galactose? This single difference makes glucose and galactose isomers. Then check the answer from the drop down menu.
Contributors
• Charles Ophardt, Professor Emeritus, Elmhurst College; Virtual Chembook | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Carbohydrates/Carbohydrates_Fundamentals/Carbohydrate_Classification.txt |
Introduction
The chemistry of carbohydrates most closely resembles that of alcohol, aldehyde, and ketone functional groups. As a result, the modern definition of a CARBOHYDRATE is that the compounds are polyhydroxy aldehydes or ketones. The chemistry of carbohydrates is complicated by the fact that there is a functional group (alcohol) on almost every carbon. In addition, the carbohydrate may exist in either a straight chain or a ring structure. Ring structures incorporate two additional functional groups: the hemiacetal and acetal.
A major part of the carbon cycle occurs as carbon dioxide is converted to carbohydrates through photosynthesis. Carbohydrates are utilized by animals and humans in metabolism to produce energy and other compounds.
Photosynthesis is a complex series of reactions carried out by algae, phytoplankton, and the leaves in plants, which utilize the energy from the sun. The simplified version of this chemical reaction is to utilize carbon dioxide molecules from the air and water molecules and the energy from the sun to produce a simple sugar such as glucose and oxygen molecules as a by product. The simple sugars are then converted into other molecules such as starch, fats, proteins, enzymes, and DNA/RNA i.e. all of the other molecules in living plants. All of the "matter/stuff" of a plant ultimately is produced as a result of this photosynthesis reaction.
Di- and Poly-Carbohydrates
• Monosaccharides contain one sugar unit such as glucose, galactose, fructose, etc.
• Disaccharides contain two sugar units. In almost all cases one of the sugars is glucose, with the other sugar being galactose, fructose, or another glucose. Common disaccharides are maltose, lactose, and sucrose.
• Polysaccharides contain many sugar units in long polymer chains of many repeating units. The most common sugar unit is glucose. Common poly saccharides are starch, glycogen, and cellulose.
Table 1: Common Carbohydrates
Name Derivation of name and Source
Monosaccharides
Glucose From Greek word for sweet wine; grape sugar, blood sugar, dextrose.
Galactose Greek word for milk--"galact", found as a component of lactose in milk.
Fructose Latin word for fruit--"fructus", also known as levulose,found in fruits and honey; sweetest sugar.
Ribose Ribose and Deoxyribose are found in the backbone structure of RNA and DNA, respectively.
Disaccharides - contain two monosaccharides
Sucrose French word for sugar--"sucre", a disaccharide containing glucose and fructose; table sugar, cane sugar, beet sugar.
Lactose Latin word for milk--"lact"; a disaccharide found in milk containing glucose and galactose.
Maltose
French word for "malt"; a disaccharide containing two units of glucose; found in germinating grains, used to make beer.
Common Polysaccharides
Starch Plants store glucose as the polysaccharide starch. The cereal grains (wheat, rice, corn, oats, barley) as well as tubers such as potatoes are rich in starch.
Cellulose The major component in the rigid cell walls in plants is cellulose and is a linear polysaccharide polymer with many glucose monosaccharide units.
Glycogen This is the storage form of glucose in animals and humans which is analogous to the starch in plants. Glycogen is synthesized and stored mainly in the liver and the muscles.
Metabolism
Metabolism occurs in animals and humans after the ingestion of organic plant or animal foods. In the cells a series of complex reactions occurs with oxygen to convert for example glucose sugar into the products of carbon dioxide and water and ENERGY. This reaction is also carried out by bacteria in the decomposition/decay of waste materials on land and in the water.
Combustion occurs when any organic material is reacted (burned) in the presence of oxygen to give off the products of carbon dioxide and water and ENERGY. The organic material can be any fossil fuel such as natural gas (methane), oil, or coal. Other organic materials that combust are wood, paper, plastics, and cloth.
The whole purpose of both processes is to convert chemical energy into other forms of energy such as heat.
Contributors
• Charles Ophardt, Professor Emeritus, Elmhurst College; Virtual Chembook
Haworth Formula
Traditionally, in carbohydrate chemistry, the furanose rings and the pyranose rings in carbohydrate molecules are shown in the planar conformation, placed on the plane perpendicular to the plane of the paper.
This representation of rings is known as the Haworth formula. eg: cyclic forms of D-glucose
To generate the Haworth formulas of the cyclic forms of a monosaccharide, use the following procedure, explained using the pyranoses of D-glucose.
Step 1: Draw the Fischer projection of the acyclic form of D-glucose. (See D,L convention)
Step 2: Number the carbon chain in 1 starting at the top.
Step 3: To generate the pyranose ring, the oxygen atom on C-5 in 1 needs to be attached to C-1 by a single bond.
In 1, C-1 is behind the plane of the paper and the hydroxy group on C-5 is in front. For the pyranose ring to be planar, both C-1 and the hydroxy group on C-5 have to be either behind or in front of the plane of the paper. C-5 is a chiral center. In order to bring the hydroxy group on C-5 to the site occupied by the \(CH_2OH\) group without changing the absolute configuration at C-5, rotate the three ligands H, OH, and CH2OH on C-5 in 1 clockwise without moving the fourth ligand. (See Fischer projection)
1 and 2 both represent D-glucose, but, in 2, unlike in 1, C-1 and the hydroxy group on C-5 are on the same side of the plane of the paper.
Step 4: Ignore that 2 is a Fischer projection and rotate it clockwise by 90º.
Step 5: Redraw the atom chain along the horizontal axis as follows.
Step 6: Add the ligands on C-2 through C-5 in 4. The ligands pointing up in 3 are pointing up in 4; those pointing down in 3 are pointing down in 4.
Step 7: Remove the hydrogen atom and the oxygen atom on C-1 and the hydrogen atom in the hydroxy group on C-5 in 5 and connect the two atoms by a single bond.
Step 8: Add the two remaining bonds to C-1 in 6.
Step 9: Attach a hydrogen atom to the bond pointing up and a hydroxy group to the bond pointing down on C-1 in 7.
Step 10: Interchange the hydrogen atom and the hydroxy group on C-1 in 8.
8 and 9 are the Haworth formulas of the pyranoses of D-glucose. If, in the acyclic form of a monosaccharide, the hydroxy group that reacts with the carbonyl carbon is not on a chiral carbon (eg: D-fructose→pyranoses), skip step 3. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Carbohydrates/Carbohydrates_Fundamentals/Carbohydrate_Overview.txt |
Carbohydrates are the most abundant class of organic compounds found in living organisms. They originate as products of photosynthesis, an endothermic reductive condensation of carbon dioxide requiring light energy and the pigment chlorophyll.
n CO2 + n H2O + energy CnH2nOn + n O2
Introduction
The formulas of many carbohydrates can be written as carbon hydrates, Cn(H2O)n, hence their name. The carbohydrates are a major source of metabolic energy, both for plants and for animals that depend on plants for food. Aside from the sugars and starches that meet this vital nutritional role, carbohydrates also serve as a structural material (cellulose), a component of the energy transport compound ATP, recognition sites on cell surfaces, and one of three essential components of DNA and RNA.
Carbohydrates are called saccharides or, if they are relatively small, sugars. Several classifications of carbohydrates have proven useful, and are outlined in the following table.
1. Carbohydrates have been given non-systematic names, although the suffix ose is generally used. The most common carbohydrate is glucose (C6H12O6). Applying the terms defined above, glucose is a monosaccharide, an aldohexose (note that the function and size classifications are combined in one word) and a reducing sugar. The general structure of glucose and many other aldohexoses was established by simple chemical reactions. The following diagram illustrates the kind of evidence considered, although some of the reagents shown here are different from those used by the original scientists.
Glucose and other saccharides are extensively cleaved by periodic acid, thanks to the abundance of vicinal diol moieties in their structure. This oxidative cleavage, known as the Malaprade reaction is particularly useful for the analysis of selective O-substituted derivatives of saccharides, since ether functions do not react. The stoichiometry of aldohexose cleavage is shown in the following equation.
HOCH2(CHOH)4CHO + 5 HIO4 ——> H2C=O + 5 HCO2H + 5 HIO3
The Configuration of Glucose
The four chiral centers in glucose indicate there may be as many as sixteen (24) stereoisomers having this constitution. These would exist as eight diastereomeric pairs of enantiomers, and the initial challenge was to determine which of the eight corresponded to glucose. This challenge was accepted and met in 1891 by the German chemist Emil Fischer. His successful negotiation of the stereochemical maze presented by the aldohexoses was a logical tour de force, and it is fitting that he received the 1902 Nobel Prize for chemistry for this accomplishment. One of the first tasks faced by Fischer was to devise a method of representing the configuration of each chiral center in an unambiguous manner. To this end, he invented a simple technique for drawing chains of chiral centers, that we now call the Fischer projection formula. Click on this link for a review.
At the time Fischer undertook the glucose project it was not possible to establish the absolute configuration of an enantiomer. Consequently, Fischer made an arbitrary choice for (+)-glucose and established a network of related aldose configurations that he called the D-family. The mirror images of these configurations were then designated the L-family of aldoses. To illustrate using present day knowledge, Fischer projection formulas and names for the D-aldose family (three to six-carbon atoms) are shown below, with the asymmetric carbon atoms (chiral centers) colored red. The last chiral center in an aldose chain (farthest from the aldehyde group) was chosen by Fischer as the D / L designator site. If the hydroxyl group in the projection formula pointed to the right, it was defined as a member of the D-family. A left directed hydroxyl group (the mirror image) then represented the L-family. Fischer's initial assignment of the D-configuration had a 50:50 chance of being right, but all his subsequent conclusions concerning the relative configurations of various aldoses were soundly based. In 1951 x-ray fluorescence studies of (+)-tartaric acid, carried out in the Netherlands by Johannes Martin Bijvoet (pronounced "buy foot"), proved that Fischer's choice was correct.
It is important to recognize that the sign of a compound's specific rotation (an experimental number) does not correlate with its configuration (D or L). It is a simple matter to measure an optical rotation with a polarimeter. Determining an absolute configuration usually requires chemical interconversion with known compounds by stereospecific reaction paths.
Models of representative aldoses may be examined by clicking on the Fischer formulas for glyceraldehyde, erythrose, threose, ribose, arabinose, allose, altrose, glucose or mannose in the above diagram.
Important Reactions
Emil Fischer made use of several key reactions in the course of his carbohydrate studies. These are described here, together with the information that each delivers.
Oxidation
As noted above, sugars may be classified as reducing or non-reducing based on their reactivity with Tollens', Benedict's or Fehling's reagents. If a sugar is oxidized by these reagents it is calledreducing, since the oxidant (Ag(+) or Cu(+2)) is reduced in the reaction, as evidenced by formation of a silver mirror or precipitation of cuprous oxide. The Tollens' test is commonly used to detect aldehyde functions; and because of the facile interconversion of ketoses and aldoses under the basic conditions of this test, ketoses such as fructose also react and are classified as reducing sugars.
When the aldehyde function of an aldose is oxidized to a carboxylic acid the product is called an aldonic acid. Because of the 2º hydroxyl functions that are also present in these compounds, a mild oxidizing agent such as hypobromite must be used for this conversion (equation 1). If both ends of an aldose chain are oxidized to carboxylic acids the product is called an aldaric acid. By converting an aldose to its corresponding aldaric acid derivative, the ends of the chain become identical (this could also be accomplished by reducing the aldehyde to CH2OH, as noted below). Such an operation will disclose any latent symmetry in the remaining molecule. Thus, ribose, xylose, allose and galactose yield achiral aldaric acids which are, of course, not optically active. The ribose oxidation is shown in equation 2 below.
1.
2.
3.
Other aldose sugars may give identical chiral aldaric acid products, implying a unique configurational relationship. The examples of arabinose and lyxose shown in equation 3 above illustrate this result. Remember, a Fischer projection formula may be rotated by 180º in the plane of projection without changing its configuration.
Reduction
Sodium borohydride reduction of an aldose makes the ends of the resulting alditol chain identical, HOCH2(CHOH)nCH2OH, thereby accomplishing the same configurational change produced by oxidation to an aldaric acid. Thus, allitol and galactitol from reduction of allose and galactose are achiral, and altrose and talose are reduced to the same chiral alditol. A summary of these redox reactions, and derivative nomenclature is given in the following table.
Derivatives of HOCH2(CHOH)nCHO
HOBr Oxidation ——> HOCH2(CHOH)nCO2H
an Aldonic Acid
HNO3 Oxidation ——> H2OC(CHOH)nCO2H
an Aldaric Acid
NaBH4 Reduction ——> HOCH2(CHOH)nCH2OH
an Alditol
Osazone Formation
1.
2.
The osazone reaction was developed and used by Emil Fischer to identify aldose sugars differing in configuration only at the alpha-carbon. The upper equation shows the general form of the osazone reaction, which effects an alpha-carbon oxidation with formation of a bis-phenylhydrazone, known as an osazone. Application of the osazone reaction to D-glucose and D-mannose demonstrates that these compounds differ in configuration only at C-2.
Chain Shortening and Lengthening
1.
2.
These two procedures permit an aldose of a given size to be related to homologous smaller and larger aldoses. The importance of these relationships may be seen in the array of aldose structurespresented earlier, where the structural connections are given by the dashed blue lines. Thus Ruff degradation of the pentose arabinose gives the tetrose erythrose. Working in the opposite direction, a Kiliani-Fischer synthesis applied to arabinose gives a mixture of glucose and mannose. An alternative chain shortening procedure known as the Wohl degradation is essentially the reverse of the Kiliani-Fischer synthesis.
Using these reactions we can now follow Fischer's train of logic in assigning the configuration of D-glucose.
1. In order to determine which of these epimers was glucose, Fischer made use of the inherent C2 symmetry in the four-carbon dissymmetric core of one epimer (B). This is shown in the following diagram by a red dot where the symmetry axis passes through the projection formula. Because of this symmetry, if the aldehyde and 1º-alcohol functions at the ends of the chain are exchanged, epimer B would be unchanged; whereas A would be converted to a different compound. By clicking on the diagram, the consequences of such an exchange will be displayed.
Ketoses
If a monosaccharide has a carbonyl function on one of the inner atoms of the carbon chain it is classified as a ketose. Dihydroxyacetone may not be a sugar, but it is included as the ketose analog of glyceraldehyde. The carbonyl group is commonly found at C-2, as illustrated by the following examples (chiral centers are colored red). As expected, the carbonyl function of a ketose may be reduced by sodium borohydride, usually to a mixture of epimeric products. D-Fructose, the sweetest of the common natural sugars, is for example reduced to a mixture of D-glucitol (sorbitol) and D-mannitol, named after the aldohexoses from which they may also be obtained by analogous reduction. Mannitol is itself a common natural carbohydrate.
Although the ketoses are distinct isomers of the aldose monosaccharides, the chemistry of both classes is linked due to their facile interconversion in the presence of acid or base catalysts. This interconversion, and the corresponding epimerization at sites alpha to the carbonyl functions, occurs by way of an enediol tautomeric intermediate. By clicking on the diagram, an equation illustrating these isomerizations will be displayed.
Because of base-catalyzed isomerizations of this kind, the Tollens' reagent is not useful for distinguishing aldoses from ketoses or for specific oxidation of aldoses to the corresponding aldonic acids. Oxidation by HOBr is preferred for the latter conversion.
Anomeric Forms of Glucose
Fischer's brilliant elucidation of the configuration of glucose did not remove all uncertainty concerning its structure. Two different crystalline forms of glucose were reported in 1895. Each of these gave all the characteristic reactions of glucose, and when dissolved in water equilibrated to the same mixture. This equilibration takes place over a period of many minutes, and the change in optical activity that occurs is called mutarotation. These facts are summarized in the diagram below.
When glucose was converted to its pentamethyl ether (reaction with excess CH3I & AgOH), two different isomers were isolated, and neither exhibited the expected aldehyde reactions. Acid-catalyzed hydrolysis of the pentamethyl ether derivatives, however, gave a tetramethyl derivative that was oxidized by Tollen's reagent and reduced by sodium borohydride, as expected for an aldehyde. These reactions will be displayed above by clicking on the diagram.
The search for scientific truth often proceeds in stages, and the structural elucidation of glucose serves as a good example. It should be clear from the new evidence presented above, that the open chain pentahydroxyhexanal structure drawn above must be modified. Somehow a new stereogenic center must be created, and the aldehyde must be deactivated in the pentamethyl derivative. A simple solution to this dilemma is achieved by converting the open aldehyde structure for glucose into a cyclic hemiacetal, called a glucopyranose, as shown in the following diagram. The linear aldehyde is tipped on its side, and rotation about the C4-C5 bond brings the C5-hydroxyl function close to the aldehyde carbon. For ease of viewing, the six-membered hemiacetal structure is drawn as a flat hexagon, but it actually assumes a chair conformation. The hemiacetal carbon atom (C-1) becomes a new stereogenic center, commonly referred to as the anomeric carbon, and the α and β-isomers are called anomers.
We can now consider how this modification of the glucose structure accounts for the puzzling facts noted above. First, we know that hemiacetals are in equilibrium with their carbonyl and alcohol components when in solution. Consequently, fresh solutions of either alpha or beta-glucose crystals in water should establish an equilibrium mixture of both anomers, plus the open chain chain form. This will be shown above by clicking on the diagram. Note that despite the very low concentration of the open chain aldehyde in this mixture, typical chemical reactions of aldehydes take place rapidly.
Second, a pentamethyl ether derivative of the pyranose structure converts the hemiacetal function to an acetal. Acetals are stable to base, so this product should not react with Tollen's reagent or be reduced by sodium borohydride. Acid hydrolysis of acetals regenerates the carbonyl and alcohol components, and in the case of the glucose derivative this will be a tetramethyl ether of the pyranose hemiacetal. This compound will, of course, undergo typical aldehyde reactions. By clicking on the diagram a second time this relationship will be displayed above.
5. Cyclic Forms of Monosaccharides
As noted above, the preferred structural form of many monosaccharides may be that of a cyclic hemiacetal. Five and six-membered rings are favored over other ring sizes because of their low angle and eclipsing strain. Cyclic structures of this kind are termed furanose (five-membered) or pyranose (six-membered), reflecting the ring size relationship to the common heterocyclic compounds furan and pyran shown on the right. Ribose, an important aldopentose, commonly adopts a furanose structure, as shown in the following illustration. By convention for the D-family, the five-membered furanose ring is drawn in an edgewise projection with the ring oxygen positioned away from the viewer. The anomeric carbon atom (colored red here) is placed on the right. The upper bond to this carbon is defined as beta, the lower bond then is alpha.
Click on the following diagram to see a model of β-D-ribofuranose.
The cyclic pyranose forms of various monosaccharides are often drawn in a flat projection known as a Haworth formula, after the British chemist, Norman Haworth. As with the furanose ring, the anomeric carbon is placed on the right with the ring oxygen to the back of the edgewise view. In the D-family, the alpha and beta bonds have the same orientation defined for the furanose ring (beta is up & alpha is down). These Haworth formulas are convenient for displaying stereochemical relationships, but do not represent the true shape of the molecules. We know that these molecules are actually puckered in a fashion we call a chair conformation. Examples of four typical pyranose structures are shown below, both as Haworth projections and as the more representative chair conformers. The anomeric carbons are colored red.
Models of these glucose, galactose, mannose and allose pyranose structures may be viewed by Clicking Here.
A practice page for examining the configurations of aldohexoses may be viewed by Clicking Here.
The size of the cyclic hemiacetal ring adopted by a given sugar is not constant, but may vary with substituents and other structural features. Aldolhexoses usually form pyranose rings and their pentose homologs tend to prefer the furanose form, but there are many counter examples. The formation of acetal derivatives illustrates how subtle changes may alter this selectivity. By clicking on the above diagram. the display will change to illustrate this. A pyranose structure for D-glucose is drawn in the rose-shaded box on the left. Acetal derivatives have been prepared by acid-catalyzed reactions with benzaldehyde and acetone. As a rule, benzaldehyde forms six-membered cyclic acetals, whereas acetone prefers to form five-membered acetals. The top equation shows the formation and some reactions of the 4,6-O-benzylidene acetal, a commonly employed protective group. A methyl glycoside derivative of this compound (see below) leaves the C-2 and C-3 hydroxyl groups exposed to reactions such as the periodic acid cleavage, shown as the last step. The formation of an isopropylidene acetal at C-1 and C-2, center structure, leaves the C-3 hydroxyl as the only unprotected function. Selective oxidation to a ketone is then possible. Finally, direct di-O-isopropylidene derivatization of glucose by reaction with excess acetone results in a change to a furanose structure in which the C-3 hydroxyl is again unprotected. However, the same reaction with D-galactose, shown in the blue-shaded box, produces a pyranose product in which the C-6 hydroxyl is unprotected. Both derivatives do not react with Tollens' reagent. This difference in behavior is attributed to the cis-orientation of the C-3 and C-4 hydroxyl groups in galactose, which permits formation of a less strained five-membered cyclic acetal, compared with the trans-C-3 and C-4 hydroxyl groups in glucose. Derivatizations of this kind permit selective reactions to be conducted at different locations in these highly functionalized molecules.
The ring size of these cyclic monosaccharides was determined by oxidation and chain cleavage of their tetra methyl ether derivatives. To see how this was done for glucose Click Here.
6. Glycosides
Acetal derivatives formed when a monosaccharide reacts with an alcohol in the presence of an acid catalyst are called glycosides. This reaction is illustrated for glucose and methanol in the diagram below. In naming of glycosides, the "ose" suffix of the sugar name is replaced by "oside", and the alcohol group name is placed first. As is generally true for most acetals, glycoside formation involves the loss of an equivalent of water. The diether product is stable to base and alkaline oxidants such as Tollen's reagent. Since acid-catalyzed aldolization is reversible, glycosides may be hydrolyzed back to their alcohol and sugar components by aqueous acid.
The anomeric methyl glucosides are formed in an equilibrium ratio of 66% alpha to 34% beta. From the structures in the previous diagram, we see that pyranose rings prefer chair conformations in which the largest number of substituents are equatorial. In the case of glucose, the substituents on the beta-anomer are all equatorial, whereas the C-1 substituent in the alpha-anomer changes to axial. Since substituents on cyclohexane rings prefer an equatorial location over axial (methoxycyclohexane is 75% equatorial), the preference for alpha-glycopyranoside formation is unexpected, and is referred to as the anomeric effect.
Glycosides abound in biological systems. By attaching a sugar moiety to a lipid or benzenoid structure, the solubility and other properties of the compound may be changed substantially. Because of the important modifying influence of such derivatization, numerous enzyme systems, known as glycosidases, have evolved for the attachment and removal of sugars from alcohols, phenols and amines. Chemists refer to the sugar component of natural glycosides as the glycon and the alcohol component as the aglycon. Two examples of naturally occurring glycosides and one example of an amino derivative will be displayed above by clicking on the diagram. Salicin, one of the oldest herbal remedies known, was the model for the synthetic analgesic aspirin. A large class of hydroxylated, aromatic oxonium cations called anthocyanins provide the red, purple and blue colors of many flowers, fruits and some vegetables. Peonin is one example of this class of natural pigments, which exhibit a pronounced pH color dependence. The oxonium moiety is only stable in acidic environments, and the color changes or disappears when base is added. The complex changes that occur when wine is fermented and stored are in part associated with glycosides of anthocyanins. Finally, amino derivatives of ribose, such as cytidine play important roles in biological phosphorylating agents, coenzymes and information transport and storage materials.
For a discussion of the anomeric effect Click Here. For examples of structurally and functionally modified sugars Click Here.
Disaccharides
When the alcohol component of a glycoside is provided by a hydroxyl function on another monosaccharide, the compound is called a disaccharide. Four examples of disaccharides composed of two glucose units are shown in the following diagram. The individual glucopyranose rings are labeled A and B, and the glycoside bonding is circled in light blue. Notice that the glycoside bond may be alpha, as in maltose and trehalose, or beta as in cellobiose and gentiobiose. Acid-catalyzed hydrolysis of these disaccharides yields glucose as the only product. Enzyme-catalyzed hydrolysis is selective for a specific glycoside bond, so an alpha-glycosidase cleaves maltose and trehalose to glucose, but does not cleave cellobiose or gentiobiose. A beta-glycosidase has the opposite activity.
In order to draw a representative structure for cellobiose, one of the glucopyranose rings must be rotated by 180º, but this feature is often omitted in favor of retaining the usual perspective for the individual rings. The bonding between the glucopyranose rings in cellobiose and maltose is from the anomeric carbon in ring A to the C-4 hydroxyl group on ring B. This leaves the anomeric carbon in ring B free, so cellobiose and maltose both may assume alpha and beta anomers at that site (the beta form is shown in the diagram). Gentiobiose has a beta-glycoside link, originating at C-1 in ring A and terminating at C-6 in ring B. Its alpha-anomer is drawn in the diagram. Because cellobiose, maltose and gentiobiose are hemiacetals they are all reducing sugars (oxidized by Tollen's reagent). Trehalose, a disaccharide found in certain mushrooms, is a bis-acetal, and is therefore a non-reducing sugar. A systematic nomenclature for disaccharides exists, but as the following examples illustrate, these are often lengthy.
• Disaccharides made up of other sugars are known, but glucose is often one of the components. Two important examples of such mixed disaccharides will be displayed above by clicking on the diagram. Lactose, also known as milk sugar, is a galactose-glucose compound joined as a beta-glycoside. It is a reducing sugar because of the hemiacetal function remaining in the glucose moiety. Many adults, particularly those from regions where milk is not a dietary staple, have a metabolic intolerance for lactose. Infants have a digestive enzyme which cleaves the beta-glycoside bond in lactose, but production of this enzyme stops with weaning. Cheese is less subject to the lactose intolerance problem, since most of the lactose is removed with the whey. Sucrose, or cane sugar, is our most commonly used sweetening agent. It is a non-reducing disaccharide composed of glucose and fructose joined at the anomeric carbon of each by glycoside bonds (one alpha and one beta). In the formula shown here the fructose ring has been rotated 180º from its conventional perspective.
To examine a model of sucrose Click Here
Additional Topics
For a brief discussion of sweetening agents Click Here. For examples of some larger saccharide oligomers Click Here.
Polysaccharides
As the name implies, polysaccharides are large high-molecular weight molecules constructed by joining monosaccharide units together by glycosidic bonds. They are sometimes called glycans. The most important compounds in this class, cellulose, starch and glycogen are all polymers of glucose. This is easily demonstrated by acid-catalyzed hydrolysis to the monosaccharide. Since partial hydrolysis of cellulose gives varying amounts of cellobiose, we conclude the glucose units in this macromolecule are joined by beta-glycoside bonds between C-1 and C-4 sites of adjacent sugars. Partial hydrolysis of starch and glycogen produces the disaccharide maltose together with low molecular weight dextrans, polysaccharides in which glucose molecules are joined by alpha-glycoside links between C-1 and C-6, as well as the alpha C-1 to C-4 links found in maltose. Polysaccharides built from other monosaccharides (e.g. mannose, galactose, xylose and arabinose) are also known, but will not be discussed here.
Over half of the total organic carbon in the earth's biosphere is in cellulose. Cotton fibers are essentially pure cellulose, and the wood of bushes and trees is about 50% cellulose. As a polymer of glucose, cellulose has the formula (C6H10O5)n where n ranges from 500 to 5,000, depending on the source of the polymer. The glucose units in cellulose are linked in a linear fashion, as shown in the drawing below. The beta-glycoside bonds permit these chains to stretch out, and this conformation is stabilized by intramolecular hydrogen bonds. A parallel orientation of adjacent chains is also favored by intermolecular hydrogen bonds. Although an individual hydrogen bond is relatively weak, many such bonds acting together can impart great stability to certain conformations of large molecules. Most animals cannot digest cellulose as a food, and in the diets of humans this part of our vegetable intake functions as roughage and is eliminated largely unchanged. Some animals (the cow and termites, for example) harbor intestinal microorganisms that breakdown cellulose into monosaccharide nutrients by the use of beta-glycosidase enzymes.
Cellulose is commonly accompanied by a lower molecular weight, branched, amorphous polymer called hemicellulose. In contrast to cellulose, hemicellulose is structurally weak and is easily hydrolyzed by dilute acid or base. Also, many enzymes catalyze its hydrolysis. Hemicelluloses are composed of many D-pentose sugars, with xylose being the major component. Mannose and mannuronic acid are often present, as well as galactose and galacturonic acid.
Starch is a polymer of glucose, found in roots, rhizomes, seeds, stems, tubers and corms of plants, as microscopic granules having characteristic shapes and sizes. Most animals, including humans, depend on these plant starches for nourishment. The structure of starch is more complex than that of cellulose. The intact granules are insoluble in cold water, but grinding or swelling them in warm water causes them to burst.
The released starch consists of two fractions. About 20% is a water soluble material called amylose. Molecules of amylose are linear chains of several thousand glucose units joined by alpha C-1 to C-4 glycoside bonds. Amylose solutions are actually dispersions of hydrated helical micelles. The majority of the starch is a much higher molecular weight substance, consisting of nearly a million glucose units, and called amylopectin. Molecules of amylopectin are branched networks built from C-1 to C-4 and C-1 to C-6 glycoside links, and are essentially water insoluble. Representative structural formulas for amylose and amylopectin will be shown above by clicking on the diagram. To see an expanded structure for amylopectin click again on the diagram. The branching in this diagram is exaggerated, since on average, branches only occur every twenty five glucose units.
Hydrolysis of starch, usually by enzymatic reactions, produces a syrupy liquid consisting largely of glucose. When cornstarch is the feedstock, this product is known as corn syrup. It is widely used to soften texture, add volume, prohibit crystallization and enhance the flavor of foods.
Glycogen is the glucose storage polymer used by animals. It has a structure similar to amylopectin, but is even more highly branched (about every tenth glucose unit). The degree of branching in these polysaccharides may be measured by enzymatic or chemical analysis.
For examples of chemical analysis of branching Click Here.
Synthetic Modification of Cellulose
Cotton, probably the most useful natural fiber, is nearly pure cellulose. The manufacture of textiles from cotton involves physical manipulation of the raw material by carding, combing and spinning selected fibers. For fabrics the best cotton has long fibers, and short fibers or cotton dust are removed. Crude cellulose is also available from wood pulp by dissolving the lignan matrix surrounding it. These less desirable cellulose sources are widely used for making paper.
In order to expand the ways in which cellulose can be put to practical use, chemists have devised techniques for preparing solutions of cellulose derivatives that can be spun into fibers, spread into a film or cast in various solid forms. A key factor in these transformations are the three free hydroxyl groups on each glucose unit in the cellulose chain, --[C6H7O(OH)3]n--. Esterification of these functions leads to polymeric products having very different properties compared with cellulose itself.
Cellulose Nitrate, first prepared over 150 years ago by treating cellulose with nitric acid, is the earliest synthetic polymer to see general use. The fully nitrated compound, --[C6H7O(ONO2)3]n--, called guncotton, is explosively flammable and is a component of smokeless powder. Partially nitrated cellulose is called pyroxylin. Pyroxylin is soluble in ether and at one time was used for photographic film and lacquers. The high flammability of pyroxylin caused many tragic cinema fires during its period of use. Furthermore, slow hydrolysis of pyroxylin yields nitric acid, a process that contributes to the deterioration of early motion picture films in storage.
Cellulose Acetate, --[C6H7O(OAc)3]n--, is less flammable than pyroxylin, and has replaced it in most applications. It is prepared by reaction of cellulose with acetic anhydride and an acid catalyst. The properties of the product vary with the degree of acetylation. Some chain shortening occurs unavoidably in the preparations. An acetone solution of cellulose acetate may be forced through a spinneret to generate filaments, called acetate rayon, that can be woven into fabrics.
Viscose Rayon, is prepared by formation of an alkali soluble xanthate derivative that can be spun into a fiber that reforms the cellulose polymer by acid quenching. The following general equation illustrates these transformations. The product fiber is called viscose rayon.
ROH
NaOH
RO(-) Na(+) + S=C=S
RO-CS2(-) Na(+)
H3O(+)
ROH
cellulose viscose solution rayon
Nonreducing Sugar
A nonreducing sugar is a carbohydrate that is not oxidized by a weak oxidizing agent (an oxidizing agent that oxidizes aldehydes but not alcohols, such as the Tollen’s reagent) in basic aqueous solution. The characteristic property of nonreducing sugars is that, in basic aqueous medium, they do not generate any compounds containing an aldehyde group.
eg: sucrose, which contains neither a hemiacetal group nor a hemiketal group and, therefore, is stable in water. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Carbohydrates/Carbohydrates_Fundamentals/Introduction_to_Carbohydrates.txt |
• Blood Glucose Test
Blood glucose levels are now measured by a procedure based upon the enzyme glucose oxidase. Since an enzyme is used, it is very specific for only D-glucose, and will not be subject to interferences from other molecules in the blood.
• Starch and Iodine
Plants store glucose as the polysaccharide starch; the cereal grains (wheat, rice, corn, oats, barley) as well as tubers such as potatoes are also rich in starch. Starch can be separated into two fractions--amylose and amylopectin. Natural starches are mixtures of amylose (10-20%) and amylopectin (80-90%).
• Sugar and Teeth
Sugar, saliva, and bacteria lead to a formidable combination that may lead to tooth decay. After eating sugar, particularly sucrose, and even within minutes of brushing your teeth, sticky glycoproteins (combination of carbohydrate and protein molecule) adhere to the teeth to start the formation of plaque. At the same time millions of bacteria known as Streptococcus mutans also adhere to the glycoprotein. Although, many oral bacteria also adhere, only the S. mutans is able to cause cavities.
Case Studies
With a couple of million people in the U. S. with diabetes, it is necessary to have a simple, specific test for the concentration of glucose in the blood. A rapid test is needed to manage the levels of insulin in diabetes mellitus. If not enough insulin is present, the blood glucose may be very elevated. On the other hand if too much insulin is present, the glucose levels are too low.
Oxidation of Glucose
Blood glucose levels are now measured by a procedure based upon the enzyme glucose oxidase. Since an enzyme is used, it is very specific for only D-glucose, and will not be subject to interferences from other molecules in the blood.
Glucose is a reducing sugar, which means that it can be oxidized. The enzyme glucose oxidase catalyzes the oxidation of beta-D-glucose to D-gluconic acid. The alpha-D-glucose is rapidly converted to the beta form so that all of the glucose is measured at one time.
Diatomic oxygen from the air is the oxidizing agent acting upon the glucose reducing agent. During the reaction the ring opens and the aldehyde on carbon # 1 is converted to the acid, D-gluconic acid. At the same time the oxygen in the presence of water is converted to hydrogen peroxide. So far all of the chemicals are colorless, so you would not be able to see the reaction taking place. Therefore another step is needed to produce a color.
Color Producing Reaction
Several methods of detection are possible, but in most cases, the hydrogen peroxide reacts with a second color producing chemical. An example is something called o-Toluidine or 2-methylaniline which reacts with the hydrogen peroxide using an enzyme called peroxidase to produce a color forming chemical.
The concentration of the glucose can be related to the intensity of color produced. The more the intensity, the higher the concentration of glucose. A simple color chart can be used to "read" the concentration of the glucose.
Starch and Iodine
Plants store glucose as the polysaccharide starch; the cereal grains (wheat, rice, corn, oats, barley) as well as tubers such as potatoes are also rich in starch. Starch can be separated into two fractions--amylose and amylopectin. Natural starches are mixtures of amylose (10-20%) and amylopectin (80-90%).
Introduction
Amylose forms a colloidal dispersion in hot water whereas amylopectin is completely insoluble. The structure of amylose consists of long polymer chains of glucose units connected by an alpha acetal linkage. Starch - Amylose shows a very small portion of an amylose chain. All of the monomer units are alpha -D-glucose, and all the alpha acetal links connect C #1 of one glucose and to C #4 of the next glucose. As a result of the bond angles in the α acetal linkage, amylose actually forms a spiral much like a coiled spring. See the graphic below, which show four views in turning from a the side to an end view.
Chemical Test for Starch or Iodine
Amylose in starch is responsible for the formation of a deep blue color in the presence of iodine. The iodine molecule slips inside of the amylose coil. Iodine - KI Reagent: Iodine is not very soluble in water, therefore the iodine reagent is made by dissolving iodine in water in the presence of potassium iodide. This makes a linear triiodide ion complex with is soluble that slips into the coil of the starch causing an intense blue-black color.
• Starch Test: Add Iodine-KI reagent to a solution or directly on a potato or other materials such as bread, crackers, or flour. A blue-black color results if starch is present. If starch amylose is not present, then the color will stay orange or yellow. Starch amylopectin does not give the color, nor does cellulose, nor do disaccharides such as sucrose in sugar.
• Iodine Test: When following the changes in some inorganic oxidation reduction reactions, iodine may be used as an indicator to follow the changes of iodide ion and iodine element. Soluble starch solution is added. Only iodine element in the presence of iodide ion will give the characteristic blue black color. Neither iodine element alone nor iodide ions alone will give the color result. This phenomenon is used in the iodine clock demonstration.
Sugar and Teeth
Sugar, saliva, and bacteria lead to a formidable combination that may lead to tooth decay. After eating sugar, particularly sucrose, and even within minutes of brushing your teeth, sticky glycoproteins (combination of carbohydrate and protein molecule) adhere to the teeth to start the formation of plaque. At the same time millions of bacteria known as Streptococcus mutans also adhere to the glycoprotein. Although, many oral bacteria also adhere, only the S. mutans is able to cause cavities.
Introduction
In the next stage, the bacteria use the fructose in a metabolism process of glycolysis to get energy. The end product of glycolysis under anaerobic conditions is lactic acid. The lactic acid creates extra acidity to decrease the pH to the extent of dissolving the calcium phosphate in the tooth enamel leading to the start of a cavity. Preventative measures include frequent brushing and flossing to prevent plaque build up. A diet rich in calcium and fluoride in the water lead to stronger tooth enamel. A diet of more complex carbon hydrates that are low in sugar and no sucrose snacks between meals is also a good preventative measure.
Only the S. mutans bacteria has an enzyme called glucosyl transferase on its surface that is able to cause the polymerization of glucose on the sucrose with the release of the fructose. The same enzyme continues to add many glucose molecules to each other to form dextran which is very similar in structure to amylose in starch. The dextran along with the bacteria adheres tightly to the tooth enamel and leads to the formation of plaque. This is just the first phase of cavity formation.
The graphic below shows only a portion of this process which shows the release of the fructose. The glucose undergoes further polymerization as stated above.
Glycolysis
In the next stage, the bacteria use the fructose in a metabolism process of glycolysis to get energy. The end product of glycolysis under anaerobic conditions is lactic acid. The lactic acid creates extra acidity to decrease the pH to the extent of dissolving the calcium phosphate in the tooth enamel leading to the start of a cavity.
Preventative measures include frequent brushing and flossing to prevent plaque build up. A diet rich in calcium and fluoride in the water lead to stronger tooth enamel. A diet of more complex carbon hydrates that are low in sugar and no sucrose snacks between meals is also a good preventative measure. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Carbohydrates/Case_Studies/Blood_Glucose_Test.txt |
The most useful carbohydrate classification scheme divides the carbohydrates into groups according to the number of individual simple sugar units. Monosaccharides contain a single unit; disaccharides contain two sugar units; and polysaccharides contain many sugar units as in polymers - most contain glucose as the monosaccharide unit.
Thumbnail Ball-and-stick model of the α-lactose molecule. (Public Domain; Ben Mills).
Disaccharides
Lactose or milk sugar occurs in the milk of mammals - 4-6% in cow's milk and 5-8% in human milk. It is also a by product in the the manufacture of cheese. Lactose is made from galactose and glucose units. The galactose and glucose units are joined by an acetal oxygen bridge in the beta orientation. To recognize galactose look for the upward projection of the -OH on carbon # 4. See details on the galactose page towards the bottom.
Lactose intolerance
Lactose intolerance is the inability to digest significant amounts of lactose, the predominant sugar of milk. This inability results from a shortage of the enzyme lactase, which is normally produced by the cells that line the small intestine. Lactase breaks down the lactose, milk sugar, into glucose and galactose that can then be absorbed into the bloodstream. When there is not enough lactase to digest the amount of lactose consumed, produce some uncomfortable symptoms. Some adults have low levels of lactase. This leads to lactose intolerance. The ingested lactose is not absorbed in the small intestine, but instead is fermented by bacteria in the large intestine, producing uncomfortable volumes of carbon dioxide gas. While not all persons deficient in lactase have symptoms, those who do are considered to be lactose intolerant.
Common symptoms include nausea, cramps, bloating, gas, and diarrhea, which begin about 30 minutes to 2 hours after eating or drinking foods containing lactose. The severity of symptoms varies depending on the amount of lactose each individual can tolerate.
Fortunately, lactose intolerance is relatively easy to treat by controlling the diet. No cure or treatment exists to improve the body's ability to produce lactase. Young children with lactase deficiency should not eat any foods containing lactose. Most older children and adults need not avoid lactose completely, but individuals differ in the amounts and types of foods they can handle. Dietary control of lactose intolerance depends on each person's learning through trial and error how much lactose he or she can handle.
Acetal Functional Group
Carbon # 1 (red on left) is called the anomeric carbon and is the center of an acetal functional group. A carbon that has two ether oxygens attached is an acetal. The Beta position is defined as the ether oxygen being on the same side of the ring as the C # 6. In the chair structure this results in a horizontal or up projection. This is the same definition as the -OH in a hemiacetal.
Compare Lactose and Maltose Acetals
The position of the oxygen in the acetal on the anomeric carbon (#1) is an important distinction for disaccharide chemistry. Lactose has a beta acetal. The Beta position is defined as the oxygen in the acetal being on the same side of the ring as the C # 6. In the chair structure this results in a horizontal projection. Maltose has an alpha acetal. The Alpha position is defined as the oxygen in the acetal being on the opposite side of the ring as the C # 6. In the chair structure this results in a downward projection. The alpha and beta acetal label is not applied to any other carbon - only the anomeric carbon of the left monosaccharide, in this case # 1 (red).
Recognize galactose and glucose
To further identify lactose and maltose, identify the presence of galactose in lactose in the left most structure by the upward -OH on the carbon # 4. Identify glucose in maltose in the left most structure by the horizontal -OH on the carbon # 4. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Carbohydrates/Disaccharides/Lactose.txt |
Sucrose or table sugar is obtained from sugar cane or sugar beets. Sucrose is made from glucose and fructose units. The glucose and fructose units are joined by an acetal oxygen bridge in the alpha orientation. The structure is easy to recognize because it contains the six member ring of glucose and the five member ring of fructose.
Introduction
To recognize glucose look for the horizontal projection of the -OH on carbon #4. The alpha acetal is is really part of a double acetal, since the two monosaccharides are joined at the hemiacetal of glucose and the hemiketal of the fructose. There are no hemiacetals remaining in the sucrose and therefore sucrose is a non-reducing sugar.
Figure \(1\): Sucrose
Sugar Processing
Sugar or more specifically sucrose is a carbohydrate that occurs naturally in every fruit and vegetable. It is the major product of photosynthesis, the process by which plants transform the sun's energy into food. Sugar occurs in greatest quantities in sugar cane and sugar beets from which it is separated for commercial use.
In the first stage of processing the natural sugar stored in the cane stalk or beet root is separated from the rest of the plant material by physical methods. For sugar cane, this is accomplished by:
1. pressing the cane to extract the juice containing the sugar
2. boiling the juice until it begins to thicken and sugar begins to crystallize
3. spinning the sugar crystals in a centrifuge to remove the syrup, producing raw sugar; the raw sugar still contains many impurities
4. shipping the raw sugar to a refinery where it is washed and filtered to remove remaining non-sugar ingredients and color
5. crystallizing, drying and packaging the refined sugar.
Beet sugar processing is similar, but it is done in one continuous process without the raw sugar stage. The sugar beets are washed, sliced and soaked in hot water to separate the sugar-containing juice from the beet fiber. The sugar-laden juice is purified, filtered, concentrated and dried in a series of steps similar to cane sugar processing.
Acetal Functional Group
Carbon # 1 (red on left) is called the anomeric carbon and is the center of an acetal functional group. A carbon that has two ether oxygens attached is an acetal. The Alpha position is defined as the ether oxygen being on the opposite side of the ring as the C # 6. In the chair structure this results in a down projection. This is the same definition as the -OH in a hemiacetal.
A second acetal grouping is defined by the green atoms. This result because the the formation reaction of the disaccharide is between the hemiacetal of glucose and the hemiketal of the fructose.
Figure \(2\): Sucrose
Invert Sugar
When sucrose is hydrolyzed it forms a 1:1 mixture of glucose and fructose. This mixture is the main ingredient in honey. It is called invert sugar because the angle of the specific rotation of the plain polarized light changes from a positive to a negative value due to the presence of the optical isomers of the mixture of glucose and fructose sugars.
Hydrolysis of Sucrose
In the hydrolysis of any di- or poly saccharide, a water molecule helps to break the acetal bond as shown in red. The acetal bond is broken, the H from the water is added to the oxygen on the glucose. The -OH is then added to the carbon on the fructose.
Figure \(1\): Hydrolosis of Sucrose
Exercise \(1\)
Is glucose - alpha or beta?
Answer
The -OH on carbon # 1 is projected down therefore, alpha.
Exercise \(2\)
Is fructose - alpha or beta?
Answer
The -OH on carbon # 1 is projected downand is on the same side of the ring as C#6, extreme right on fructose therefore, beta. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Carbohydrates/Disaccharides/Sucrose.txt |
The most useful carbohydrate classification scheme divides the carbohydrates into groups according to the number of individual simple sugar units. Monosaccharides contain a single unit; disaccharides contain two sugar units; and polysaccharides contain many sugar units as in polymers - most contain glucose as the monosaccharide unit.
Thumbnail Ball-and-stick model of the α-D-glucose molecule, \(C_6H_{12}O_6\). (Public Domain; Ben Mills).
Monosaccharides
Fructose is more commonly found together with glucose and sucrose in honey and fruit juices. Fructose, along with glucose are the monosaccharides found in disaccharide, sucrose. Fructose is classified as a monosaccharide, the most important ketose sugar, a hexose, and is a reducing sugar.
Introduction
An older common name for fructose is levulose, after its levorotatory property of rotating plane polarized light to the left (in contrast to glucose which is dextrorotatory). Bees gather nectar from flowers which contains sucrose. They then use an enzyme to hydrolyze or break apart the sucrose into its component parts of glucose and fructose. High Fructose Corn Syrup
Ring Structure for Fructose
The chair form of fructose follows a similar pattern as that for glucose with a few exceptions. Since fructose has a ketone functional group, the ring closure occurs at carbon # 2. In the case of fructose a five membered ring is formed. The -OH on carbon #5 is converted into the ether linkage to close the ring with carbon #2. This makes a 5 member ring - four carbons and one oxygen.
Steps in the ring closure (hemiketal synthesis)
1. The electrons on the alcohol oxygen are used to bond the carbon #2 to make an ether (red oxygen atom).
2. The hydrogen (green) is transferred to the carbonyl oxygen (green) to make a new alcohol group (green).
The ring structure is written with the orientation depicted on the left for the monosaccharide and is consistent with the way the glucose is depicted.
Hemiketal Functional Group
The anomeric carbon is the center of a hemiketal functional group. A carbon that has both an ether oxygen and an alcohol group (and is attached to two other carbons is a hemiketal.
Compare Alpha and Beta Fructose
The Beta position is defined as the -OH being on the same side of the ring as the C # 6. In the ring structure this results in a upwards projection for the -OH on carbon # 2. The Alpha position is defined as the -OH being on the opposite side of the ring as the C # 6. In the ring structure this results in a downward projection for the -OH on carbon # 2. The alpha and beta label is not applied to any other carbon - only the anomeric carbon, in this case # 2.
Figure: Compare Alpha and Beta Fructose
Compare Glucose and Fructose in the Chair Structures
The six member ring and the position of the -OH group on the carbon (#4) identifies glucose from the -OH on C # 4 in a down projection in the Haworth structure). Fructose is recognized by having a five member ring and having six carbons, a hexose. Both glucose and fructose may be either alpha or beta on the anomeric carbon, so this is not distinctive between them.
Problems
Which carbon in the structure on the in Figure 2 is the anomeric carbon?
Furanose
If the molecule of the cyclic form of a monosaccharide has an oxygen-containing five-membered ring, the compound is called a furanose e.g. two of the four cyclic forms of D-fructose.
The ring in a furanose is called the furanose ring.
Galactose
Galactose is more commonly found in the disaccharide, lactose or milk sugar. It is found as the monosaccharide in peas. Galactose is classified as a monosaccharide, an aldose, a hexose, and is a reducing sugar.
Galactosemia - Genetic Enzyme Deficiency
One baby out of every 18,000 is born with a genetic defect of not being able to utilize galactose. Since galactose is in milk as part of lactose, it will build up in the blood and urine. Undiagnosed it may lead to mental retardation, failure to grow, formation of cataracts, and in sever cases death by liver damage. The disorder is caused by a deficiency in one or more enzymes required to metabolize galactose. The treatment for the disorder is to use a formula based upon the sugar sucrose rather than milk with lactose. The galactose free diet is critical only in infancy, since with maturation another enzyme is developed that can metabolize galactose.
Ring Structure for Galactose
The chair form of galactose follows the same pattern as that for glucose. The anomeric carbon is the center of a hemiacetal functional group. A carbon that has both an ether oxygen and an alcohol group is a hemiacetal.
Figure \(1\): Compare Alpha and Beta Galactose in the Chair form below.
he Beta position is defined as the -OH being on the same side of the ring as the C # 6. In the chair structure this results in a horizontal projection (Haworth - an upwards projection). The Alpha position is defined as the -OH being on the opposite side of the ring as the C # 6. In the chair and Haworth structure this results in a downward projection.
Compare Glucose and Galactose in the Chair Structures
The position of the -OH group on the carbon (#4) is the only distinction between glucose and galactose. Glucose is defined as the -OH on C # 4 in a horizontal projection in the chair form, (down in the Haworth structure). Galactose is defined as the -OH on C # 4 in a upward projection in the chair form,(also upward in the Haworth structure). Both glucose and galactose may be either alpha or beta on the anomeric carbon, so this is not distinctive between them.
Exercise \(1\)
Which carbon in the structure on the left is the anomeric carbon on galactose?
Contributors
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Carbohydrates/Monosaccharides/Fructose.txt |
Glucose is by far the most common carbohydrate and classified as a monosaccharide, an aldose, a hexose, and is a reducing sugar. It is also known as dextrose, because it is dextrorotatory (meaning that as an optical isomer is rotates plane polarized light to the right and also an origin for the D designation. Glucose is also called blood sugar as it circulates in the blood at a concentration of 65-110 mg/dL of blood.
Introduction
Glucose is initially synthesized by chlorophyll in plants using carbon dioxide from the air and sunlight as an energy source. Glucose is further converted to starch for storage.
Figure \(1\): Ring Structure for Glucose
Up until now we have been presenting the structure of glucose as a chain. In reality, an aqueous sugar solution contains only 0.02% of the glucose in the chain form, the majority of the structure is in the cyclic chair form. Since carbohydrates contain both alcohol and aldehyde or ketone functional groups, the straight-chain form is easily converted into the chair form - hemiacetal ring structure. Due to the tetrahedral geometry of carbons that ultimately make a 6 membered stable ring , the -OH on carbon #5 is converted into the ether linkage to close the ring with carbon #1. This makes a 6 member ring - five carbons and one oxygen.
Steps in the ring closure (hemiacetal synthesis):
1. The electrons on the alcohol oxygen are used to bond the carbon #1 to make an ether (red oxygen atom).
2. The hydrogen (green) is transferred to the carbonyl oxygen (green) to make a new alcohol group (green).
The chair structures are always written with the orientation depicted below to avoid confusion.
Figure \(2\): Hemiacetal Functional Group. Carbon # 1 is now called the anomeric carbon and is the center of a hemiacetal functional group. A carbon that has both an ether oxygen and an alcohol group is a hemiacetal.
Compare Alpha and Beta Glucose in the Chair Structures
The position of the -OH group on the anomeric carbon (#1) is an important distinction for carbohydrate chemistry. The Beta position is defined as the -OH being on the same side of the ring as the C #6. In the chair structure this results in a horizontal projection. The Alpha position is defined as the -OH being on the opposite side of the ring as the C #6. In the chair structure this results in a downward projection. The alpha and beta label is not applied to any other carbon - only the anomeric carbon, in this case #1.
Figure \(3\): Compare Alpha and Beta Glucose in the Haworth Structures. The Beta position is defined as the -OH being on the same side of the ring as the C # 6. In the Haworth structure this results in an upward projection. The Alpha position is defined as the -OH being on the opposite side of the ring as the C # 6. In the Haworth structure this also results in a downward projection.
Contributors
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook
Ribose
Ribose and its related compound, deoxyribose, are the building blocks of the backbone chains in nucleic acids, better known as DNA and RNA. Ribose is used in RNA and deoxyribose is used in DNA. The deoxy- designation refers to the lack of an alcohol, -OH, group as will be shown in detail further down. Ribose and deoxyribose are classified as monosaccharides, aldoses, pentoses, and are reducing sugars.
Ring Structure for Ribose
The chair form of ribose follows a similar pattern as that for glucose with one exception. Since ribose has an aldehyde functional group, the ring closure occurs at carbon # 1, which is the same as glucose. See the graphic on the left. The exception is that ribose is a pentose, five carbons. Therefore a five membered ring is formed. The -OH on carbon #4 is converted into the ether linkage to close the ring with carbon #1. This makes a 5 member ring - four carbons and one oxygen.
Steps in the ring closure (hemiacetal synthesis)
1. The electrons on the alcohol oxygen are used to bond the carbon #1 to make an ether (red oxygen atom).
2. The hydrogen (green) is transferred to the carbonyl oxygen (green) to make a new alcohol group (green).
The chair structures are always written with the orientation depicted above to avoid confusion. Carbon # 1 is now called the anomeric carbon and is the center of a hemiacetal functional group. A carbon that has both an ether oxygen and an alcohol group is a hemiacetal.
Compare Ribose and Deoxyribose Structures
The presence or absence of the -OH group on carbon (#2) is an important distinction between ribose and deoxyribose. Ribose has an alcohol at carbon # 2, while deoxyribose does not have the alcohol group. See red -OH and H in the structures below. The Beta position is defined as the -OH being on the same side of the ring as the C # 6. In the ring structure this results in a upward projection. The Alpha position is defined as the -OH being on the opposite side of the ring as the C # 6. In the ring structure this results in a downward projection. The alpha and beta label is not applied to any other carbon - only the anomeric carbon, in this case # 1.
Contributors
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook
Oligosaccharides
An oligosaccharide is a carbohydrate whose molecule, upon hydrolysis, yields two to ten Monosaccharid molecules. Oligosaccharides are classified into subclasses based on the number of monosaccharide molecules that form when one molecule of the oligosaccharide is hydrolyzed. Oligosaccharides can have many functions including cell recognition and cell binding. For example, glycolipids have an important role in the immune response.
Thumbnail: Structure of galactooligosaccharide. (Public Domain; Klaas1978) | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Carbohydrates/Monosaccharides/Glucose_%28Dextrose%29.txt |
The most useful carbohydrate classification scheme divides the carbohydrates into groups according to the number of individual simple sugar units. Monosaccharides contain a single unit; disaccharides contain two sugar units; and polysaccharides contain many sugar units as in polymers - most contain glucose as the monosaccharide unit.
Thumbnail: Schematic two-dimensional cross-sectional view of glycogen: A core protein of glycogenin is surrounded by branches of glucose units. The entire globular granule may contain around 30,000 glucose units. By Mikael Häggström, used with permission (Public Domain).
Polysaccharides
Polysaccharides are carbohydrate polymers consisting of tens to hundreds to several thousand monosaccharide units. All of the common polysaccharides contain glucose as the monosaccharide unit. Polysaccharides are synthesized by plants, animals, and humans to be stored for food, structural support, or metabolized for energy.
The major component in the rigid cell walls in plants is cellulose. Cellulose is a linear polysaccharide polymer with many glucose monosaccharide units. The acetal linkage is beta which makes it different from starch. This peculiar difference in acetal linkages results in a major difference in digestibility in humans. Humans are unable to digest cellulose because the appropriate enzymes to breakdown the beta acetal linkages are lacking. Indigestible cellulose is the fiber which aids in the smooth working of the intestinal tract.
Animals such as cows, horses, sheep, goats, and termites have symbiotic bacteria in the intestinal tract. These symbiotic bacteria possess the necessary enzymes to digest cellulose in the GI tract. They have the required enzymes for the breakdown or hydrolysis of the cellulose; the animals do not, not even termites, have the correct enzymes. No vertebrate can digest cellulose directly.
Even though we cannot digest cellulose, we find many uses for it including: Wood for building; paper products; cotton, linen, and rayon for clothes; nitrocellulose for explosives; cellulose acetate for films. The structure of cellulose consists of long polymer chains of glucose units connected by a beta acetal linkage. The graphic on the left shows a very small portion of a cellulose chain. All of the monomer units are beta-D-glucose, and all the beta acetal links connect C # 1 of one glucose to C # 4 of the next glucose.
Acetal Functional Group
Carbon # 1 is called the anomeric carbon and is the center of an acetal functional group. A carbon that has two ether oxygens attached is an acetal. The Beta position is defined as the ether oxygen being on the same side of the ring as the C # 6. In the chair structure this results in a horizontal or up projection. This is the same definition as the -OH in a hemiacetal.
Compare Cellulose and Starch Structures
Cellulose: Beta glucose is the monomer unit in cellulose. As a result of the bond angles in the beta acetal linkage, cellulose is mostly a linear chain. Starch: Alpha glucose is the monomer unit in starch. As a result of the bond angles in the alpha acetal linkage, starch-amylose actually forms a spiral much like a coiled spring.
Fiber in the Diet
Dietary fiber is the component in food not broken down by digestive enzymes and secretions of the gastrointestinal tract. This fiber includes hemicelluloses, pectins, gums, mucilages, cellulose, (all carbohydrates) and lignin, the only non-carbohydrate component of dietary fiber. High fiber diets cause increased stool size and may help prevent or cure constipation. Cereal fiber, especially bran, is most effective at increasing stool size while pectin has little effect. Lignin can be constipating.
Fiber may protect against the development of colon cancer, for populations consuming high fiber diets have a low incidence of this disease. The slow transit time (between eating and elimination) associated with a low fiber intake would allow more time for carcinogens present in the colon to initiate cancer. But constipated people do not have a higher incidence of colon cancer than fast eliminators, so fiber's role in colon cancer remains unclear.
Dietary fiber may limit cholesterol absorption by binding bile acids. High fiber diets lower serum cholesterol and may prevent cardiovascular disease. Some fibers, such as pectin and rolled oats, are more effective than others, such as wheat, at lowering serum cholesterol. Dietary fiber is found only in plant foods such as fruits, vegetables, nuts, and grains. Whole wheat bread contains more fiber than white bread and apples contain more fiber than apple juice, which shows that processing food generally removes fiber. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Carbohydrates/Polysaccharides/Cellulose.txt |
Polysaccharides are carbohydrate polymers consisting of tens to hundreds to several thousand monosaccharide units. All of the common polysaccharides contain glucose as the monosaccharide unit. Polysaccharides are synthesized by plants, animals, and humans to be stored for food, structural support, or metabolized for energy.
Introduction
Glycogen is the storage form of glucose in animals and humans which is analogous to the starch in plants. Glycogen is synthesized and stored mainly in the liver and the muscles. Structurally, glycogen is very similar to amylopectin with alpha acetal linkages, however, it has even more branching and more glucose units are present than in amylopectin. Various samples of glycogen have been measured at 1,700-600,000 units of glucose.
The structure of glycogen consists of long polymer chains of glucose units connected by an alpha acetal linkage. The graphic on the left shows a very small portion of a glycogen chain. All of the monomer units are alpha-D-glucose, and all the alpha acetal links connect C # 1 of one glucose to C # 4 of the next glucose.
The branches are formed by linking C #1 to a C #6 through an acetal linkages. In glycogen, the branches occur at intervals of 8-10 glucose units, while in amylopectin the branches are separated by 12-20 glucose units.
Acetal Functional Group
Carbon # 1 is called the anomeric carbon and is the center of an acetal functional group. A carbon that has two ether oxygens attached is an acetal. The Alpha position is defined as the ether oxygen being on the opposite side of the ring as the C # 6. In the chair structure this results in a downward projection. This is the same definition as the -OH in a hemiacetal.
Starch vs. Glycogen
Plants make starch and cellulose through the photosynthesis processes. Animals and human in turn eat plant materials and products. Digestion is a process of hydrolysis where the starch is broken ultimately into the various monosaccharides. A major product is of course glucose which can be used immediately for metabolism to make energy. The glucose that is not used immediately is converted in the liver and muscles into glycogen for storage by the process of glycogenesis. Any glucose in excess of the needs for energy and storage as glycogen is converted to fat.
Starch
Polysaccharides are carbohydrate polymers consisting of tens to hundreds to several thousand monosaccharide units. All of the common polysaccharides contain glucose as the monosaccharide unit. Polysaccharides are synthesized by plants, animals, and humans to be stored for food, structural support, or metabolized for energy.
Introduction
Plants store glucose as the polysaccharide starch. The cereal grains (wheat, rice, corn, oats, barley) as well as tubers such as potatoes are rich in starch. Starch can be separated into two fractions--amylose and amylopectin. Natural starches are mixtures of amylose (10-20%) and amylopectin (80-90%).
Amylose forms a colloidal dispersion in hot water, while amylopectin is soluble it is demanding of more extensive heating than amylose. The structure of amylose consists of long polymer chains of glucose units connected by an alpha acetal linkage. The graphic on the left shows a very small portion of an amylose chain. All of the monomer units are alpha -D-glucose, and all the alpha acetal links connect C #1 of one glucose and C #4 of the next glucose.
Acetal Functional Group
Carbon # 1 is called the anomeric carbon and is the center of an acetal functional group. A carbon that has two ether oxygens attached is an acetal. The Alpha position is defined as the ether oxygen being on the opposite side of the ring as the C # 6. In the chair structure this results in a downward projection. This is the same definition as the -OH in a hemiacetal.
As a result of the bond angles in the alpha acetal linkage, amylose actually forms a spiral much like a coiled spring. Amylose is responsible for the formation of a deep blue color in the presence of iodine, which slips inside of the amylose coil.
Amylopectin
The graphic on the left shows very small portion of an amylopectin-type structure showing two branch points [drawn closer together than they should be]. The acetal linkages are alpha connecting C #1 of one glucose to C #4 of the next glucose.
The branches are formed by linking C #1 to a C #6 through an acetal linkages. Amylopectin has 12-20 glucose units between the branches. Natural starches are mixtures of amylose and amylopectin. In glycogen, the branches occur at intervals of 8-10 glucose units, while in amylopectin the branches are separated by 10-12 glucose units. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Carbohydrates/Polysaccharides/Glycogen.txt |
Hydrogenase enzymes catalyze the reversible oxidation of molecular hydrogen to protons and electrons [H2 ↔ 2H+ + 2e-] [1].The two most studied classes are Ni-Fe hydrogenases and Fe-only hydrogenases. In general, Ni-Fe hydrogenase enzymes consume molecular hydrogen as a fuel source, and Fe-only hydrogense enzymes produce molecular hydrogen [2].
Representation of Fe-only Hydrogenase
Crystal structure of the hydrogenase was obtained from a sulfate-reducing microorganism to 1.6 Å resolution (fig 1). Desulfovibrio desulfuricans (DdH) is a 53 kDa protein located in the periplasm [3]. Crystal structure of the enzyme was obtained from Clostridium pasteurianum an anaerobic soil microorganism to 1.8 Å resolution (fig 2). This enzyme, CpI, is a 61 kDa protein found in the cytoplasm [4]. DdH is a dimer with three 4Fe-4S cubanes and a H-cluster (fig 3)[5]. CpI is a monomer with three 4Fe-4S cubanes, one 2Fe-2S cluster, and a H-cluster (fig 4)[5].
Figure 1: The DdH Fe-only hydrogenase domain structure. Fe atoms light green, sulfurs in black, nitrogen in blue, oxygen in red, and carbon in gray [4]. fig (2) The CpI Fe-only hydrogenase domain structure. Fe atoms are shown in rust, sulfurs in yellow, nitrogen in blue, carbon in black, oxygen in red; purple indicates the sulfur-bridging moiety of unknown composition [3]. fig (3) The DdH Fe-only hydrogenase contains 3 [4Fe-4S] cubanes and a H-Cluster[5].fig (4) The CpI Fe-only hydrogenase contains 3 [4Fe-4S] cubanes the 2[4Fe–4S] cubanes, 1 [2Fe-2S] cluster, and a H-Cluster[5].
Point group symmetry
The overall structure of CpI resembles a mushroom consisting of four domains: the large active site domain forms "cap" and three smaller domains form "stem". The molecular point group for all the structure is C1(fig 4)[8]. The "stem" domains bind four iron-sulfur clusters and are termed FS4A-FS4B, FS4C and FS2. FS2 has D2h symmetry(fig 4B). The FS4A-FS4B domain has D2d symmetry(fig 4A). The FS4C domain is placed between the FS2 and FS4A-FS4B domains and consists of two helices linked by a loop that binds a single [Fe4S4] cluster via one His and three Cys residues [9].
Electronic Structure of the 2Fe Subunit
In order to understand the reactivity of Fe only hydrogenase it is important to know its oxidation state .When the enzyme is isolated in air, we observe an oxidized, inactive state (Hoxair)also known as the EPR silent state. Further reductive treatment results in an oxidized, active state(Hox). The Hox is a low-spin state with a rhombic g = 2.10 signal in EPR. When the Fe-only hydrogenase is fully reduced, another EPR-silent state (Hred) is obtained. DFT calculations show that there are different oxidation states of 2Fe subunit in the system for these three intermediate states (Hox air, Hox and Hred) during catalytic H2 evolution. Calculations are as follows (i) the 2Fe subunit in the Hox air state is most likely a Fe(II)-Fe(II) complex with a hydroxyl (OH-) group bonded at the Fed i.e. Fe(II)-Fe(II)(OH) (ii) a Fe(II)-Fe(I)(vacant) complex is the best candidate for the 2Fe subunit in the Hox state. (iii) the 2Fe subunit in the Hred is a mixture with the major component probably being a protonated Fe(I)-Fe(I) complex. This protonated complex is most likely to mix with its self-arranged form, Fe(II)-Fe(II) hydride.
Few questions arises here.1.Why Fe(II)-Fe(I)(vacant) state is most probable in the system? 2. We know from general inorganic chemistry that Fe(II) and F(III) are stable complexes (due to 3d5 (half occupied) or 3d6 (t2g states occupied) configurations), but Fe(I) is an unstable complex. Then, why the low oxidation states, such as Fe(I)-Fe(I), are stable while the high oxidation states, for example Fe(III)-Fe(III) and Fe(III)-Fe(II), do not exist[6]. I am considering MO theory and back bonding concept to answer above questions which are important for understanding the reactivity of Fe hydrogenase .
Metals with lower oxidation states have a larger electron density. This electron density allows the metal to donate π-electrons from their d orbital to the anitibonding orbital of its ligand, π-back bonding (fig 5). In this situation the π- back bonding stabilizes the bond between the iron and the carbon and destabilizes the bond between the carbon and the oxygen. Also, FTIR studies show that the oxidation state of Fe(II) must be lower than Fe(I) because we observe a negative shift in the band produced in FTIR spectra[6]. Thus, Fe(II)-Fe(I) oxidation state is most probable for the iron subcluster.
Fig 6 is the interaction diagram between the Fe valence orbitals (3d, 4s, 4p) and the 5σ, 2δorbitals of CO and CN ligands (the interaction between the Fe valence orbitals and the S 2p orbital is omitted here). The energy levels of the 2Fe subunit are shown in the middle. It was found that all the redox states (Fe(II)-Fe(II), Fe(II)- Fe(I), and Fe(I)-Fe(I)) are very similar except for the occupation of a frontier orbital, labeled as eg-2δ. It is unoccupied in Fe(II)-Fe(II), half-occupied in Fe(II)-Fe(I), and fully occupied in Fe(I)-Fe(I) state. Fully filled eg-2δ orbital makes Fe(I)-Fe(I) complex stable. For Fe(III)-Fe(III) and Fe(III)-Fe(II) states, electrons in even stronger bonding orbitals of t2g-2δmust be depleted, which is strongly energetically disfavored. This is the reason why Fe(III)- Fe(III) or Fe(III)-Fe(II) species do not exist [6].
Reactivity of Fe only hydrogenase
When enzyme is isolated in air it changes its state from Fe(II)-Fe(I)(OH) to Fe(II)-Fe(I)(vacant). Actually, an exchangeable water molecule is bound to the active site labeled “vacant” in the figure. The enzyme then accepts an electron and a proton so that Fe(II)-Fe(I) is oxidized to protonated Fe(I)-Fe(I) Complex.The enzyme changes configuration, and a hydride is transferred into the active site of Fe(II) by reduction. The subsequent addition of an electron and a proton allows for the formation of molecular hydrogen, and the loss of H2 returns the system back to its native active form [6].
Conclusion
Fe-only Hydrogenase is a very important area of research. Understanding the mechanism of Fe-only Hydrogenase could open the doors for energy sciences development . New developments in the research of Fe-only hydrogenase has peaked interest for use of enzymes in the production of hydrogen. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Enzymes/Case_Studies/Fe-only_Hydrogenase.txt |
Horseradish Peroxidase is a metalloenzyme that exists in the root of the horseradish plant. There are a large number of peroxidase isoenzymes of horseradish with the most common being the C type.1 This type will be discussed in the remaining report because of the extensive research that has been completed on it.
Introduction
Horseradish peroxidase uses hydrogen peroxide to oxidize both organic and inorganic compounds.1 Horseradish peroxidase along with other heme peroxidases are brightly colored especially under the near-ultraviolet light.2 This property of heme peroxidases make them useful for attaching to “transparent” proteins so that they can be seen under different wavelengths. The heme group that is in horseradish peroxidase is simpler than those in mammalians and therefore makes it an excellent starting point in the in-depth study of heme peroxidases and their functions.2 It has been found that horseradish peroxidase when combined with other compounds is highly reactive toward human tumor cells. A better understanding of horseradish peroxidase could lead to a new targeted cancer therapy.1
Structure
Horseradish peroxidase C has two metal centers, one of iron heme group and two calcium atoms.1 The structure is shown in Figure 1. The heme group has a planar structure with the iron atom held tightly in the middle of a porphyrin ring which is comprised of four pyrrole molecules.3 Iron has two open bonding sites, one above and one below the plane of the heme group. The heme group has a histidine (enzyme) attached in the proximal histidine residue (His170) which is located below the heme group in Figure 1. The second histidine residue in the distal side of the heme group, above the heme group, is vacant in the resting state.1 This site is open for hydrogen peroxide to attach during reduction-oxidation reactions. An oxygen atom will bond to this vacant site during activation. The iron atom’s sixth octahedral position is considered the active site of the enzyme. During the enzyme reaction, the bonding of the hydrogen peroxide to the iron atom creates an octahedral configuration around the iron atom. Other small molecules can also bond to the distal site, creating the same octahedral configuration.1
Figure 2 shows the three-dimensional structure of horseradish peroxidase.4 The iron heme is in the center of the enzyme shown in black with the iron atom as the red sphere. The two calcium atoms are black spheres and lie within the helical regions of the enzyme, with one in the distal region and one in the proximal region. The α-Helical and β-sheet regions of the enzyme are shown as the multicolored helical structures. According to Veitch, “Each calcium site is seven-coordinate with oxygen-donor ligands provided by a combination of amino acid side-chain carboxylates (Asp), hydroxyl groups (Ser, Thr), backbone carbonyls and a structural water molecule (distal site only).”1 Both the heme group and the calcium atoms are crucial to the enzyme working properly and the loss of one would result in instability.
Symmetry and Raman Spectrometry
Pure iron, at room temperature, has a body-centered cubic structure. However, according to the RCSB Protein Data Bank, x-ray diffraction shows that horseradish peroxidase has an orthorhombic structure.4 An orthorhombic structure is where all of the angles in the unit cell equal 90o and each of the three sides do no equal each other. In the case of horseradish peroxidase the three side lengths are: a=40.28 Å, b=67.46 Å, and c=117.11 Å.4 X-ray diffraction also provided the data to create the three-dimensional structure in Figure 2.
The immediate ligand environment of the iron atom in the resting state boasts a point group of D4d. The molecule is not linear nor does it have tetrahedral, icosahedral, or octahedral symmetry (at least in the rest state). The molecule has a C4 rotation axis and has 4 C2 axes perpendicular to the C4 axis.There is not a σh plane due to the fifth nitrogen atom which is part of the His170 residue that is bonded to the bottom of the iron atom.There are 4 σv planes that contain the C4 axis which leads to a point group symmetry of D4d. When the metalloenzyme is not in the resting state the sixth octahedral position of the iron atom is filled which will lead to a change in point group designation.Figure 2 shows the structure of the entire metalloenzyme.It can be seen that there is no symmetry within this structure and horseradish peroxidase as a whole has a point group of C1. There is no principal axis, mirror plane, or center of inversion in the enzyme.
Resonance Raman techniques are difficult to use with horseradish peroxidase due to the reactivity and photolability of the enzyme. This causes the horseradish peroxidase enzyme to break down into a second phase (HRP-II), ferric, and ferrous species.5 Scientists W. Anthony Oertling and Gerald T. Babcock attempted to mitigate this problem by lowering to cryogenic temperatures, pulsing a near-UV laser, and mixing hydrogen peroxide in the enzyme very quickly. Although some of the old problems still arose, they were able to get a Raman spectrum of the horseradish peroxidase. Horseradish peroxidase has three intermediate compounds all of which have a different color under optical spectroscopy.2 Because of this, the different stages of the redox cycle can be identified and a better understand of what constitutes each part of the cycle can be found.
Reactions
Figure 3 shows the five oxidation states of horseradish peroxidase.6 During the redox reaction, the hydrogen peroxide bonds to the vacant octahedral position on the iron atom which initiates the reaction. There are three different intermediate horseradish peroxidase compounds that form during the reaction. They are created as shown in the figure, with either an addition of an electron or a reaction with hydrogen peroxide. The reduction of compound I to compound II and compound II back to the rest state is carried out by reduction substrates. They are usually phenols or aromatic amines.2 To fully understand how the structure is changing during these stages, x-ray diffraction data must be taken. This, however, has posed a problem because electrons that are stirred up by the x-rays will alter the reduction-oxidation stage of the active site.6 | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Enzymes/Case_Studies/Horseradish_Peroxidase.txt |
Enzymes are catalysts that drive reaction rates forward. Most, but not all, are made up of amino acid chains called proteins that accelerate the rate of reactions in chemical systems. Their functionality depends on how the proteins are folded as to what they bind and react with. For protein-based catalysts, amino acid polarization lies at the core of catalytic activity.
Enzymatic Kinetics
Enzymes exist in all biological systems in abundant numbers, but not all of their functions are fully understood. Enzymes are important for a variety of reasons, most significantly because they are involved in many vital biochemical reactions. Increasing the reaction rate of a chemical reaction allows the reaction to become more efficient, and hence more products are generated at a faster rate. These products then become involved in some other biological pathway that initiates certain functions of the human body. This is known as the catalytic efficiency of enzymes, which, by increasing the rates, results in a more efficient chemical reaction within a biological system.
Introduction
An enzyme's active sites are usually composed of amino acid residues; depending on which amino acid residues are present, the specificity of the substrate can vary greatly. Depending on the pH level, the physical properties (mainly the electric charge) of an enzyme can change. A change in the electric charge can alter the interaction between the active site amino acid residues and the incoming substrate. With that said, the substrate can bind to the active site via hydrogen bonding or van der Waals forces. Once the substrate binds to the active site it forms an enzyme-substrate complex that is then involved in further chemical reactions.
In order for an enzyme to be active and be energetically favorable to allow a chemical reaction to proceed forward, a substrate must bind to an enzyme's "active site". An active site can be thought of as a lock and the substrate as a key; this is known as the lock and key model. A key (substrate) must be inserted and turned (chemical reaction), then the lock (enzyme) opens (production of products). Note that an enzyme might have more than one active site. Another theory on the active site-substrate relationship is the induced fit theory, which is quite opposite of the lock and key theory (where the active site is seemingly inflexible). In the induced fit theory, the active site of the enzyme is very flexible, and only changes its conformation when the substrate binds to it.
Enzymes work as a catalyst by lowering the Gibbs free energy of activation of the enzyme-substrate complex. Below are two figures showing a basic enzymatic reaction with and without a catalyst:
Figure 1: The energies of the stages of a chemical reaction. Uncatalyzed (dashed line), substrates need a lot of activation energy to reach a transition state, which then decays into lower-energy products. When enzyme catalyzed (solid line), the enzyme binds the substrates (ES), then stabilizes the transition state (ES) to reduce the activation energy required to produce products (EP) which are finally released. from Wikipedia.
Notice the difference of the Gibbs energy with the presence of a catalyst; it is higher in value when a catalyst is absent. The Gibbs energy can tell us whether or not a reaction is spontaneous judging from its value. In this case ∆G, the Gibbs free energy, can be conceptualized as the probability of a reaction occurring in nature without any interference. With a lower (more negative) Gibbs energy value, the probability of reaction is higher, and vice versa. Enzymes are here to increase the probability of the chemical reaction via many mechanisms, with one of the more prominent explanations being that it adopts a more favorable conformation.
The efficiency of the enzyme can be determined as follows: consider a simple enzymatic reaction:
Figure 2: An enzyme catalyzes the reaction of two substrates and to form one product. from Wikipedia.
German biochemist Leonor Michaelis and Canadian biochemist Maud Menten derived an equation describing this system, later known as the "Michaelis-Menten Equation", shown below:
$v_0 = \dfrac{V_{max}[S]}{K_M + [S]} \tag{1}$
This equation gives the rate of the reaction at a given substrate concentration, assuming a known Vmax, which is the maximum rate the reaction can proceed at, and KM, the Michaelis constant. However, in a practical application of the Michaelis-Menten, V0 is often measured, and Vmax is observed as a saturation or plateau in a data plot. Because the substrate concentration is known, KM is usually the calculated value of interest.
For $K_M$, assume $V_0= \dfrac{V_{max}}{2}$:
$\dfrac{V_{max}}{2} = \dfrac{V_{max}[S]}{K_M + [S]} \tag{2}$
$(K_M + [S]) \dfrac{V_{max}}{2} = V_{max}[S] \tag{3}$
$K_M + [S] = \dfrac{V_{max}[S]}{\dfrac{V_{max}}{2}} \tag{4}$
$K_M + [S] = 2[S] \tag{5}$
$K_M = [S] \tag{6}$
The Michaelis constant can be thought of as the rate at which the substrate becomes unbound from the enzyme, which can either occur in the events of substrate-enzyme complex becoming the product, or the substrate becomes unbound to the enzyme. KM can be shown as an equation.
$K_M = \dfrac{k_{-1} + k_2}{k_1} \tag{7}$
Whereas k-1 is the rate constant at which the substrate becomes unbound to the enzyme, resulting in the dissociation of the enzyme-substrate complex, k2 is the rate constant where the substrate-enzyme complex disappears and turns into product, and K1 is the rate constant for the formation of the the substrate-enzyme complex formation. Therefore, KM can be viewed as the rate of substrate-enzyme complex disappearance divided by the rate of substrate-enzyme complex formation, which is the level at which half of the substrate is bound to the enzyme. KM is a useful indicator for the presence of an inhibitor because we can look for changes in KM and compare to our control (biological systems that we know have zero inhibitor presence). KM is a dependent variable, and its value can change due to many reasons, including the pH level of the system, temperature, or any other condition that might affect a chemical reaction. A small KM indicates that the substrate has a high affinity for the enzyme.
The Michaelis-Menten equation is most useful in measuring enzyme efficiency if v0 is plotted against [S], as follows:
Figure 3: Diagram of reaction speed and Michaelis-Menten constant. from Wikipedia.
Vmax is the maximum rate at which the reaction can run, regardless of [S], meaning that even if you add more substrate, the reaction cannot go any faster. That is because at Vmax all of the active sites on the enzyme are occupied. After all the explanations on various forms of enzyme kinetic equations, we arrive at our conclusion of catalytic efficiency. Referring back to Fig 3, we have:
$V_o = k_2 \left(\dfrac{[E]_o[S]}{\dfrac{k_{-1} + k_2}{k_1} + [S]}\right) \tag{8}$
Notice $k_2$ describes an irreversible reaction as opposed to an equilibrium expression, when compared to k-1 and k1. k2 here is also known as kcat, the catalytic efficiency of enzyme. From the previous discussion, v0 is the measured reaction rate, which is the product formation over time, so it can be concluded that an equation would look like the following:
$v_0 = \dfrac{d[P]}{dt} = k_2[E]_0 \tag{9}$
Where [E]0 is the total enzyme concentration.
It is also known that VMax is observed when all of the enzyme-substrate complex disappear and turn into products, so we can make the following assumption:
$V_{max} = k_2[E]_0 \tag{10}$
and after rearrangement, we have this equation:
$k_{cat} = k_2 = \dfrac{V_{max} }{[E]_0} \tag{11}$
That is the equation for calculating catalytic efficiency, to be used after we obtain data from experiments and after using the Michaelis-Menten equation. With a larger kcat , the enzyme is efficient because less enzyme is needed.
Problems
1. What is the rate of product formation if k2 is 4.3 min-1 and [ES] is 2.3 x 10-2 M?
2. Find the formation of ES if k1= 3.3 x 103 min-1, k-1 = 1.1 x 10-10 min-1 and [E] and [S] are 1.2 x 10-6 M and 6.3 x 10-2 M respectively.
3. Given that k-1 = 8 x 104 s-1, k2 = 9 x 105 s-1, k1 = 7 x 106 M-1s-1 find KM. What does the answer tell us about the affinity of the substrate?
4. Using the KM value from above, find v0 if we determine experimentally the Vmax value to be 1.5 x 10-4 M min-1 and the [S] is 5.1 x 10-4 M.
5. Judging from KM from problem 1, we hypothesize that [E]0 is half of [S], find k2 using the Vmax value given above. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Enzymes/Enzymatic_Kinetics/Catalytic_Efficiency_of_Enzymes.txt |
Enzyme assays are used are used to study the rates of enzyme catalyzed reactions.
Contributors and Attributions
• Matthew Mesias, Janelle Defiesta (UCD)
Enzyme Inhibition
Enzymes are proteins that speed up the rate of a reaction by providing an alternate route to overcoming the activation energy. The graph below shows the path of a reaction both with and without the presence of an enzyme.
Contributors and Attributions
• Peter Abboud, Julie Choe, Kristopher Restel
General Enzymatic Kinetics
Enzymes are catalysts, most are proteins, that bind temporarily to one or more of the reactants of the reaction they catalyze. In doing so, they lower the amount of activation energy needed and thus speed up the reaction.
• Jai Pal
Michaelis-Menten Kinetics
Two 20th century scientists, Leonor Michaelis and Maud Leonora Menten, proposed the model known as Michaelis-Menten Kinetics to account for enzymatic dynamics. The model serves to explain how an enzyme can cause kinetic rate enhancement of a reaction and explains how reaction rates depends on the concentration of enzyme and substrate.
Introduction
The general reaction scheme of an enzyme-catalyzed reaction is as follows:
$E + S \xrightarrow[ ]{k_1}[ ES ] \xrightarrow[ ] {k_2} E + P$
The enzyme interacts with the substrate by binding to its active site to form the enzyme-substrate complex, ES. That reaction is followed by the decomposition of ES to regenerate the free enzyme, E, and the new product, P.
To begin our discussion of enzyme kinetics, let's define the number of moles of product (P) formed per time as V. The variable, V, is also referred to as the rate of catalysis of an enzyme. For different enzymes, V varies with the concentration of the substrate, S. At low S, V is linearly proportional to S, but when S is high relative to the amount of total enzyme, V is independent of S. Concentrations is important in determining the initial rate of an enzyme-catalyzed reaction. A more thorough explanation of enzyme rates can be found here: Definition of Reaction Rate.
To understand Michaelis-Menten Kinetics, we will use the general enzyme reaction scheme shown below, which includes the back reactions in addition the the forward reactions:
$E + S \xrightarrow[ ]{k_1}[ ES ] \xrightarrow[ ] {k_2} E + P$
$E + S \xleftarrow[ ]{k_3}[ ES ] \xleftarrow[ ] {k_4} E + P$
The table below defines each of the rate constants in the above scheme.
Table 1: Model parameters
Rate Constant Reaction
$k_1$ The binding of the enzyme to the substrate forming the enzyme substrate complex.
$k_2$ Catalytic rate; the catalysis reaction producing the final reaction product and regenerating the free enzyme. This is the rate limiting step.
$k_3$ The dissociation of the enzyme-substrate complex to free enzyme and substrate .
$k_4$ The reverse reaction of catalysis.
Substrate Complex
$E + S \xrightarrow[ ]{k_1} ES$ $v_o = k_1[E][S]$
$ES \xrightarrow[ ]{k_2} E + S$ $v_o = k_2[ES]$
$ES \xrightarrow[ ]{k_3} E + P$ $v_o = k_3[ES]$
$E + P \xrightarrow[ ]{k_4} ES$ $v_o = k_4[E][P]=0$
The ES complex is formed by combining enzyme E with substrate S at rate constant k1. The ES complex can either dissociate to form EF (free enzyme) and S, or form product P at rate constant k2 and k3, respectively. The velocity equation can be derived in either of the 2 methods that follow.
Method 1: The Rapid Equilibrium Approximation
E, S, and the ES complex can equilibrate very rapidly. The instantaneous velocity is the catalytic rate that is equal to the product of ES concentration and k2 the catalytic rate constant.
$v_o = k_2[E - S]$
The total enzyme concentration (ET) is equal to the concentration of free enzyme E (EF) plus the concentration of the bound enzyme in ES complex:
$[E]_T = [E_F] + [ES]$
$K_s = \dfrac{k_2}{k_1} = \dfrac{[E][S]}{[ES]}$
$K_s \dfrac{([E_o] - [ES])[S]}{[ES]}$
$[ES] = \dfrac{[E_o][S]}{K_s + [S]}$
$v_o = \left(\dfrac{dP}{dt}\right)_o = k_3[ES]$
$v_o = \left(\dfrac{dP}{dt} \right)_o = \dfrac{k_3[Eo][S]}{Ks + [S]}$
At high substrate concentrations, $[S] >> K_s$ we get:
$v_o = \left(\dfrac{dP}{dt}\right)_o = k_3[E_o] = V_{max}$ (1)
Method 2: The Steady-State Approximation
The figure above shows the relatively low and constant concentration of the enzyme-substrate complex due to the complex's slow formation and rapid consumption. Note the falling substrate concentration and the rising product concentration.
Figure 1: Change in concentrations over time for enzyme E, substrate S, complex ES and product P. from Wikipedia.
The rates of formation and breakdown of the E - S complex are given in terms of known quantities:
• The rate of formation of E-S = $k_1[E][S]$
(with the assumption that [P] =0)
• The rate of breakdown of E-S = $k_2 [ES] + k_3[ES] = (K_2 + K_3) [ES]$
At steady state,
$\dfrac{d[ES]}{dt} = k_1[E][S] +k_2[ES] + k_3 [ES] =0$
Therefore, rate of formation of E-S is equal to the rate of breakdown of E-S
So,
$k_1[E][S] = (k_2 + k_3)[ES]$
Dividing through by $k_1$:
$[E] [S] = \dfrac{(k_2 + k_3)}{k_1} [E-S]$
Substituting $\dfrac{(k_2 + k_3)}{k_1}$ with kM:
$[E] [S] = K_M [ES]$
$k_M = \dfrac{breakdown [ES]}{formation [ES]}$
$K_m$ implies that half of the active sites on the enzymes are filled. Different enzymes have different $K_m$ values. They typically range from 10-1 to 10-7 M. The factors that affect $K_m$ are:
• pH
• temperature
• ionic strengths
• the nature of the substrate
Substituting [EF] with [ET]-[ES]: ET = [ES] + [EF]
([ET] - [ES]) [S] = kM [ES]
[ET] [S] -[ES][S] = kM [ES]
[ET] [S] = [ES] [S] + kM [ES]
[ET] [S] = [ES] ([S] + kM)
Solving for [ES]:
[ES] = $\dfrac{([E_T] [S])}{([S] + k_M)}$
The rate equation from the rate limiting step is:
Vo = $\dfrac{dP}{dt}$ = k2[ES]
Multiplying both sides of the equation by k2:
$k_2 [ES] = k_2 (\dfrac{([E_T][S])}{(K_M + [S])}$
$V_o = k_2 (\dfrac{([E_T][S])}{(K_M + [S])}$
When S>>KM, vo is approximately equal to k2[ET]. When the [S] great, most of the enzyme is found in the bound state ([ES]) and Vo = Vmax
We can then substitue k2[ET] with Vmaxto get the Michaelis Menten Kinetic Equation:
vo =$\dfrac{(v_{max}[S])}{(k_M+ [S])}$
Reaction Order Note
When $[S] << K_m$,
$v = \dfrac{V_{max}[S]}{K_m}$
This means that the rate and the substrate concentration are directly proportional to each other. The reaction is first-order kinetics.
When $[S] >> K_m$,
$v = V_{max}$
This means that the rate is equal to the maximum velocity and is independent of the substrate concentration. The reaction is zero-order kinetics.
Figure 2: Diagram of reaction speed and Michaelis-Menten kinetics. from Wikipedia.
Then, at
$v = \dfrac{V_{max}}{2}$, $K_m = [S]$
$v = \dfrac{V_{max}}{2} = \dfrac{V_{max}[S]}{K_m + [S]}$
Therefore, $K_m$ is equal to the concentration of the substrate when the rate is half of the maximum velocity. From the Michaelis Menten Kinetic equation, we have many different ways to find $K_m$ and $V_{max}$ such as the Lineweaver-Burk plot, Hanes-Woolf plot, and Eadie-Hofstee plot, etc.
Lineweaver-Burk Plot
For example, by taking the reciprocal of the Michaelis Menten Kinetics Equation, we can obtain the Lineweaver-Burk double reciprocal plot:
$v_o= \dfrac{(V_{max}[S])}{(K_M +[S])}$
$\dfrac{1}{v} = \dfrac{(k_M + [S])}{v_{max} [S]}$
$\dfrac{1}{v} = \left( \dfrac{K_m}{V_{max}} \right) \left(\dfrac{1}{[S]} \right) + \dfrac{1}{V_{max}}$
Apply this to equation for a straight line $y = mx + b$ and we have:
$y = \dfrac{1}{v}$
$x = \dfrac{1}{[S]}$
$m = {slope} = \dfrac{K_m}{V_{max}}$
$b = {y-intercept} = \dfrac{1}{V_{max}}$
When we plot $y = \dfrac{1}{v}$ versus $x = \dfrac{1}{[S]}$, we obtain a straight line.
$x-intercept = \dfrac{-1}{K_m}$
$y-intercept = \dfrac{1}{V_{max}}$
$slope = \dfrac{K_m}{V_{max}}$
Figure 3. An example of a Lineweaver-Burke plot. from Wikipedia.
Eadie-Hofstee
Another way to calculate these values ($k_M$, $V_{max}$) and represent enzyme kinetics:
$V_o = \dfrac{(V_{max}[S])}{(K_M +[S])}$
vo (kM +[S]) = vmax[S]
vo kM + vo[S] = vmax [S]
vo [S] = -vo kM = vmax [S]
Dividing through by [S] $v_o = -k_m \dfrac{v_o}{[S]} + v_{max}$
Figure 4
Contributors and Attributions
• Han Le, Sandy Algaze, Eva Tan | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Enzymes/Enzymatic_Kinetics/Enzyme_Assays.txt |
Contributors and Attributions
• Abhinav Nath (Atkins group) University of Washington
Ping-pong mechanisms
The simplest of enzymes will involve one substrate binding to the enzyme and producing a product plus the enzyme. However, the majority of enzymes are more complex and catalyze reactions involving multiple substrates. Binding of two substrates can occur through two mechanisms: sequential mechanism and non-sequential mechanism. In sequential mechanisms both substrates bind the enzyme and the reaction proceeds to form products which are then released from the enzyme. This mechanism can be further subdivided into random and ordered reactions. For random reactions the order in which the substrates bind does not matter. In ordered reactions one substrate must bind the enzyme before the second substrate is able to bind. Non-Sequential mechanism does not require both substrates to bind before releasing the first product. This page will focus on the non-sequential mechanism, which is also known as the "ping-pong" mechanism. It is called this because the enzyme bounces back and forth from an intermediate state to its standard state.The enzyme acts like a ping-pong ball, bouncing from one state to another.
The Mechanism
Ping-pong mechanism, also called a double-displacement reaction, is characterized by the change of the enzyme into an intermediate form when the first substrate to product reaction occurs. It is important to note the term intermediate indicating that this form is only temporary. At the end of the reaction the enzyme MUST be found in its original form. An enzyme is defined by the fact that it is involved in the reaction and is not consumed. Another key characteristic of the ping-pong mechanism is that one product is formed and released before the second substrate binds. The figure below explains the Ping Pong mechanism through an enzymatic reaction.
This image shows that as substrate A binds to the enzyme, enzyme-substrate complex EA forms. At this point, the intermediate state, E* forms. P is released from E* , then B binds to E*. B is converted to Q, which is released as the second product. E* becomes E, and the process can be repeated. Often times, E* contains a fragment of the original substrate A.This fragment can alter the function of the enzyme, gets attached to substrate B, or both.
Here is another diagram showing this same reaction:
Example \(1\): Chymotrypsin
An example of the ping-pong mechanism would be the action of chymotrypsin. When reacted with p-nitrophenyl acetate (A), the reaction of chymotrypsin is seen to occur in two steps. In the first step, the substrate reacts extremely fast with the enzyme, leading to the formation of a small amount of p-nitrophenolate (P). In the second step, the substrate-enzyme interaction results in the formation of acetate ion (Q). The action of chymotrypsin is a ping-pong reaction because the binding of the two substrates causes the enzyme to switch back and forth between two states. Please refer to the section Chymotrypsin and pre-steady-state enzyme kinetics for more details on the action of chymotrypsin.
Example \(2\): Pyruvate Carboxylase
Another example of an enzyme that exhibits a ping-pong mechanism is pyruvate carboxylase. This enzyme catalyzes the addition of carbon dioxide to pyruvate in order to form oxaloacetate. (leads to gluconeogenesis) This biotin-containing enzyme works by binding CO2 (A) to form carboxybiotin (EA). The biotin swings over towards pyruvate (E*P) and releases CO2. (P, due to the fact that it had been moved from its original binding site) Pyruvate (B), in close proximity to CO2, attacks the partial positive of Carbon in CO2 (E*B). Oxaloacetate is formed within the enzyme (EQ) and gets released (Q). While this attack is occurring, biotin swings back to its initial position, (E* --> E) and is ready to bind another CO2.
Further Reading
An important factor to understand about the ping-pong mechanism is that when plotting a 1/v and 1/[A] plot at varying concentrations of B, a series of parallel lines are seen. In this case A is the first substrate and B being the second substrate.
Refer to these sections on enzyme kinetics and michaelis-menten kinetics to get a better understanding of what this type of plot means.
Sample Questions
1. The form in which the ping pong mechanism binds substrates is identified as which type of mechanism?
2. What are two characteristics of an enzyme that catalyzes a reaction through the ping-pong mechanism?
3. The following diagram shows the mechanism of glutamate-aspartate aminotransferase: Would this mechanism be considered a ping-pong/double-displacement reaction? Why or why not?
Answers
1. The ping-pong mechanism is a non-sequential mechanism. A product is released after the first substrate is bound.
2. One, a product is seen before the second substrate is bound. Two, binding of the first substrate causes the enzyme to change into an intermediate form that will bind the second substrate. Three, the plot of 1/v vs. 1/[A] as [B] changes will be parallel lines.
3. Yes! This would definitely be considered a ping-pong mechanism. First, we can see that there is an E' state which is indicative of a ping-pong mechanism. Pyridoxal is a coenzyme bound to glutamate-aspartate aminotransferase that accepts an amino group from glutamate and becomes pyridoxamine while releasing alpha-ketoglutarate. Pyridoxamine bound to the enzyme will then donate its amino group to oxaloacetate to regenrate pyridoxal as well as aspartate.
References
1. Chang, Raymond. 2005. Physical Chemistry for the Biosciences. Sausalito (CA): University Science Books. p. 372-377.
2. Cleland, Wallace. "Derivation of Rate Equations for Multisite Ping-Pong Mechanisms with Ping-Pong Reactions at One or More Sites." Journal of Biological Chemistry 248.24 (1973): 8353-355. Print.
3. Garrett, R., and Charles M. Grisham. "Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway." Biochemistry. Belmont, CA: Brooks/Cole, Cengage Learning, 2010. 664-66. Print.
4. Garrett, Reginald and Charles M. Grisham. "Enzymes-Kinetics and Specificity". Biochemistry, Fourth Edition. Belmont, CA: Brooks/Cole, Cengage Learning, 2010. p. 406-407.
Contributors and Attributions
• Melissa Hill, Laura Lan, Andrew Jilwan | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Enzymes/Enzymatic_Kinetics/Michaelis-Menten_Kinetics_1.txt |
Sigmoidal kinetic profiles are the result of enzymes that demonstrate positive cooperative binding. cooperativity refers to the observation that binding of the substrate or ligand at one binding site affects the affinity of other sites for their substrates. For enzymatic reactions with multiple substrate binding sites, this increased affinity for the substrate causes a rapid and coordinated increase in the velocity of the reaction at higher $[S]$ until $V_{max}$ is achieved. Plotting the $V_0$ vs. $[S]$ for a cooperative enzyme, we observe the characteristic sigmoidal shape with low enzyme activity at low substrate concentration and a rapid and immediate increase in enzyme activity to $V_{max}$ as $[S]$ increases. The phenomenon of cooperativity was initially observed in the oxygen-hemoglobin interaction that functions in carrying oxygen in blood. Positive cooperativity implies allosteric binding – binding of the ligand at one site increases the enzyme’s affinity for another ligand at a site different from the other site. Enzymes that demonstrate cooperativity are defined as allosteric. There are several types of allosteric interactions: (positive & negative) homotropic and heterotropic
.
Figure 1: Rate of Reaction (velocity) vs. Substrate Concentration.
Positive and negative allosteric interactions (as illustrated through the phenomenon of cooperativity) refer to the enzyme's binding affinity for other ligands at other sites, as a result of ligand binding at the initial binding site. When the ligands interacting are all the same compounds, the effect of the allosteric interaction is considered homotropic. When the ligands interacting are different, the effect of the allosteric interaction is considered heterotropic. It is also very important to remember that allosteric interactions tend to be driven by ATP hydrolysis.
Hill Coefficient
The degree of cooperativity is determined by Hill equation (Equation 1) for non-Michaelis-Menten kinetics. The Hill equation accounts for allosteric binding at sites other than the active site. $n$ is the "Hill coefficient." When n < 1, there is negative cooperativity; When n = 1, there is no cooperativity; When $n > 1$, there is positive cooperativity
$\theta = \dfrac{[L]^n}{K_d+[L]^n} = \dfrac{[L]^n}{K_a^n+[L]^n} \label{1}$
where
• $\theta$ is the fraction of ligand binding sites filled
• $[L]$ is the ligand concentration
• $K_d$ is the apparent dissociation constant derived from the law of mass action (equilibrium constant for dissociation)
• $K_a$ is the ligand concentration producing half occupation (ligand concentration occupying half of the binding sides), that is also the microscopic dissociation constant
• $n$ is the Hill coefficient that describes the cooperativity
Taking the logarithm of both sides of the equation leads to an alternative formulation of the HIll Equation.
$\log \left( \dfrac{\theta}{1-\theta} \right) = n\log [L] - \log K_d \label{2}$
Currently, there are 2 models for illustrating cooperativity: the concerted model and the sequential model
• The concerted model illustrates cooperativity by assuming that proteins have two or more subunits, and that each part of the protein molecule is able to exist in either the relaxed (R) state or the tense (T) state - the tense state of a protein molecule is favored when it doesn't have any substrates bound. All aspects, including binding and dissociation constants are the same for each ligand at the respective binding sites. This model can also be referred to as the Monod-Wyman-Changeux model, as named after its founders.
• The sequential model aims to demonstrate cooperativity by assuming that the enzyme/protein molecule affinity is relative and changes as substrates bind. Unlike the concerted model, the sequential model accounts for different species of the protein molecule.
Contributors and Attributions
• Tinuke Fashokun
Turnover Number
In enzyme kinetics, we are interested to know how many maximum molecules of substrate can be converted into product per catalytic site of a given concentration of enzyme per unit time.
\[ k_{cat} =\dfrac{ V_{max}}{E_t} \]
with
• The units of Turn over number (kcat) are \(k_{cat}\) = (moles of product/sec)/ (moles of enzyme) or sec-1.
Contributors and Attributions
• Kiranpreet Kaur | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Enzymes/Enzymatic_Kinetics/Sigmoid_Kinetics.txt |
Introduction
Human immunodeficiency virus (HIV) is a retrovirus, which is a class of viruses that carry genetic information in RNA.There are two types of HIV, HIV-1 and HIV-2, with HIV-1 being the most predominant, it is commonly called just HIV. Both types of HIV damage a person’s body by destroying specific blood cells, called CD4+ T cells, which are crucial to helping the body fight diseases in the immune system. This can lead to immune deficiency, which is when the infection with the virus progressively deteriorates the immune system and is considered deficient when it no longer works to help fight infection and disease.2
According to the 2006 Morbidity and Mortality Weekly Report, published by the Center for Disease Control there were approximately 1.1 million United State Citizens are affected by HIV.3 There was an estimated number of 56, 300 people that newly contracted HIV.4 Although the annual incidence for HIV has remained constant throughout recent years, the prevalence has increased each year. These drugs developed for HIV treatment are based on the mechanism HIV uses, including proteases, to infect its host.7 The HIV-1 protease is synthesized from the gag and pol genes along with other proteins.8 Retroviruses, such as HIV-1, are able to reverse transcribe because of the reverse transcriptase which is transcribed by the pol gene.9 HIV-1 RNA contains many genes, specifically gag and pol, that encode for many proteins.10 The open reading frames of gag and pol genes overlap in HIV-1.11 Studies have found that the initial cleavages are made by the immature protease dimer in the membrane of the infected cell during virus budding, or replication. Once these intramolecular cleavages are made a more active gag-pol processing intermediate is released, which becomes the active protease.8
Structure
The mechanism of HIV-1 protease is still yet to be fully understood. The main way the mechanism are studied is through the use of mimicry substrates and simulations. HIV-1 protease has been studied intensely using various inhibitors, observing partial steps of the process. Since the main target of these inhibitors is to bind to the Asp-25 of the catalytic triad, each inhibitor would vary in its mechanism to accomplish this.14 Further investigation would then take place of the various proposed mechanisms in attempts to synthesize new drugs that would act in a similar fashion.
The first part of the mechanism begins with the substrate binding onto the protease. Figure 2 accents the key amino acids in HIV-1 protease that assists in substrate binding. It is predicted that a substrate first binds via a hydrogen bond to aspartic acid 30 on one chain. Once this initial bond is made, the binding is then further stabilized by bondage to the glycine rich region in the flap of the same monomer. A salt bridge is then formed from the substrate to glutamic acid 35 of the other monomer. This completes binding of the substrate to the protease.15 At this point, waters molecules that are found at the tips of the flaps at isoleucine 50 on each monomer dissociates from the protease.15,16 The release of the water molecule results in a structural conformation change of the protease, changing it from semi-open to closed, tightening the space between the protease and substrate.15
HIV protease has variable states that it exists in, such as the two states mentioned above--the semi-open and closed state. These states depend on whether a substrate is bound to it. In its unbound state, the protease’s glycine rich flaps (shown in grey in Figure 1) are in a semi open state. Figure 1 depicts the protease in a closed flap state, which occurs when a ligand is bound to it (ligand not shown). An open state is thought to be the least frequent of the three states.17
Once in the tightened state, aspartic acid 25 and 25’ hydrogen binds to their adjacent glycine, and then becomes supported by the following threonine. Originally, there is a water molecule bound between the aspartic acids. One of the aspartic acid exists in a deprotonated state and the other one is protonated. The water molecule stabilizes the aspartates in this form.When the substrate binds to the protease, it causes conformational changes that brings the substrate to the position of the water molecule, and the water molecule acts as a nucleophile to the substrate. The oxygen of the water attacks the carbonyl group of the substrate peptide bond that is by the active site as the nitrogen picks up the hydrogen of the protonated aspartic acid. What results is an hydroxl group is added to the carbonyl group as an amine is formed on other side of the peptide bond, leaving a hydrogen atom behind to stablize the two aspartates. This is proposed to occur in a concerted fashion. This mechanism is outlined in Figure 3.1,18
Figure 2. HIV-1 Protease with Accented Substrate Binding Assistant Amino Acids.13,15 Aspartic acid 30 is shown in pink; Glycine 48, 49, and 51 are shown in white; Isoleucine 50 and 50’ are shown in yellow; and glutamic acid 35’ is shown in green. (Primes distinguish amino acids from each monomer.)
Figure 3. Proposed Proteolytic Mechanism.1,18, Boxed molecules in “a” are showing the target peptide bond of the substrate and the water molecule hydrogen bonding between the two aspartic acids. The rest are the catalytic aspartic acids in the active site. “b” shows the proposed concerted mechanism between the water, peptide bond and aspartic acids. “c” shows the end products.
Treatment
With a disease this prevalent, medication is key in trying to extend the afflicted’s life. As mentions above, since a mutation to the aspartic acid in the active site of HIV-1 protease renders pro-viruses that are unable to form completely and infect other sites, the protease has been one of the targets for therapy. These drugs are referred to as protease inhibitors.14 A current Food and Drug Administration approved drug against this HIV-1 protease is nelfinavir mesylate, 2-[2-Hydroxy-3-(3-hydroxy-2-methyl-benzoylamino)-4-phenyl sulfanyl-butyl]-decahydro-isoquinoline-3-carboxylic acid tert-butylamide; C32H45N3 O4S (“Viracept”R). Figure 3 shows the drug fitting into the protease. At the center of the drug, a hydroxy group binds with the catalytic aspartic acid (boxed in red), while the other four groups (boxed in white) stabilizes the drug to the protease, making its bond more favorable than its natural substrate. This compounds accomplishes this by making various hydrophobic interactions and hydrogen bonds.19
This drug has a high drug efficiency. In order to prevent 50 percent of the HIV-1 infected cells from becoming necrotic, a dosage of 14nM is required.19 Although it is a high potent drug, there also a few side effects that come along with it. Side effects include fever, back pain, rash sweating, vomiting, and diarrhea based on a study of 62 HIV infected children ages 3 months to 13 years. Fourteen out of the 62 had diarrhea as a side effect and less than 6% of the study group had the other side effects.20
Due to the high mutation rate of HIV-1, often, multiple drugs are combined as a treatment in attempts to retard its spread as much as possible. A commonly seen drug paired with protease inhibitors is reverse transcriptase inhibitor. Protease inhibitors prevents the protease transcribed by the gag-pol gene and reverse transcriptase inhibitors prevents the reverse transcriptase transcribed by the pol gene. This combination targets two essential proteins that have been shown to stop HIV-1’s life cycle if these genes have been mutated. By targeting both proteins, HIV-1 activity is seen to decrease more than just one. An example of a reverse transcriptase inhibitor is Efavirenz. Efavirenz, in combination with nelfinavir mesylate has shown to increase the immune cell count and decrease the seen HIV-1 molecules in the blood plasma. The side effect of this drug are similar to those of nelfinavir mesylate.21 The effects of these developed drugs are the main reason HIV-1 infected people can live on life longer than they would have been able to in the past.
Figure 3. HIV-1 Protease with nelfinavir mesylate.22 This is a top down view of the protease showing how the drug fits into the protease. Light blue molecules are carbons, red molecules are oxygens, blue molecules are nitrogens and yellow molecules are sulfurs. White boxed areas show the four main pockets the inhibitor lays in and the red boxed area shows the binding to the catalytic aspartic acid. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Enzymes/HIV.txt |
Metalloproteases (metallo, metal) are members of a clan of proteases that contain a metal ion at their active site which acts as a catalyst in the hydrolysis peptide binds.1 The metallic core of each enzyme is the location of the specific reaction performed by the enzyme, in the case of metalloproteases, the cleavage of peptide bonds within proteins. The most common metal ion metalloproteases utilize is a zinc ion (Zn2+).2 Other transition metals have been found at active sites, such as Co2+ and Mn2+, and some have been used to restore function in zinc-metalloproteases in which the Zn2+ core has been removed.3 Generally, metal ions are bound in a nearly tetrahedral conformation at the active site. Three amino acid ligands, usually charged residues, associate with the metal core along with one water molecule which is used for hydrolysis.4
There are two major divisions of metalloproteases: metalloendopeptidases and metalloexopeptidases. Each division is named for the region of the hydrolysis-targeted protein at which the reaction takes place.5,6 Within these divisions are nested more highly specified target sites and conserved catalytic residues wholly dependent on the nature of the enzyme.1,2,7
Metalloendopeptidase: Thermolysin
Background
Thermolysin (TLN; EC 3.4.24.27, Figure 1) is a 34.6 kDa Zn2+-endopeptidase secreted by the bacterium Bacillus thermoproteolyticus.8,9 TLN and TLN-like proteins are used by bacteria to break down exogenous proteins for nutrition and as virulence factors aiding in host colonization and tissue degradation.10,11 TLN is active in the hydrolysis of internal peptide bonds on the N-terminal side of large hydrophobic amino acids, between leucine, isoleucine, or phenylalanine. Thermolysin was the first metalloproteases to be completely sequenced.12 Commercially, TLN is used as a nonspecific protease (within its cleavage site specificity) in peptide sequencing and is used in the production of the artificial sweetener aspartame.13
Figure 1. General structure of thermolysin (PDBID 3DNZ). Alpha-helices in blue, beta-sheets in red, Zn2+ ion in yellow, active site side chains in magenta, Ca2+ ions in green.8
Structure
The overall structure of the protein consists of 316 amino acid residues organized into two domains separated by the active site.12 The N-terminal domain is predominately composed of β-sheets, while the C-terminal domain is primarily composed of α-helices. The two large domains are separated by a central α-helix. One Zn2+ is bound via three residues in the active site within a conserved binding motif of HEXXH (His 142, His 146, Glu 166) located on the C-terminal side (Figure 2).14,15 The tetrahedral binding of Zn2+ is rounded out by a nucleophilic water molecule. This water molecule is capable of alternating between two distinct association positions, W1 and W2, which are in turn stabilized by His 231 and Glu 143, respectively.16 Four Ca2+ ions are associated with the enzyme that aide in thermostability.12
Figure 2. Active site of thermolysin.
Catalytic Residue Importance
Zinc: The bound Zn2+ is responsible for catalyzing peptide hydrolysis and stabilizing the various intermediates of the reaction. Although normally bound in a tetrahedral structure, during catalysis Zn2+ assumes a pentacoordinate geometry between the original three residues (His142, His146, and Glu166), the oxygen of the nucleophilic water, and the carbonyl oxygen of the substrate.7,14,17 The formation of a gem-diolate intermediate is stabilized by Zn2+.14,17 Removal of Zn2+ yields an inactive enzyme. Exogenous addition of other divalent transition metals, specifically Zn2+, Co2+, Fe2+, and Mn2+, results in the regaining of 100%, 200%, 60%, and 10% enzymatic activity.3 Zn2+ is also responsible for the polarization of the carbonyl bond of the substrate and the enhancement of nucleophilicity of the catalytic water molecule.
Glu143: Glu143 is responsible for the polarization of the catalytic water molecule leading to an enhancement of nucleophilicity. Additionally, Glu143 abstracts a proton from the water molecule and transfers it to the amide leaving group. In site-directed mutagenesis experiments involving the neutral protease of B. stearothermophilus, a protease with an amino acid sequence identical to that of thermolysin, Glu143Asp and Glu143Gln substitutions resulted in no catalytic activity of the enzyme.12,18 The mechanism proposed by Mock and colleagues suggests a diminished catalytic role of Glu143, which is instead used solely for charge stabilization with no association with the nucleophilic water molecule.19,20 Mutagenesis studies show that Glu143Asp substitutions result in inactive enzymes, despite the same side chain charge that would provide electrostatic stability.18
His231: His231 is responsible for substrate stabilization during hydrolysis. The carbonyl oxygen of the peptide is hydrogen bonded to N1 of His231 in the intermediate step of hydrolysis. Mutagenesis experiments involving His231Phe and His231Ala show 430- and 500-fold reductions in catalytic activity with no significant change in Km.21 These findings led to a proposed TLN-mechanism with an emphasis on the role of His231 as a general base, but this mechanism is less favored due to the residual activities of His231-substituted mutants compared to inactive mutants generated by Glu143 mutagenesis studies.18,19,21
Tyr157: The importance of Tyr157 has been debated in several mechanisms.18,22 Site-directed mutagenesis studies show an 80% drop in catalytic activity of Tyr157Trp mutants, and it has been suggested that the hydroxyl-H of Ty157 stabilizes the carboxylate-O of the peptide substrate during hydrolysis.17,23 Energetics modeling suggests a primary role for Tyr157 in transition state stabilization and substrate binding with an increase in the activation barrier energy of 2.7 kcal/mol and a decrease of binding affinity of 0.5 kcal/mol.24
Asp226: Asp226 has been suggested to stabilize the catalytic His231 through H-bonding of the Asp226 carboxylate to N3 of His231.17,22 Mutagenesis of Asp226Ala shows a relatively small decrease in catalytic activity of 40-fold and energetics modeling suggest an increase in overall ΔG‡ 2.2 kcal/mol.23,24
Mechanism
Scheme 1. Mechanism of peptide hydrolysis by thermolysin.24
Several mechanisms for TLN mediated peptide cleavage have been proposed with varying emphasis on the importance of the roles of the catalytic residues, Glu143 and His231, as well as several catalysis-associated residues, Tyr157 and Asp226. The generally accepted mechanism for TLN-mediated hydrolysis proceeds via the two-step process depicted in Scheme 1.24 Briefly:
1. The active site houses the catalytic Zn2+ ion bound to three residues and a catalytic water (W)
2. An incoming peptide substrate displaces the hydrogens of W1 towards Glu143, while the oxygen of W (OW) remains associated with Zn2+; Zn2+ associates with the carbonyl oxygen (OP) of the substrate forming the enzyme-substrate complex (ES)
3. Polarizing actions on Zn2+ by Glu143 allow nucleophilic attack of carbonyl carbon (CP) by OW forming Transition State 1 (TS1); stabilized by Asp226 at N3, His231 N1 (originally protonated) stabilizes the substrate via hydrogen bonding at the carbonyl oxygen
4. The intermediate (INT) gem-diolate is formed as Glu143 breaks the HW1-OW bond and the amide of the substrate (NP) hydrogen bonds HW2
5. The NP-CP bond of the substrate begins to break as the CP-OP bond regains double bond character and the tetrahedral CP gem-diolate intermediate is pushed to Transition State 2 (TS2)
6. The NP-CP is fully broken and the Glu143-bound HW2 bond begins to break as the new N-terminal hydrolyzed peptide is released (PROD)
7. The active site is hydrated and is free for new peptide docking
The catalytic activity of TLN is dependent on both temperature and pH.25 The pKas of TLN are 5.0 and 8.25, and maximum catalytic activity has been measured at pH 7.2.25,26 The thermostability of TLN conferred by the four Ca2+ ions allows an increase in catalytic activity at temperatures approaching 40°C, with no significant loss in activity or conformation alteration until temperatures exceed 70°C.25,27
Inhibition and Inhibitors
Chelating agents such as EDTA have been shown to completely inactivate TLN and other metalloproteases by removal of the metal ion.3 In addition to direct removal of the catalytic metal ion, substrate and transition state analogs have been synthesized that greatly decrease the catalytic activity of TLN and TLN-like proteins. Transition state analogs that resemble the mechanistic transition states of the normal hydrolysis reaction catalyzed by TLN have been both isolated and synthesize. Phosphoramidates, a group of phosphoryl group containing amino acid or peptide compounds have been shown to mimic the tetrahedral intermediate of the TLN mechanism.28,29 Overall inhibition has been shown to vary from five to 1000-fold decrease in catalytic activity through use of transition state analogs.28,30 Holmquist and Vallee combined substrate analog inhibition with anionic ligands that tightly bind active site metals such as R-S- and R-P-O-.30 By combining two approaches, catalytic activity of TLN was decreased 10,000-fold. This compared to ~800-fold decrease when transition state analogs were combined with metal-binding ligands, suggesting a stronger inhibition effect of the substrate analog.30
Metalloexopeptidase: Carboxypeptidase A
Background
Carboxypeptidase A (carboxypolypeptidase; CPA; EC 3.4.17.1) is a 35 kD metalloenzyme within the zinc hydrolase family and as such contains a Zn2+ ion cofactor located within its active site.31,32 Originally isolated from bovine pancreas tissue in 1929, CPA is an exopeptidase that catalyzes the hydrolysis of C-terminal esters and peptides with large hydrophobic side chains.31,32 Biologically, CPA facilitates the breakdown of proteins during metabolism, while proposed commercial applications include its use the hydrolysis of cheese whey protein and the production of phenylalanine-free protein hydrolysates for use by individuals with phenylketonuria.33,34
Figure 3: General structure of carboxypeptidase A (PDBID 1YME). Alpha-helices in blue, beta-sheets in red, Zn2+ ion in yellow, active site side chains in cyan.
Figure 4: Carboxypeptidase A active site.32
Structure
The structure of CPA was first determined in 1967 using x-ray diffraction, making it one earliest proteins to have been characterized using the technique (Figure 3).35 CPA consists of a single chain containing 307 amino acids and a single Zn2+ ion in its active site.35 Zn2+ is stabilized within the active site through interactions with His69, Glu72, and His196 and is additionally bound by a catalytic water molecule that has been shown to interact with Glu270 (Figure 4).36 Mutagenesis studies have indicated that the additional residues Arg127, Tyr248, Arg71, Asn144, and Arg145 form an outer shell, which includes Glu270, around the active site and contribute to the catalytic function of CPA through the stabilization of substrate molecules.36 In both proposed mechanisms for the catalytic activity of CPA, Glu270 plays an important role, either acting as a general base-general acid or as a nucleophile.36
Mechanisms
Two mechanisms have been suggested for CPA catalyzed hydrolysis—the promoted-water pathway, also known as the general base-general acid pathway, and the nucleophilic, or anhydride, pathway—with experimental evidence existing for both mechanisms.36
Promoted-water pathway
Scheme 2: Promoted-water (general base-general acid) pathway. Active site substituent side chains in black, substrate in red, water in blue.36
In the enzyme substrate (ES) complex, the Zn2+ tetrahedron consists of a water molecule, His69, Glu72, and His196 bound to the ion while the water molecule in the near attack position is maintained through hydrogen bonding with Glu270 and Ser197. The substrate position is maintained through interactions with Arg127, Asn144, Arg145, Tyr248, and Arg71. The water molecule attacks the scissile carbonyl carbon of the substrate molecule through nucleophilic addition with Glu270 acting as a general base, leading to the first transition state (TS1) and the formation of the tetrahedral intermediate (TI). Following the formation of the TI, the leaving group is protonated and the peptide bond is cleaved with Glu270 now severing as a general acid, forming the second transition state (TI2) and finally the enzyme product (EP) complex. An oxyanion hole, created by the polarization of the scissile carbonyl carbon of the substrate in the ES complex by Arg127 and the presence of Zn2+, helps stabilize the charge generated on the carbonyl oxygen during TS1 and TI.36
Nucleophilic pathway
Scheme 3: Nucleophilic pathway. Active site substituent side chains in black, substrate in red, water in blue.36
In the enzyme substrate (ES) complex, the Zn2+ tetrahedron consists of His69, Glu72, His196, and the scissile carbonyl oxygen of the substrate molecule. This direct binding by Zn2+ polarizes the carbonyl oxygen, facilitating the nucleophilic attack by the carboxylate side chain of Glu270, which is in the near attack position. The substrate position is maintained through interactions with Arg127, Asn144, Arg145, Tyr248, and Arg71. The nucleophilic attack by Glu270 on the scissile carbonyl carbon leads to the first transition state (TS1) and the formation of the acyl enzyme intermediate (AE). A water molecule present in the active site nucleophilically attacks the carboxylate carbon of Glu270, resulting in deacylation and the transition through the second transition state (TS2) and finally the enzyme product (EP) complex.36
Computational evidence suggests that in regards to proteolysis, the promoted-water pathway is the only feasible pathway of the two. However, both pathways are feasible in esterolysis reactions, with the promoted-water pathway having the lower kinetic barrier. This suggests that under normal conditions the promoted-water pathway is favored, whereas at low temperatures, the formation of the tetrahedral intermediate by the promoter-water pathway and the formation of the acyl enzyme intermediate by the nucleophilic pathways are comparable. The second deacylation step in the nucleophilic pathway presents too high of a barrier however to be viable versus the promoted-water pathway. This then suggests that in regards to both proteolysis and esterolysis, the promoted-water pathway is the dominant pathway of CPA.36
Inhibitors and Inhibition
Ultraviolet-visible radiation (400 W, λ=250-750 nm) has been shown to cause uncompetitive inhibition of CPA with the decrease in enzymatic activity indirectly proportional to the irradiation time, with total enzymatic inactivation after 20 minutes of exposure. Additionally, exposure times of greater than 24 minutes are suspected to adversely affect the structure of CPA, resulting in the formation of protein aggregates.37
Active site-directed inhibitors of CPA, which are characterized by the presence of a terminal carboxylate, a hydrophobic side chain, and a zinc-binding group, have been identified, among which include the enantiomers of 2-benzyl-5-hydroxy-4-oxopentanoic acid. (L)-2-benzyl-5-hydroxy-4-oxopentanoic acid interacts through its carboxylate by forming hydrogen bonds with Arg145, Arg127, and Tyr248 and through its terminal hydroxyl group by forming a hydrogen bond with Ser197.38
Transition state analog inhibitors of CPA have also been found, which when bound with the enzyme active site create a pseudo-transition state complex. Both (R)-2-benzyl-3-nitropropanoic acid and (R)-2-benzyl-5-nitro-4-oxopentanoic acid are capable of inhibiting CPA by forming complexes with their respective nitro groups, Glu270, Arg127, and Zn2+.39,40
Contributors and Attributions
• Hank Kimbrough (Truman State University)
• Christopher Tracy (Truman State University) | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Enzymes/Metalloproteases.txt |
Fats and lipids are important because they serve as energy source, as well as a storage for energy in the form of fat cells. They also have a major cellular function as structural components in cell membranes. These membranes in association with carbohydrates and proteins regulate the flow of water, ions, and other molecules into and out of the cells. Hormone steroids and prostaglandins are chemical messengers between body tissues. Vitamins A, D, E, and K are lipid soluble and regulate critical biological processes; other lipids add in vitamin absorption and transportation. Lipids act as a shock absorber to protect vital organs and insulate the body from temperature extremes.
Thumbnail: This fluid lipid bilayer cross section is made up entirely of phosphatidylcholine. (Public Domain; Bensaccount).
Contributors
• Charles Ophardt, Professor Emeritus, Elmhurst College; Virtual Chembook
Applications of Lipids
Every cell is enclosed by a membrane which gives structure to the cell and allows for the passage of nutrients and wastes into and out of the cell. The purpose of the bilayer membrane is to separate the cell contents from the outside environment. The outside of the cell is mostly water and the inside of the cell is mostly water. The cell membrane may be coated with other molecules such as carbohydrates and proteins, which serve as receptor sites for other messenger molecules. Interaction with the cell membrane allows for molecular communication signals to pass from outside to inside of the cell.
Introduction
Cell membranes are composed of two classes of molecules: lipids and proteins. The proteins serve as enzymes, carry molecules, and provide the membrane with distinctive functional properties. Details of proteins and enzyme structures are given elsewhere. The lipids provide the structural integrity for the cell. The lipids found in the membrane consist of two parts: hydrophilic (water soluble) and hydrophobic (water insoluble). The hydrophobic portion of the lipids is the non-polar long hydrocarbon chains of two fatty acids. The fatty acids are present as esters bonded to glycerol. The third-OH group on glycerol is ester bonded to phosphate hence the term phospholipid. The phosphate ester portion of the molecule is polar or even ionic and hence is water soluble. A simple interaction of several phospholipids is shown in the graphic on the left.
There are two common phospholipids found in the bilayer:
1. Lecithin contains the amino alcohol, choline.
2. Cephalins contain the amino alcohols serine or ethanolamine.
The arrangement of phospholipids in cell membranes has been deduced by X-Ray diffraction data. The phospholipids are arranged as a bilayer (two molecules thick). The phospholipids are stacked with the non-polar hydrocarbon chains pointed inward while the polar ends act as the external surface as shown in graphic on the left. The structure of the bilayer is another application of the solubility principle of "likes dissolve likes".
Most of the fatty acids in the membrane are unsaturated because this allows the membrane to be more flexible (cis bonds are bent) to allow certain molecules through the membrane. However, the interaction of the hydrophobic inside of the layer acts as a barrier for ionic and polar molecules from entering the inside of the cell. In animal cells cholesterol is inserted between the non-polar chains, and makes up about 20% of the molecules of the membrane. This helps to make the membrane more rigid and adds strength.
Lipid Bilayer Graphic: Red/white spheres represent water molecules on the outside surfaces of the bilayer which are hydrophilic (water loving). The gray spheres represent the non-polar hydrocarbon chains, which are hydrophobic or water hating. The purple spheres represent individual phospholid molecules.
Contributors
• Charles Ophardt, Professor Emeritus, Elmhurst College; Virtual Chembook | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Lipids/Applications_of_Lipids/Lipid_Bilayer_Membranes.txt |
Fatty acids are merely carboxylic acids with long hydrocarbon chains. The hydrocarbon chain length may vary from 10-30 carbons (most usual is 12-18). The non-polar hydrocarbon alkane chain is an important counter balance to the polar acid functional group. In acids with only a few carbons, the acid functional group dominates and gives the whole molecule a polar character. However, in fatty acids, the non-polar hydrocarbon chain gives the molecule a non-polar character.
• Hydrogenation of Unsaturated Fats and Trans Fat
Saturated fats have a chain like structure which allows them to stack very well forming a solid at room temperature. Unsaturated fats are not linear due to double bonded carbons which results in a different molecular shape because the sp2 carbons are trigonal planar. This causes the fat molecules to poorly stack resulting in fats that are liquid at room temperature. Unsaturated fats can be converted to saturated fats via hydrogenation reactions.
• Introduction to Fatty Acids
Fatty acids are carboxylic acids with long hydrocarbon chains. There are two groups of fatty acids--saturated and unsaturated. Recall that the term unsaturated refers to the presence of one or more double bonds between carbons as in alkenes. A saturated fatty acid has all bonding positions between carbons occupied by hydrogens. The melting points for the saturated fatty acids follow the boiling point principle observed previously.
• Prostaglandins
Prostaglandins were first discovered and isolated from human semen in the 1930s by Ulf von Euler of Sweden. Thinking they had come from the prostate gland, he named them prostaglandins. It has since been determined that they exist and are synthesized in virtually every cell of the body. Prostaglandins, are like hormones in that they act as chemical messengers, but do not move to other sites, but work right within the cells where they are synthesized.
Thumbnail: A ball-and-stick diagram of arachidonic acid. (Public Domain; SubDural12). Arachidonic acid is a polyunsaturated fatty acid present in the phospholipids of membranes of the body's cells, and is abundant in the brain, muscles, and liver.
Fatty Acids
In the late 1970’s the lipid hypothesis came in to existences stating that eating saturated fats leads to elevated LDL (Low Density Lipoprotein) which was perceived to be "bad cholesterol." This will result in coronary heart disease which is hardening and narrowing of arteries resulting in heart attack. Fats were eventually classified in to 2 categories: “healthy fats” and “unhealthy fats”. Unhealthy fats where perceived to be saturated fats and healthy fats where perceived to be unsaturated fats.
A meta-analysis of 72 studies with over 103,052 people have found no validity in the lipid hypothesis. The conclusion of the Meta-Analysis was,“In contrast to current recommendations, this systematic review found no evidence that saturated fat increases the risk of coronary disease, or that polyunsaturated fats have a cardio protective effect.”[1] Dietary fats play a critical role in human health. They help keep cells healthy, help with brain development, help with the use of fat soluble vitamins, and they help cushion organs protecting them against blunt trauma. Fats come in multiple forms, saturated, unsaturated and trans fats just to name a few.
Saturated fats are solid at room temperature due to their molecular shape. The term saturated is in reference to an sp3 carbon chain that has its remaining sp3 orbitals bonded with hydrogen atoms. Thus the term “saturated”. It’s “saturated” with hydrogen. Saturated fats have a chain like structure which allows them to stack very well forming a solid at room temperature. Unsaturated fats are not linear due to double bonded carbons which results in a different molecular shape because the sp2 carbons are trigonal planar, not tetrahedral (sp3 carbons) as the carbons are in saturated fats. This change in structure will cause the fat molecules to not stack very well resulting in fats that are liquid at room temperature. Butter is mostly saturated fat, that’s why it’s solid at room temperature. Olive Oil is liquid at room temperature, thus it’s an unsaturated fat. An unsaturated fat can be made in to a saturated fat via hydrogenation reactions.
Hydrogenation Reaction
Unsaturated fatty acids may be converted to saturated fatty acids by the relatively simple hydrogenation reaction. Recall that the addition of hydrogen to an alkene (unsaturated) results in an alkane (saturated). A simple hydrogenation reaction is:
$\ce{H_2C=CH_2 + H_2 \rightarrow CH_3CH_3}$
alkene plus hydrogen yields an alkane
Vegetable oils are commonly referred to as "polyunsaturated". This simply means that there are several double bonds present. Vegetable oils may be converted from liquids to solids by the hydrogenation reaction. Margarines and shortenings are "hardened" in this way to make them solid or semi-solids.
Figure 1: Hydrogenation of a oleic fatty acid
Vegetable oils which have been partially hydrogenated, are now partially saturated so the melting point increases to the point where a solid is present at room temperature. The degree of hydrogenation of unsaturated oils controls the final consistency of the product. What has happened to the healthfulness of the product which has been converted from unsaturated to saturated fats?
Trans Fat
A major health concern during the hydrogenation process is the production of trans fats. Trans fats are the result of a side reaction with the catalyst of the hydrogenation process. This is the result of an unsaturated fat which is normally found as a cis isomer converts to a trans isomer of the unsaturated fat. Isomers are molecules that have the same molecular formula but are bonded together differently. Focusing on the sp2 double bonded carbons, a cis isomer has the hydrogens on the same side. Due to the added energy from the hydrogenation process, the activation energy is reached to convert the cis isomers of the unsaturated fat to a trans isomer of the unsaturated fat. The effect is putting one of the hydrogens on the opposite side of one of the carbons. This results in a trans configuration of the double bonded carbons. The human body does not recognize trans fats.
Although trans fatty acids are chemically "monounsaturated" or "polyunsaturated," they are considered so different from the cis monounsaturated or polyunsaturated fatty acids that they can not be legally designated as unsaturated for purposes of labeling. Most of the trans fatty acids (although chemically still unsaturated) produced by the partial hydrogenation process are now classified in the same category as saturated fats.
The major negative is that trans fat tends to raise "bad" LDL- cholesterol and lower "good" HDL-cholesterol, although not as much as saturated fat. Trans fat are found in margarine, baked goods such as doughnuts and Danish pastry, deep-fried foods like fried chicken and French-fried potatoes, snack chips, imitation cheese, and confectionery fats.
Contributors
• Charles Ophardt, Professor Emeritus, Elmhurst College; Virtual Chembook,
• Antonio Rodriguez | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Lipids/Fatty_Acids/Hydrogenation_of_Unsaturated_Fats_and_Trans_Fat.txt |
Fatty acids are merely carboxylic acids with long hydrocarbon chains. The hydrocarbon chain length may vary from 10-30 carbons (most usual is 12-18). The non-polar hydrocarbon alkane chain is an important counter balance to the polar acid functional group. In acids with only a few carbons, the acid functional group dominates and gives the whole molecule a polar character. However, in fatty acids, the non-polar hydrocarbon chain gives the molecule a non-polar character.
Introduction
The most common fatty acids are listed. Note that there are two groups of fatty acids--saturated and unsaturated. Recall that the term unsaturated refers to the presence of one or more double bonds between carbons as in alkenes. A saturated fatty acid has all bonding positions between carbons occupied by hydrogens. The melting points for the saturated fatty acids follow the boiling point principle observed previously. Melting point principle: as the molecular weight increases, the melting point increases. This observed in the series lauric (C12), palmitic (C16), stearic (C18). Room temperature is 25oC, Lauric acid which melts at 44o is still a solid, while arachidonic acid has long since melted at -50o, so it is a liquid at room temperature.
Table 1: Common Fatty Acids
Acid Name Structure Melting Point
SATURATED
Lauric CH3(CH2)10COOH +44
Palmitic CH3(CH2)14COOH +63
Stearic CH3(CH2)16COOH +70
UNSATURATED
Oleic CH3(CH2)7CH=CH(CH2)7COOH +16
Linoleic CH3(CH2)4(CH=CHCH2)2(CH2)6COOH -5
Linolenic CH3CH2(CH=CHCH2)3(CH2)6COOH -11
Arachidonic CH3(CH2)4(CH=CHCH2)4(CH2)2COOH -50
Melting Points of Saturated vs. Unsaturated Fatty Acids
Note that as a group, the unsaturated fatty acids have lower melting points than the saturated fatty acids. The reason for this phenomenon can be found by a careful consideration of molecular geometries. The tetrahedral bond angles on carbon results in a molecular geometry for saturated fatty acids that is relatively linear although with zigzags. See graphic on the left. This molecular structure allows many fatty acid molecules to be rather closely "stacked" together. As a result, close intermolecular interactions result in relatively high melting points.
On the other hand, the introduction of one or more double bonds in the hydrocarbon chain in unsaturated fatty acids results in one or more "bends" in the molecule. The geometry of the double bond is almost always a cis configuration in natural fatty acids. and these molecules do not "stack" very well. The intermolecular interactions are much weaker than saturated molecules. As a result, the melting points are much lower for unsaturated fatty acids.
Percent Fatty Acid Present in Triglycerides
Fat or Oil
Saturated
Unsaturated
Palmitic Stearic Oleic Linoleic Other
Animal Origin
Butter 29 9 27 4 31
Lard 30 18 41 6 5
Beef 32 25 38 3 2
Vegetable Origin
Corn oil 10 4 34 48 4
Soybean 7 3 25 56 9
Peanut 7 5 60 21 7
Olive 6 4 83 7 -
Contributors
• Charles Ophardt, Professor Emeritus, Elmhurst College; Virtual Chembook
Prostaglandins
Prostaglandins were first discovered and isolated from human semen in the 1930s by Ulf von Euler of Sweden. Thinking they had come from the prostate gland, he named them prostaglandins. It has since been determined that they exist and are synthesized in virtually every cell of the body. Prostaglandins, are like hormones in that they act as chemical messengers, but do not move to other sites, but work right within the cells where they are synthesized.
Introduction
Prostaglandins are unsaturated carboxylic acids, consisting of of a 20 carbon skeleton that also contains a five member ring. They are biochemically synthesized from the fatty acid, arachidonic acid. See the graphic on the left. The unique shape of the arachidonic acid caused by a series of cis double bonds helps to put it into position to make the five member ring. See the prostaglandin in the next panel
Prostaglandin Structure
Prostaglandins are unsaturated carboxylic acids, consisting of of a 20 carbon skeleton that also contains a five member ring and are based upon the fatty acid, arachidonic acid. There are a variety of structures one, two, or three double bonds. On the five member ring there may also be double bonds, a ketone, or alcohol groups. A typical structure is on the left graphic.
Functions of Prostaglandins
There are a variety of physiological effects including:
1. Activation of the inflammatory response, production of pain, and fever. When tissues are damaged, white blood cells flood to the site to try to minimize tissue destruction. Prostaglandins are produced as a result.
2. Blood clots form when a blood vessel is damaged. A type of prostaglandin called thromboxane stimulates constriction and clotting of platelets. Conversely, PGI2, is produced to have the opposite effect on the walls of blood vessels where clots should not be forming.
3. Certain prostaglandins are involved with the induction of labor and other reproductive processes. PGE2 causes uterine contractions and has been used to induce labor.
4. Prostaglandins are involved in several other organs such as the gastrointestinal tract (inhibit acid synthesis and increase secretion of protective mucus), increase blood flow in kidneys, and leukotriens promote constriction of bronchi associated with asthma.
Effects of Aspirin and other Pain Killers
When you see that prostaglandins induce inflammation, pain, and fever, what comes to mind but aspirin. Aspirin blocks an enzyme called cyclooxygenase, COX-1 and COX-2, which is involved with the ring closure and addition of oxygen to arachidonic acid converting to prostaglandins. The acetyl group on aspirin is hydrolzed and then bonded to the alcohol group of serine as an ester. This has the effect of blocking the channel in the enzyme and arachidonic can not enter the active site of the enzyme. By inhibiting or blocking this enzyme, the synthesis of prostaglandins is blocked, which in turn relives some of the effects of pain and fever. Aspirin is also thought to inhibit the prostaglandin synthesis involved with unwanted blood clotting in coronary heart disease. At the same time an injury while taking aspirin may cause more extensive bleeding.
Contributors
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Lipids/Fatty_Acids/Introduction_to_Fatty_Acids.txt |
Glycerides and waxes are lipids with have an ester as the major functional group and include: waxes, triglycerides, and phospholipids.
• Phosphoglycerides or Phospholipids
Phospholipids are similar to the triglycerides with a couple of exceptions. Phospholglycerides are esters of only two fatty acids, phosphoric acid and a trifunctional alcohol - glycerol (IUPAC name is 1,2,3-propantriol). The fatty acids are attached to the glycerol at the 1 and 2 positions on glycerol through ester bonds. There may be a variety of fatty acids, both saturated and unsatured, in the phospholipids.
• Triglycerides
Triglycerides are esters of fatty acids and a trifunctional alcohol - glycerol. The properties of fats and oils follow the same general principles as already described for the fatty acids. The important properties to be considered are: melting points and degree of unsaturation from component fatty acids.
Thumbnail: An example of a phosphatidylcholine, a type of phospholipid in lecithin. Red - choline and phosphate group; Black - glycerol; Green - unsaturated fatty acid; Blue - saturated fatty acid. (Public Domain; ).
Glycerides
Phospholipids are similar to the triglycerides with a couple of exceptions. Phospholglycerides are esters of only two fatty acids, phosphoric acid and a trifunctional alcohol - glycerol (IUPAC name is 1,2,3-propantriol). The fatty acids are attached to the glycerol at the 1 and 2 positions on glycerol through ester bonds. There may be a variety of fatty acids, both saturated and unsatured, in the phospholipids.
Introduction
The third oxygen on glycerol is bonded to phosphoric acid through a phosphate ester bond (oxygen-phosphorus double bond oxygen). In addition, there is usually a complex amino alcohol also attached to the phosphate through a second phosphate ester bond. The complex amino alcohols include choline, ethanolamine, and the amino acid-serine. The properties of a phospholipid are characterized by the properties of the fatty acid chain and the phosphate/amino alcohol. The long hydrocarbon chains of the fatty acids are of course non-polar. The phosphate group has a negatively charged oxygen and a positively charged nitrogen to make this group ionic. In addition there are other oxygen of the ester groups, which make on whole end of the molecule strongly ionic and polar.
Phospholipids are major components in the lipid bilayers of cell membranes. There are two common phospholipids:
• Lecithin contains the amino alcohol, choline.
• Cephalins contain the amino alcohols serine or ethanolamine
Lecithin
Lecithin is probably the most common phospholipid. It is found in egg yolks, wheat germ, and soybeans. Lecithin is extracted from soy beans for use as an emulsifying agent in foods. Lecithin is an emulsifier because it has both polar and non-polar properties, which enable it to cause the mixing of other fats and oils with water components. See more discussion on this property in soaps. Lecithin is also a major component in the lipid bilayers of cell membranes.
Lecithin contains the ammonium salt of choline joined to the phosphate by an ester linkage. The nitrogen has a positive charge, just as in the ammonium ion. In choline, the nitrogen has the positive charge and has four methyl groups attached.
Cephalins
Cephalins are phosphoglycerides that contain ehtanolamine or the amino acid serine attached to the phosphate group through phosphate ester bonds. A variety of fatty acids make up the rest of the molecule. Cephalins are found in most cell membranes, particularly in brain tissues. They also iimportant in the blood clotting process as they are found in blood platelets.
Note: The MEP coloration of the electrostatic potential does not show a strong red color for the phosphate-amino alcohol portion of the molecule as it should to show the strong polar property of that group. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Lipids/Glycerides/Phosphoglycerides_or_Phospholipids.txt |
Triglycerides are esters of fatty acids and a trifunctional alcohol - glycerol (IUPAC name is 1,2,3-propantriol). The properties of fats and oils follow the same general principles as already described for the fatty acids. The important properties to be considered are: melting points and degree of unsaturation from component fatty acids.
Introduction
Since glycerol has three alcohol functional groups, three fatty acids must react to make three ester functional groups. The three fatty acids may or may not be identical. In fact, three different fatty acids may be present. The synthesis of a triglyceride is another application of the ester synthesis reaction. To write the structure of the triglyceride you must know the structure of glycerol and be given or look up the structure of the fatty acid in the table.
Table \(1\):The common fats and oils including fatty acid content are listed below.
Fat or Oil Saturated Unsaturated
Palmitic Stearic Oleic Linoleic Other
Animal Origin
Butter 29 9 27 4 31
Lard 30 18 41 6 5
Beef 32 25 38 3 2
Vegetable Origin
Corn oil 10 4 34 48 4
Soybean 7 3 25 56 9
Peanut 7 5 60 21 7
Olive 6 4 83 7 -
Synthesis of a Triglyceride
Since glycerol, (IUPAC name is 1,2,3-propantriol), has three alcohol functional groups, three fatty acids must react to make three ester functional groups. The three fatty acids may or may not be identical. In fact, three different fatty acids may be present. nThe synthesis of a triglyceride is another application of the ester synthesis reaction. To write the structure of the triglyceride you must know the structure of glycerol and be given or look up the structure of the fatty acid in Table \(1\) - find lauric acid.
Glycerol
The simplified reaction reveals the process of breaking some bonds and forming the ester and the by product, water. Refer to the graphic on the left for the synthesis of trilauroylglycerol. First, the -OH (red) bond on the acid is broken and the -H (red) bond on the alcohol is also broken. Both join to make HOH, a water molecule. Secondly, the oxygen of the alcohol forms a bond (green) to the acid at the carbon with the double bond oxygen. This forms the ester functional group. This process is carried out three times to make three ester groups and three water molecules.
Structure of a Triglyceride
As you can see from the graphic on the left, the actual molecular model of the triglyceride does not look at all like the line drawing. The reason for this difference lies in the concepts of molecular geometry. Trilauroylglycerol. All of the above factors contribute to the apparent "T" shape of the molecule.
Problems
Quiz: Which acid (short chain or fatty) would most likely be soluble in water?
... in hexane?
1 Practice writing out a triglyceride of stearic acid. Again look up the formula of stearic acid and use the structure of glycerol.
2. Write down your answers. Then check the answers from the drop down menu.
What is the molecular geometry of all three carbons in glycerol (look at model above)?
What is the molecular geometry of the carbon at the center of the ester group?
What is the molecular geometry of the single bond oxygen?
Outside Links
• Snell, Foster D. "Soap and glycerol." J. Chem. Educ. 1942, 19, 172. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Lipids/Glycerides/Triglycerides.txt |
• Sphingolipids
Sphingolipids are a second type of lipid found in cell membranes, particularly nerve cells and brain tissues. They do not contain glycerol, but retain the two alcohols with the middle position occupied by an amine.
• Wax
A wax is a simple lipid which is an ester of a long-chain alcohol and a fatty acid. The alcohol may contain from 12-32 carbon atoms. Waxes are found in nature as coatings on leaves and stems. The wax prevents the plant from losing excessive amounts of water.
Thumbnail: Sphingolipids consist of a long-chain base (green) amide linked to a fatty acid (black). Various modifications can be made to the basic structure (red) including desaturations (4, 8 and n-9), hydroxylations (2 & 4) and headgroups (R). (CC SA BY 4.0; Jonathan E. Markham at the Department of Biochemistry, University of Nebraska-Lincoln).
Non-glyceride Lipids
Sphingolipids are named after the sphinx in Greek mythology, part woman and part lion, who devoured all who could not answer her riddles. Spingolipids appeared to Johann Thudichum in 1874 as part of the dangerous riddle of the brain. Sphingolipids are a second type of lipid found in cell membranes, particularly nerve cells and brain tissues. They do not contain glycerol, but retain the two alcohols with the middle position occupied by an amine.
Introduction
Sphingosine has three parts, a three carbon chain with two alcohols and amine attached and a long hydrocarbon chain. In sphingomyelin, the base sphingosine has several other groups attached as shown in the graphic on the left. A fatty acid is attached to the amine through amide bond. Phosphate is attached through a phosphate ester bond, and again through a phosphate ester bond to choline. The human brain and spinal cord is made up of gray and white regions. The white region is made of nerve axons wrapped in a white lipid coating, the myelin sheath, which provides insulation to allow rapid conduction of electrical signals. Multiple sclerosis caused by a gradual degradation of the myelin sheath.
Sphingomyleins are located throughout the body in nerve cell membranes. They make up about 25 % of the lipids in the myelin sheath that surrounds and insulates cells of the central nervous system. Niemann-Pick disease is caused by a deficiency of an enzyme that breaks down excessive sphingomyelin, which then builds up on the liver, spleen, brain, and bone marrow. An effected child usually dies within several years.
Glycolipids and Cerebrosides
Glycolipids are complex lipids that contain carbohydrates. Cerebrosides are an example which contain the sphingosine backbone attached to a fatty acid and a carbohydrate. The carbohydrates are most often glucose or galactose. Those that contain several carbohydrates are called gangliosides. The example on the left is shown with glucose. Glucocerebroside has the specific function to be in the cell membranes of macrophages, (cells that protect the body by destroying foreign microorganisms. Galactocerebroside is found almost exclusively in the membranes of brain cells.
There are several genetic diseases resulting from the absence of specific enzymes which breakdown the glycolipids. Tay-Sachs, which mainly effects Jewish children, results in a build up of gangliosides and result in death in several years. Gaucher's disease results in the excessive build up of glucocerebroside resulting in severe anemia and enlarged liver and spleen.
Contributors
• Charles Ophardt, Professor Emeritus, Elmhurst College; Virtual Chembook | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Lipids/Non-glyceride_Lipids/Sphingolipids.txt |
A wax is a simple lipid which is an ester of a long-chain alcohol and a fatty acid. The alcohol may contain from 12-32 carbon atoms. Waxes are found in nature as coatings on leaves and stems. The wax prevents the plant from losing excessive amounts of water. Carnuba wax is found on the leaves of Brazilian palm trees and is used in floor and automobile waxes. Lanolin coats lambs, wool. Beeswax is secreted by bees to make cells for honey and eggs. Spermaceti wax is found in the head cavities and blubber of the sperm whale. Many of the waxes mentioned are used in ointments, hand creams, and cosmetics (read the ingredients lists).
Introduction
Paraffin wax, used in some candles, is not based upon the ester functional group, but is a mixture of high molecular weight alkanes. Ear wax is a mixture of phospholipids and esters of cholesterol. The waxes with their component alcohols and fatty acids are listed below.
Waxes
Wax Alcohol Fatty Acid
Carnuba CH3(CH2)28CH2-OH CH3(CH2)24COOH
Beeswax CH3(CH2)28CH2-OH CH3(CH2)14COOH
Spermacetic CH3(CH2)14CH2-OH CH3(CH2)14COOH
Ester Synthesis
Simple esters are made from an organic acid and an alcohol. The ester functional group is of primary importance in the biochemical group of compounds called waxes, triglycerides, and phospholipids. The simplified reaction reveals the process of breaking some bonds and forming the ester and the by product, water. Refer to the graphic on the left for the synthesis of carnuba wax.
First, the -OH (red) bond on the acid is broken and the -H (red) bond on the alcohol is also broken. Both join to make HOH, a water molecule. Secondly, the oxygen of the alcohol forms a bond (green) to the acid at the carbon with the double bond oxygen. This forms the ester functional group. The long carbon chains do not participate in the reaction, but are just part of the final molecule.
Lipstick
Lipstick consists of a suspension of coloring agents in high molecular weight hydrocarbons, waxes, and/or fats. The color usually comes from a dye precipitated by a metal ion such as Fe (III), Ni(II), or Co(II) ions. An ingredients list may be: dye (4-8%); castor oil, paraffin, or fats to dissolve dye (50%); lanolin (25%); carnauba and/or beeswax as a stiffening agent (36%); perfume (1.5%). The lipstick is made by first dispersing the dye in the castor oil. Then the other waxes and lanolin are added as the mixture is heated and stirred. The molten waxes are then cast in suitable forms to harden.
Eye Makeup
Eyebrow pencils are very much like lipstick but contain lamp black (carbon soot) as a black coloring agent. A different mixture of waxes may be used to give the desired melting point. Brown pencils are made by adding iron oxide (rust) as a pigment. A water-resistant mascara has a mixture of waxes, fats, oils, and soap. Other coloring agents in addition to blacks and browns may be chromic oxide (dark green) and ultramarine (blue pigment of sodium and aluminum silicate).
Questions
1. For practice write the structures of beeswax and spermacetic wax using the information in the table.
2. Based upon the structure are waxes likely to be soluble in water?
Contributors
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Lipids/Non-glyceride_Lipids/Wax.txt |
One major class of lipids is the steroids, which have structures totally different from the other classes of lipids. The main feature of steroids is the ring system of three cyclohexanes and one cyclopentane in a fused ring system as shown below. There are a variety of functional groups that may be attached. The main feature, as in all lipids, is the large number of carbon-hydrogens which make steroids non-polar.
Introduction
Steroids include such well known compounds as cholesterol, sex hormones, birth control pills, cortisone, and anabolic steroids.
Cholesterol
The best known and most abundant steroid in the body is cholesterol. Cholesterol is formed in brain tissue, nerve tissue, and the blood stream. It is the major compound found in gallstones and bile salts. Cholesterol also contributes to the formation of deposits on the inner walls of blood vessels. These deposits harden and obstruct the flow of blood. This condition, known as atherosclerosis, results in various heart diseases, strokes, and high blood pressure.
Much research is currently underway to determine if a correlation exists between cholesterol levels in the blood and diet. Not only does cholesterol come from the diet, but cholesterol is synthesized in the body from carbohydrates and proteins as well as fat. Therefore, the elimination of cholesterol rich foods from the diet does not necessarily lower blood cholesterol levels. Some studies have found that if certain unsaturated fats and oils are substituted for saturated fats, the blood cholesterol level decreases. The research is incomplete on this problem.
Structures of Sex Hormones
Sex hormones are also steroids. The primary male hormone, testosterone, is responsible for the development of secondary sex characteristics. Two female sex hormones, progesterone and estrogen or estradiol control the ovulation cycle. Notice that the male and female hormones have only slight differences in structures, but yet have very different physiological effects.
Testosterone promotes the normal development of male genital organs ans is synthesized from cholesterol in the testes. It also promotes secondary male sexual characteristics such as deep voice, facial and body hair. Estrogen, along with progesterone regulates changes occurring in the uterus and ovaries known as the menstrual cycle. For more details see Birth Control. Estrogen is synthesized from testosterone by making the first ring aromatic which results in mole double bonds, the loss of a methyl group and formation of an alcohol group.
Adrenocorticoid Hormones
The adrenocorticoid hormones are products of the adrenal glands ("adrenal" means adjacent to the renal (kidney). The most important mineralocrticoid is aldosterone, which regulates the reabsorption of sodium and chloride ions in the kidney tubules and increases the loss of potassium ions. Aldosterone is secreted when blood sodium ion levels are too low to cause the kidney to retain sodium ions. If sodium levels are elevated, aldosterone is not secreted, so that some sodium will be lost in the urine. Aldosterone also controls swelling in the tissues.
Cortisol, the most important glucocortinoid, has the function of increasing glucose and glycogen concentrations in the body. These reactions are completed in the liver by taking fatty acids from lipid storage cells and amino acids from body proteins to make glucose and glycogen.
In addition, cortisol and its ketone derivative, cortisone, have the ability to inflammatory effects. Cortisone or similar synthetic derivatives such as prednisolone are used to treat inflammatory diseases, rheumatoid arthritis, and bronchial asthma. There are many side effects with the use of cortisone drugs, so there use must be monitored carefully.
Contributors
Thumbnail: Ball-and-stick model of the cholesterol molecule, a compound essential for animal life that forms the membranes of animal cells. (Public Domain; Jynto). | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Lipids/Steroids.txt |
Medicinal Chemistry is the science that deals with the discovery or design of new therapeutic chemicals and their development into useful medicines. It may involve synthesis of new compounds, investigations of their relationships between the structure of natural or synthetic compounds and their biological activities, elucidations of their interactions with receptors of various kinds, including enzymes and DNA, the determination of their absorption, transport, and distribution properties, and studies of the metabolic transformations of these chemicals into other chemicals.
Medicinal chemistry, in its crudest sense, has been practiced for several thousand years. Man has searched for cures of illnesses by chewing herbs, berries roots, and barks. Some of these early clinical trials were quite successful, however, not until the last 100 year has knowledge of the active constituents of these natural sources been known. The earliest written records of the Chinese, Indian, South American, and Mediterranean cultures described the therapeutic effects of various plant concoctions. If the approach to drug discovery continued as in ancient times, few diseases would be treatable today. Natural products make up a small percentage of drugs on the current market. Typically, when a natural product is found to be active, it is chemically modified in order to improve its properties. As a result of advances made in synthesis and separation methods and biochemical techniques since the late 1940s, a more rational approach to drug discovery has been possible, namely, one which involves the element of design.
• Adrenergic Drugs
The compounds ordinarily classified as central stimulants are drugs that increase behavioral activity, thought processes, and alertness or elevate the mood of an individual. These drugs differ widely in their molecular structures and in their mechanism of action. Thus, describing a drug as a stimulant does not adequately describe its medicinal chemistry. The convulsions induced by a stimulant such as strychnine, for example, are very different from agitation induced by amphetamine.
• Analgesics and Anti-Inflammatory Agents
The anti-inflammatory, analgesic, and antipyretic drugs are a heterogeneous group of compounds, often chemically unrelated (although most of them are organic acids), which nevertheless share certain therapeutic actions and side effects. The prototype is aspirin; hence these compounds are often referred to as aspirin-like drugs. All aspirin-like drugs are antipyretic, analgesic, and anti-inflammatory, but there are important differences in their activities.
• Anticancer Drugs
When fighting cancer, the entire population of neoplastic cells must be eradicated in order to obtain desired results. The logical outgrowth of these concepts has been the attempt to achieve total cell-kill by the use of several chemotherapeutic agents concurrently or in rational sequences. The resulting prolonged survival of patients with acute lymphocytic leukemia through the use of such multiple-drug regimens has encouraged the application of these principles the treatment of other neoplasms.
• Antihistamines and Local Anesthetics
Histamine is 2-(4-imidazolyl)ethylamine and is a hydrophilic molecule comprised of an imadazole ring and an amino group connected by two methylene groups. It arises in vivo by decarboxylation of the amino acid histadine. Histamine is a neurotransmitter in the CNS and a typical problem with some antihistamines is drowsiness. The effort has been to produce compounds that do not enter the brain very well.
• Antimicrobial Drugs
An antibiotic is any substance produced by a microorganism that is excreted to harm or kill another microorganism. Technically, antibiotics are microbial or fungal products. But these substances can be synthesized and mass produced in the laboratory to use against harmful microorganisms in the environment. Antibiotics can further be grouped under the broader heading of chemotherapuetic agents, chemical agents used to treat disease. Good chemotherapuetic agents are able to kill or inhibit the tar
• Basic Aspects of Drug Activity
While there are several types of exeptions, the effects of most drugs result from their interaction with functional macromolecular components of the organism. Such interaction alters the function of the pertinent cellular component and thereby initiates the series of biochemical and physiological changes that are characteristic of the response to the drug. The term receptor is used to denote the component of the organism with which the chemical agent interacts.
• Cardiovascular Drugs
Cardiovascular disease constitutes the largest single cause of death in the industrialized countries. As with cancer, which is a distant second in terms of mortality, cardiovascular disease morbidity increases with age, accounting for about two-thirds of all deaths in persons over 75. Even though some diseases affect primarily the heart and other diseases effect the vascular system, they cannot be divorced from each other. This obvious interdependence makes a unified imperative.
• Cholinergic Drugs I - Nicotinic and Muscarinic Receptors
A cholinergic drug is any of various drugs that inhibit, enhance, or mimic the action of the neurotransmitter acetylcholine within the body. Acetylcholine stimulation of the parasympathetic nervous system helps contract smooth muscles, dilate blood vessels, increase secretions, and slow the heart rate. Some cholinergic drugs, such as muscarine, pilocarpine, and arecoline, mimic the activity of acetylcholine in stimulating the parasympathetic nervous system. These drugs have few therapeutic uses.
• Colinergic Drugs II - Anticholinesterase Agents & Acetylcholine Antagonists
Acetylcholine is inactivated by the enzyme acetylcholinesterase (enlarged), which is located at cholinergic synapses and breaks down the acetylcholine molecule into choline and acetate. Three particularly well-known drugs, neostigmine, physostigmine, and diisopropyl fluorophosphate, inactivate acetylcholinesterase so that it cannot hydrolyze the acetylcholine released at the nerve ending. As a result, acetylcholine increases in quantity with successive nerve impulses.
• GABA
In most instances of natural neuron inhibition GABA is the inhibitory transmitter. GABA has the specific effect of opening anion channels in nerves, allowing large numbers of chloride ions to diffuse into the terminal fibril.
• Psychoactive Drugs
A sedative drug decreases activity, moderates excitement, and calms the recipient. A hypnotic drug produces drowsiness and facilitates the onset and maintenance of a state of sleep that resembles natural sleep in its electrocephalographic characteristics and from which the recipient may be easily aroused; the effect is sometimes called hypnosis.
Medicinal Chemistry
The compounds ordinarily classified as central stimulants are drugs that increase behavioral activity, thought processes, and alertness or elevate the mood of an individual. These drugs differ widely in their molecular structures and in their mechanism of action. Thus, describing a drug as a stimulant does not adequately describe its medicinal chemistry. The convulsions induced by a stimulant such as strychnine, for example, are very different from the behavioral stimulation and psychomotor agitation induced by a stimulant such as amphetamine.
A. Adrenergic Concepts.
The three main catacholamines (chatecol is ortho-dihydroxybenzene) are epinephrine EP, norepinephrine NE, and dopamine DA. A host of physiological and metabolic responses follows stimulation of sympathetic nerves in mammals is usually mediated by the neurotransmitter norepinephrine. As part of the response to stress, the adrenal medulla is also stimulated, resulting in elevation of the concentrations of EP and NE in the circulation. The actions of these two catecholamine are very similar at some sites but differ in significantly at others. For example, both compounds stimulate the myocardium; however, EP dilates blood vessels to skeletal muscle, whereas NE has a minimal constricting effect on them. DA is found predominantly in the basal ganglia of the CNS and is found in very low levels in peripheral tissues.
Synthesis of Catecholamines
The synthesis of the neurotransmitters DA and NE and EP and the hormones NE and EP takes place by a pathway that involves 5 enzymes (see figure below). Tyrosine is generally considered the starting point, although phenylalanine hydroxylase can hydroxylate phenylalanine to tyrosine in the event that there is a tyrosine deficiency. Tyrosine hydroxylase (structure) is the rate-limiting enzyme in this pathway. Its addition of the 3-OH yielding L-3, 4-dihydroxyphenylalanine (L-DOPA) requires O2, tetrahydropteridine, and Fe2+ as cofactors. One of the oxygen atoms in O2 is incorporated into an organic substrate and the other is reduced to water. Because this is the rate-limiting step, inhibition of this enzyme is the most likely way to reduce NE, DA, or EP levels significantly. Particularly are the a-methyltyrosine analogs, especially those containing an iodine atom in the benzene ring. The drug a -methyltyrosine is useful in the management of malignant hypertension and in pheochromocytoma. The latter is a chromaffin cell tumor that produces and spills copious amounts of NE and EP into the circulation.
DOPA is then converted to dopamine by the enzyme DOPA decarboxylase. The cofactor for this enzyme is pyridoxal (the aldahyde form of pyridoxine, vitamin B6). The copper-containing enzyme dopamine-beta-monooxygenase then converts dopamine to NE and in the end norepinephrine N-methyltransferase converts NE to EP.
Genetic defaults in, or complete absence of, the first of these 5 enzymes (Phenylalanine Hydroxylase) leads to a disease called phenylketoneuria PKU, which will lead to severe mental disorder if not treated at an early stage after birth.
Adrenergic Receptors
Research experiments using different drugs that mimic the action of norepinephrine on sympathetic effector organs have shown that there are two major types of adrenergic receptors, alpha receptors and beta receptors. The beta receptors in turn are divided into beta1 and beta 2 receptors because certain drugs affect only some beta receptors. Also, there is a less distinct division of alpha receptors into alpha1 and alpha 2 receptors.
Just as in the muscarinic receptor, and most other G protein-coupled receptors that bind biogenic amines, the adrenergic receptors possess an aspartate residue in the third transmembrane domain. The aspartate residue appears to interact with the amine residue of norepinephrine and other adrenergic ligands. Conserved serine residues in TM5 may play a role in the binding of adrenergic ligands through hydrogen bond interactions. In addition, aromatic amino acid residues, such as a phenylalanine in TM6, may contribute to the binding of ligands through pi - pi interactions.
Norepinephrine and epinephrine, both of which are secreted into the blood by the adrenal medulla, have somewhat different effects in exciting the alpha and beta receptors. Norepinephrine excites mainly alpha receptors but excites the beta receptors to a less extent as well. On the other hand, epinephrine excites both types of receptors approximately equally. Therefore, the relative effects of norepinephrine and epinephrine on different effector organs are determined by the types of receptors in the organs. If they are all beta receptors, epinephrine will be the more effective excitant. It should be emphasised that not all tissues have both of these receptors. Usually they are associated with only one type of receptor or the other.
The relative potencies of the various receptors are:
a -adrenergic receptors: EP > NE
b -adrenergic receptors: EP = NE
b 1-adrenergic receptors (heart): EP = NE
b 2-adrenergic receptors (most other tissues): EP >> NE
EP dilates blood vessels (relaxes smooth muscle) in skeletal muscle and liver vascular beds; NE constricts the same vascular beds. EP decreases resistance in the hepatic and skeletal vascular smooth muscle beds; NE increases resistance. In contrast to their opposite effects on vascular smooth muscle of the liver and skeletal muscle, both EP and NE cause vasoconstriction (contraction of smooth muscle) in blood vessels supplying the skin and mucosa. EP decreases diastolic blood pressure; NE increases diastolic blood pressure. EP relaxes bronchial smooth muscle; NE has little effect. Both EP and NE stimulate an increased rate of beating when applied directly to a heart muscle removed from the body and isolated from nervous input. In contrast, NE given intravenously causes a profound reflex bradycardia due to a baroreceptor/vagal response (and increased release of acetylcholine onto the heart) in response to the vasopressor effect of NE.
a -receptor b -receptor
vasocontriction vasodilation (b 2)
iris dilation cardioacceleration (b 1)
intestinal relaxation intestinal relaxation (b 2)
intestinal sphincter contraction uterus relaxation(b 2)
bladder sphincter contraction bronchiodilation (b 2)
The binding of the adrenergic receptor causes a series of reactions that eventually results in a characteristic response.
Two of the proteins that are phosphorylated in this process breakdown glycogen and stop glycogen synthesis.
Catabolism of Catecolamines
There are three main ways in which catacolamines are removed from a receptor - recycling back into the presynaptic neuron by an active transport reuptake mechanism, degredation to inactive compounds through the sequential actions of catecholamine-O-methyltransferase (COMT) and monoamine oxidase (MAO), and simple diffusion (see figure below).
Schematic representation of an adrenergic junction. Copyright © 1996-1997 Merck & Co., Inc., Whitehouse Station, NJ, USA. All rights reserved.
Monoamine Oxidase
MAO catalyzes the oxidative deamination of catecholamines, serotonin, and other monoamines. It is one of several oxydase-type enzymes who's coenzyme is the flavin-adenine-dinucleatide (FAD) covalently bound as a prosthetic group. The isoallozazine ring system is viewed as the catalytically functional component of the enzyme. In this view N-5 and C-4a is where the redox reaction takes place. Although the whole region undoubtedly participates.
Norepinephrine (NE) is the neurotransmitter of most postganglionic sympathetic fibers and many central neurons (eg, locus ceruleus, hypothalamus). Upon release, NE interacts with adrenergic receptors. This action is terminated largely by the re-uptake of NE back into the prejunctional neurons. Tyrosine hydroxylase and MAO regulate intraneuronal NE levels. Metabolism of NE occurs via MAO and catechol-O-methyltransferase to inactive metabolites (eg, normetanephrine, 3-methoxy-4-hydroxyphenylethylene glycol, 3-methoxy-4-hydroxymandelic acid).
B. Stimulants
Epinephrine
Epinephrine is a potent stimulator of both a and b -adrenergic receptors, and its effects on target organs are thus complex. Most of the effects which occur after injection are listed in the table on a and b -receptors shown above. Particularly prominent are the actions on the heart and the vascular and other smooth muscle.
Epinephrine is one of the most potent vasopressor drugs known. Given intravenously it evokes a characteristic effect on blood pressure, which rises rapidly to a peak that is proportional to the dose. The increase is systolic pressure is greater than diastolic pressure, so that the pulse pressure increases. As the response wanes, the mean pressure falls below normal before returning to normal. The mechanism of the rise in blood pressure due to epinephrine is three fold; a direct myocardial stimulation that increases the strength of ventricular contraction; and increased heart rate; and most important, vasoconstriction in many vascular beds, especially the in the vessels of the skin, mucosa, and kidney, and constriction in the veins. Due to this increased blood pressure and to powerful b 2-receptor vasodilator action that is partially counterbalanced by vasoconstrictor action on the a receptors that are also present, blood flow to the skeletal muscles and central nervous system is increased.
The effects of epinephrine on the smooth muscles of different organs and systems depend upon the type of adrenergic receptor in the muscle. It has powerful bronchiodilatior action, most evident when bronchial muscle is contracted as in bronchial asthma. In such situations, epinephrine has a striking therapeutic effect as a physiological antagonist to the constrictor influences since it is not limited to specific competitive antagonism such as occurs with antihistaminic drugs against histamine-induced bronchiospasm.
Epinephrine has a wide variety of clinical uses in medicine and surgery. In general, these are based on the actions of the dug on blood vessels, heart, and bronchial muscle. The most common uses of epinephrine are to relieve respiratory distress due to bronchiospasm and to provide rapid relief of hypersensitivity reactions to drugs and other allergens. Its cardiac effects may be of use in restoring cardiac rhythm in patients with cardiac arrest. It is also used as a topical hemostatic on bleeding surfaces.
Norepinephrine
Norepinephrine is the chemical mediator liberated by mammalian postgangionic adrenergic nerves. It differs from epinephrine only by lacking the methyl substitution in the amino group. Norepinephrine constitutes 10 to 20% of the catecholamine content of human adrenal medulla. Norepinephrine is a potent agonist at a receptors and has little action on b 2 receptors; however, it is somewhat less potent than epinephrine on the a receptors of most organs. Most of the effects which occur after injection are listed in the table on a and b -receptors shown above Norepinephrine has only limited therapeutic value.
Amphetamine and Methamphetamine
Amphetamine, racemic b-phenylisopropylamine, has powerful CNS stimulant actions in addition to the peripheral a and b actions common to indirectly acting sympathomimetic drugs. Unlike epinephrine, it is effective after oral administration and its effects last for several hours. Although amphetamine and methamphetamine are almost structurally identical to norepinephrine and epinephrine, these drugs have an indirect sympathomimetic action rather than directly exciting adrenergic effector receptors. Their effect is to cause release of norepinephrine from its storage vesicles in the sympathetic nerve endings The release of norepinephrine in turn causes the sympathetic effects.
Ephedrine
Ephedrine occurs naturally in various plants. It was used in China for at least 2000 years before being introduced into Western medicine in 1924. Its central actions are less pronounced than those of the amphetamines. Ephedrine stimulates both a and b receptors and has clinical uses related to both these types of action. The drug owes part of its peripheral action to the release of norepinephrine, but it also has direct effects of receptors.
Since ephedrine contains two chiral carbon atoms, four compounds are possible. Clinically, D-ephedrine is used to a large extent as an anti-asthmatic and, formerly, as a presser amine to restore low blood pressure as a result of trauma. L-pseudo-ephedrine is used primarily as a nasal decongestant.
Ephedrine differs from epinephrine mainly in its efficacy after oral administration, its much longer duration of action, its more pronounced central actions, and its much lower potency. Cardiovascular effects of ephedrine are in many ways similar to those of epinephrine, but they persist about ten times as long. The drug elevates the systolic and diastolic pressure in man, and pulse pressure increases. Bronchial muscle relaxation is less prominent but more sustained with ephedrine than with epinephrine.
The main clinical uses of ephedrine are in bronchiospasm, as a nasal decongestant, and certain allergic disorders, The drug is also used, although perhaps unwisely, as a weight loss agent.
MAOIs
The monoamine oxidase inhibitors (MAOIs) comprise a chemically heterogeneous group of drugs that have in common the ability to block oxidative deamination of naturally occurring monoamines. These drugs have numerous other effects, many of which are still poorly understood. For example, they lower blood pressure and were at one time used to treat hypertension. Their use in psychiatry has also become very limited as the tricyclic antidepressants have come to dominate the treatment of depression and allied conditions. Thus, MAOIs are used most often when tricyclic antidepressants give unsatisfactory results. In addition, whereas severe depression may not be the primary indication for these agents, certain neurotic illnesses with depressive features, and also with anxiety and phobias, may respond especially favorably.
Two main problems are associated with the MAOIs. The first is that an amino acid called "tyramine" may cause a hypertensive reaction in some people taking MAOIs. Therefore, foods containing tyramine must be avoided. Alcohol and caffeine must also be eliminated from the diet. Certain medications may react dangerously when combined with MAOIs. Therefore, it is crucial to tell the prescribing doctor about medications (including over-the-counter) you are taking. The second problem associated with MAOIs is the possibility of side effects. MAOIs not only inhibit MAO but other enzymes as well, and they interfere with the hepatic metabolism of many drugs. The dietary restrictions and side effects deter many people from staying on MAOIs.
Phenelzine is the hydrazine analog of phenylethylamine, a substrate of MAO. This and several other MAOIs, such as isocarboxazide, are structurally related to amphetamine and were synthesized in an attempt to enhance central stimulant properties.
Cocaine
Cocaine blocks the reuptake of dopamine by presynaptic neurons. More about this can be found under the topic Illegal Drugs.
Dopamine
Dopamine is the immediate metabolic precursor of NE and EP; it is a central neurotransmitter and possesses important intrinsic pharmacological properties. DA is a substrate for both MAO and COMT and thus is ineffective when administered orally.
Parkinson's Disease. Parkinson's disease can be characterized as having a DA deficiency in the brain. The pathology can be traced to certain large neurons in the substantia nigra in the basal ganglia, whose degeneration is directly related to DA deficiency. One of the principle roles of the basal ganglia is to control complex patterns of motor activity. When there is damage to the basal ganglia one's writing becomes crude.
Logic would dictate that increasing brain levels of DA should ameliorate symptoms of Parkinson's disease. Direct parental DA administration is useless since the compound does not penetrate the Blood-brain barrier. It is shown that oral dosing with L-DOPA can successfully act as a pro-drug to the extent it enters the brain and is then decarboxylated to DA there. The clinical results in terms of decreased tremors and rigidity are dramatic. However, there are complications which produce intense side effects including nausea and vomiting, that are presumably due to chemoreceptor trigger zone stimulation by large amounts of DA produced peripherally. The reason for this situation is the relatively high peripheral levels of decarboxylase enzyme compared with brain concentrations. Thus 95% of a given oral dose was converted to DA before reaching the brain to be decarboxylated there. This can be prevented using L-DOPA in combination with a drug called carbidopa (more).
Amantadine, introduced as an antiviral agent for the influenza was unexpectedly found to cause symptomatic improvement of patients with parkinsonism. Amantadine is a basic amine like dopamine, but the lipophilic nature of the cage structure enhances its ability to cross the blood brain barrier. This drug acts by releasing dopamine from intact dopaminergic terminals that remain in the nigrostraeatum of patients with Parkinson's disease. Because of this facilitated release of dopamine it appears that the therapeutic efficacy of amantadine is enhanced by the concurrent administration of levodopa. Amantadine has also been shown to delay the re-uptake of dopamine by neural cells, and it may have anticholinergic effects as well. A three dimensional view of amantadine may provide a better understanding of the structure.
The above therapies are based on the manipulation of endogenous stores of dopamine. Dopamine agonists can stimulate the receptor directly and are of therapeutic value. Some of the drugs acting as dopaminergic agonists include the ergot alkaloid derivative bromocriptine. Bromocriptine is used particularly when L-DOPA therapy fails during the advanced stages of the disease. Bromocriptine is a derivative of lysergic acid (a precursor for LSD). Its structure is shown below. The addition of the bromine atom renders this alkaloid a potent dopamine agonist and virtually all of its actions result from stimulation of dopamine receptors.
Schizophrenia. Schizophrenia results from excessive excitement of a group of neurons that secrete dopamine in the behavioral centers of the brain, including in the frontal lobes. Therefore drugs used to treat this disorder decrease the level of dopamine excreted from these neurons or antagonize dopamine. We will discuss these drugs in detail later under the topic Psychoactive Drugs.
Strychnine
Strychnine does not directly affect adrenergic mechanisms, and technically should not be listed in this category. However, its stimulating affects are a result of adrenergic mechanisms. In addition, strychnine has no demonstrated therapeutic value, despite a long history of unwarranted popularity. However, the mechanism of action of strychnine is thoroughly understood, and it is a valuable pharmacological tool for studies of inhibition in the CNS. Poisoning with strychnine results in a predictable sequence of dramatic symptoms that may be lethal unless interrupted by established therapeutic measures.
Strychnine is the principle alkaloid present in nux vomica, the seeds of a tree native to India, Stychnos nux-vomica. The structural formula for strychnine is:
Strychnine produces excitation of all portions of the CNS. This effect, however, does not result from direct synaptic excitation. Strychnine increases the level of neuronal excitability by selectively blocking inhibition. Nerve impulses are normally confined to appropriate pathways by inhibitory influences. When inhibition is blocked by strychnine, ongoing neuronal activity is enhanced and sensory stimuli produce exaggerated reflex effects.
Strychnine is a powerful convulsant, and the convulsion has a characteristic motor pattern. Inasmuch as strychnine reduces inhibition, including the reciprocal inhibition existing between antagonistic muscles, the pattern of convulsion is determined by the most powerful muscles acting at a given joint. In most laboratory animals, this convulsion is characterized by tonic extension of the body and of all limbs.
The convulsant action of strychnine is due to interference with post synaptic inhibition that is mediated by glycine. Glycine is an important inhibitory transmitter to motorneurons and interneurons in the spinal cord, and strychnine acts as a selective, competitive antagonist to block the inhibitory effects of glycine at all glycine receptors. Competitive receptor-binding studies indicate that both strychnine and glycine interact with the same receptor complex, although possibly at different sites.
The first symptoms of strychnine poisoning that is noticed is stiffness of the face and neck muscles. Heightened reflex excitability soon becomes evident. Any sensory stimulus may produce a violent motor response. In the early stages this is a coordinated extensor thrust, and in the later stages it may be a full tetanic convulsion. All voluntary muscles, including those of the face, are soon in full contraction. Respiration ceases due to the contraction of the diaphragm and the thoracic and abdominal muscles. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Medicinal_Chemistry/Adrenergic_Drugs.txt |
A. Aspirin-like Drugs
The anti-inflammatory, analgesic, and antipyretic drugs are a heterogeneous group of compounds, often chemically unrelated (although most of them are organic acids), which nevertheless share certain therapeutic actions and side effects. The prototype is aspirin; hence these compounds are often referred to as aspirin-like drugs. All aspirin-like drugs are antipyretic, analgesic, and anti-inflammatory, but there are important differences in their activities. For example, acetaminophen is antipyretic and analgesic but is only weakly anti-inflammatory. The reason for the differences are not clear; variations in the sensitivity of enzymes in the target tissues may be important.
When employed as analgesics, these drugs are usually effective only against pain of low-to-moderate intensity, particularly that associated with inflammation. Aspirin drugs do not change the perception of sensory modalities other than pain. The type of pain is important; chronic postoperative pain or pain arising from inflammation is particularly well controlled by aspirin-like drugs, whereas pain arising from the hollow viscera is usually not relieved.
As antipyretics, aspirin-like drugs reduce the body temperature in feverish states. Although all such drugs are antipyretics and analgesics, some are not suitable for either routine or prolonged use because of toxicity; phenylbutaxone is an example. This class of drugs finds its chief clinical application as anti inflammatory agents in the treatment of musculoskelatal disorders, such as rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. In general, aspirin-like drugs provide only symptomatic relief from the pain and inflammation associated with the disease and do not arrest the progression of pathological injury.
Chemistry of Action
There has been substantial progress in elucidating the mechanism of action of aspirin-like drugs, and it is now possible to understand why such heterogeneous agents have the same basic therapeutic activities and often the same side effects. Indeed, their therapeutic activity appears to depend to a large extent upon the inhibition of a defined biochemical pathways responsible for the biosynthesis of prostaglandins (see figure below) and related autacoids.
Aspirin-like drugs inhibit the conversion of arachidonic acid to the unstable endoperoxide intermediate, PGG2, which is catalyzed by the cyclooxygenase. Individual agents have differing modes of inhibitory activity on the cyclooxygenase. Aspirin itself acetylates a serine at the active site of the enzyme. Platelets are especially susceptible to this action because (unlike most other cells) they are incapable of regenerating the enzyme, presumably because they have little or no capacity for protein biosynthesis. In practical terms this means that a single dose of aspirin will inhibit the platelet cyclooxygenase for the life of the platelet (8 to 10 days); in man a dose as small as 40 mg per day is sufficient to produce this effect. In contrast to aspirin, salicylic acid has no acetylating capacity and is almost inactive against cyclooxygenase in vitro. Nevertheless, it is as active as aspirin in reducing the synthesis of prostaglandins in vivo. The basis of this action and, thus, of the anti-inflammatory effect of salicylic acid is not clearly understood. Since aspirin is rapidly hydrolyzed to salicylic acid in vivo (half-life in human plasma, approximately 15 minutes), the acetylated and nonacetylated species probably act as pharmacologically distinct entities.
Most of the other common aspirin-like drugs are irreversible inhibitors of the cyclooxygenase, although there are some exceptions. For indomethacin, the mode of inhibition is particularly complex and probably involves a site on the enzyme different from that which is acetylated by aspirin.
Pain
Prostaglandins are associated particularly with the development of pain that accompanies injury or inflammation. Large doses of PGE2 or PGF2a , given to women by injection to induce abortion, cause intense local pain. Prostaglandins can also cause headache and vascular pain when infused intravenously in man. While the doses of prostaglandins required to elicit pain are high in comparison with the concentrations expected in vivo, induction of hyperalgesia occurs when minute amounts of PGE1 are given intradermally to man. Furthermore, in experiments in man where separate infusions of PGE1, bradykinin, or histamine caused no pain, marked pain was experienced when PGE1 was added to bradykinin or histamine. When PGE1 was infused with histamine, itching was also noted.
Fever
The hypothalamus regulates the set point at which body temperature is maintained. In fever, this set point is elevated, and aspirin-like drugs promote its return to normal. These drugs do not influence body temperature when it is elevated by such factors as exercise or increases in the surrounding temperature.
Fever may be a result of infection, tissue damage, inflammation, graft rejection, malignancy, or other disease states. A multitude of microorganisms can cause fever. There is evidence that bacterial endotoxins act by stimulating the biosynthesis and release by neutrophils and other cells of an endogenous pyrogen, a protein with a molecular weight of 10,000 to 20,000. The current view is that the endogenous pyrogen passes from the general circulation into the central nervous system, where it acts upon discrete sites within the brain, especially the preoptic hypothalamic area. There is evidence that the resultant elevation of body temperature is mediated by the release of prostaglandins and that aspirin-like drugs suppress the effects of endogenous pyrogen by inhibiting the synthesis of these substances. The evidence includes the ability of prostaglandins, especially PGE2, to produce fever when infused into the cerebral ventricles or when infected into the hypothalamus. Fever is a frequent side effect of prostaglandins when they are administered to a women as abortifacients. Moreover some studies have demonstrated an increase in prostaglandin-like substances in the cerebrospinal fluid when endogenous pyrogen is injected intravenously. The fever produced by the administration of pyrogen, but not that by prostaglandins, is reduced by aspirin-like drugs.
Side Effects
Aspirin is very useful, but it has many side effects and therefore must be used carefully. Like most powerful drugs, an overdose of aspirin or salicylates can be fatal. If a child or adult takes an overdose of aspirin, induce vomiting to empty the unabsorbed medication from the stomach (if the person is still awake and conscious). Obtain emergency medical care right away.
The most common side effects of aspirin are heartburn and other symptoms of stomach irritation such as indigestion, pain, nausea, and vomiting. The stomach irritation may lead to bleeding from the stomach, which may cause black stools. These symptoms may be reduced by taking aspirin with meals, with an antacid, with a glass of milk, or by taking enteric-coated or timed-release aspirin. Also, it is best not to take aspirin with alcohol or coffee (or other beverages containing caffeine, such as tea or cocoa and many soft drinks). Alcohol and caffeine make the stomach more sensitive to irritation. The non aspirin salicylate preparations sometimes are less irritating to the stomach and may be substituted for aspirin by your doctor.
A few people develop asthma, hay fever, nasal congestion, or hives from aspirin or non-steroid anti-inflammatory drugs (NSAIDs). These people should never take aspirin, nor should people who have active stomach or duodenal ulcers. Anyone who has ever had a peptic ulcer should be very careful about taking aspirin because it can lead to a recurrence.
Aspirin is known to interfere with the action of the platelets. As a result, some people who take a lot of aspirin experience easy bruising of the skin. Therefore, people who have major bleeding problems should not take aspirin. Also, keep in mind that aspirin should not be taken for 10-14 days before surgery (including surgery in the mouth) to avoid excessive bleeding during or after the operation. These side effects probably depend on aspirin-like drugs' ability to block endogenous prostaglandin biosynthesis. Platelet function appears to be disturbed because aspirin-like drugs prevent the formation by the platelets of thrombozane A2 (TXA2), a potent aggregating agent. This accounts for the tendency of these drugs to increase the bleeding time.
Aspirin increases oxygen consumption by the body, increasing carbon dioxide production-an effect that stimulates respiration. Therefore, overdose with aspirin is often characterized by marked increases in respiratory rate, which cause the overdosed individual to appear to pant. This occurrence results in other, severe, metabolic consequences.
Prolongation of gestation by aspirin-like drugs has been demonstrated in both experimental animals and the human female. Furthermore, prostaglandins of the E and F series are potent uterotropic agents , and their biosynthesis by the uterus increases dramatically in the hours before parturition. It is thus hypothesized that prostaglandins play a major role in the initiation and progression of labor and delivery.
High doses of salicylate may cause ringing in the ears and slight deafness. Sometimes, however, these symptoms indicate mild overdose, which could become more serious.
Aspirin and NSAIDs sometimes affect the normal function of the kidneys and aspirin-like drugs promote the retention of salt and water by reducing the prostaglandin-induced inhibition of both the reabsorption of chloride and the action of antidiuretic hormone. This may cause edema in some patients with arthritis who are treated with an aspirin-like drug.
Recent reports have said there could be a link between the use of aspirin and the development of Reye's syndrome. Reye's syndrome is a rare but possibly fatal disease seen most often in children and teenagers. It usually affects those recovering from chicken pox or a viral illness such as the flu. These reports have raised concern in pediatricians (doctors who specialize in treating children) and parents of children with arthritis who need to take large doses of aspirin to control their disease.
Aspirin
In the U.S., about 10 to 20 thousand tons of aspirin are consumed each year; it is our most popular analgesic. Aspirin is one of the most effective analgesic, antipyretic, and anti-inflammatory agents.
Chemical structure
Acetaminophen (Tylenol)
Acetaminophen is an effective alternative to aspirin as an analgesic and antipyretic agent. However, its anti-inflammatory effect is minor and not clinically useful. It is commonly felt that acetaminophen may have fewer side effects than aspirin, but it should be noted that an acute overdose may produce severe or even fatal liver damage. Acetaminophen does not inhibit platelet aggregation and therefore is not useful for preventing vascular clotting.
Side effects are usually fewer than those of aspirin; the drug produces less gastric distress and less ringing in the ears. However, as stated previously, overdose can lead to severe damage of the liver.
Acetaminophen has been proved to be a reasonable substitute for aspirin when analgesic or antipyretic effectiveness is desired, especially in patients who cannot tolerate aspirin. This might include patients with peptic ulcer disease of gastric distress or those in whom the anticoagulant action of aspirin might be undesirable.
Aspirin is often combined with acetaminophen in a single tablet for relief of arthritis and other painful conditions. Sometimes other drugs such as caffeine, an antihistamine, nasal drying agents, and sedatives are also added. Although some of these preparations may have special uses for certain acute conditions such as a cold or a headache, they should not be taken for a chronic (long-term) form of arthritis. If a combination is required, each drug should be prescribed separately. The dose of each should be adjusted individually to achieve the greatest benefit with the fewest side effects.
Researchers attribute the pain-relieving activity of acetaminophen to the drug's ability to elevate the pain threshold, although the precise mechanisms involved in this process have not been clearly identified. The antipyretic, or fever-reducing, effect of acetaminophen is far better understood. Research shows that the drug inhibits the action of fever-producing agents on the heat-regulating centers of the brain by blocking the formation and release of prostaglandins in the central nervous system. However, unlike aspirin and other NSAIDs, acetaminophen has no significant effect on the prostaglandins involved in other body processes.
Despite claims to the contrary, stomach upset and hepatic toxicity are statistically as much a problem with acetaminophen as with aspirin-like drugs. Acetaminophen is normally metabolized in the liver and kidney by P450 enzymes. No toxicity is observed with therapeutic doses, however, after ingestion of large quantities (>2,000 mg/kg), a highly reactive metabolite, N-acetyl-p-benzoquinoneimine, is generated (see figure below). This species is electrophilic intermediate which is conjugated with glutathion to a non-toxic compound. Overdosing depletes glutithione and N-acetyl-p-benzoquinone reacts with nucleophilic portions (sulfhdryl groups) of critical liver cell protein. This results in cellular dysfunction and hepatic and renal toxicity. Antidote treatment consists of amino acid supplements to replenish glutathione. The P450 metabolizing enzymes differ somewhat in character between the liver and kidney. Factors that enhance renal toxicity include chronic liver disease, possibly gender, concurrent renal insults, and conditions that alter the activity of P450-metabolizing enzyme systems.
Other Aspirin-Like Drugs
Other aspirin-like drugs include diflunisal, phenylbutazone, apazone, indomethacine, sulindac, fenamates, tolmetin, ibuprofen (see figure below), and piroxicam.
Gold
Gold is not of course an aspirin-like drug. However, its end effect is similar to aspirin, so it will be briefly considered here. Gold in elemental form has been employed for centuries as an antipruritic (anti itch medication) to relieve the itching palm. At present, gold treatment includes different forms of gold salts used to treat rheumatoid arthritis and related diseases. In some people, it helps relieve joint pain and stiffness, reduce swelling and bone damage, and reduce the chance of joint deformity and disability.
The significant preparations of gold are all compounds in which the gold is attached to sulfur. The three prominent drugs are aurothioglucose, auranofin, and gold sodium thiomalate.
It takes months for gold compounds to leave the body. This means that side effects to gold therapy may take some time to resolve. Sometimes side effects even appear after the last gold injection. Rash and a metallic taste in the mouth are side effects of gold injections that may not seem serious at first. However, they are early warning signs for more serious reactions. If either of these side effects develop, the health care provider should be contacted promptly.
Some side effects may cause multiple symptoms, not all of which may occur. Side effects with multiple symptoms are:
1. Low platelet count (thrombocytopenia). Most often this appears to be an immunological disturbance that results in an accelerated degradation of platelets. Symptoms may include: black, tarry stools, blood in urine, stool, or vomit, small red dots on the skin, nose bleeds, unusual bruising or bleeding.
2. Anaphylaxis. Symptoms include sudden onset of the following soon after a gold injection: trouble swallowing, tightness in the throat, fainting, trouble breathing, wheezing, hives, swelling of the face, usually the lips or around the eyes, liver damage, abdominal pain for more than a few days, light colored stools, albuminuria, yellow eyes or skin, ulcerative colitis, severe abdominal pain or cramps, diarrhea that lasts more than a few days, blood in stool.
B. Opiates, Opiate Antagonists, and Opiate Receptors
The term opiate refers to any natural or synthetic drug that exerts actions upon the body similar to those induced by morphine, the major pain-relieving agent obtained from the opium poppy (Papaver somniferum). They were so highly regarded in the nineteenth century as remedies for pain, anxiety, cough, and diarrhea that some physicians referred to them as G.O.M.- `God's Own Medicine'. Opiates interact with what appear to be several closely related receptors, and they share some of the properties of certain naturally occurring peptides, the enkephalins, endorphins, and dynorphins.
Opiates
The term opium refers to the crude resinous extract obtained from the opium poppy. Crude opium contains a wide variety of ingredients, including morphine and codeine, both of which are widely used in medicine. The bulk of the ingredients of opium, however, consists of such organic substances as resins, oils, sugars, and proteins that account for more than 75 % of the weight of the opium but exert little pharmacological activity. Morphine is the major pain relieving drug found in opium, being approximately 10% of the crude exudate. Codeine is structurally close to morphine (see figs below), although it is much less potent and amounts to only 0.5% of the opium extract. Heroin does not occur naturally but is a semisynthetic derivative produced by a chemical modification of morphine that increases the potency (see figs. below). It takes only 3 mg. of heroin to produce the same analgesic effect as 10 mg of morphine. However, at these equally effective doses, it may be difficult to distinguish between the effects of the two compounds.
filled structure of morphine
Mechanism of Action
Studies of the binding of opioid drugs and peptides to specific sites in brain and other organs have suggested the existence of perhaps as many as eight types of receptors. In the CNS, there is reasonably firm evidence for four major categories of receptors, designated m, k, d , and s . To add confusion, there may well be subtypes of each of these receptors. Although there is considerable variation in binding characteristics and anatomical distribution among different species, inferences have been drawn from data that attempt to relate pharmacological effects to interactions with a particular constellation of receptors. For example, analgesia has been associated with both m and k receptors, while dysphoria or psychotomimetic (alteration of behavior or personality) effects have been ascribed to s receptors; based primarily on their localization in limbic regions of the brain, d receptors are thought to be involved in alterations of affective behavior. The actions of opioid drugs that are currently available have usually been interpreted with respect to the participation of only three types of receptors - m, k, and s ; at each, a given agent may act as an agonist, a partial agonist, or an antagonist (see table ). The m receptor is thought to meduate supraspinal analgesia, respiratory depression, euphoria, and physical dependance; the k receptor, spinal analgesia, miosis, and sedation; the s receptor, dysphoria, hallucinations, and respiratory and vasomotor stimulation.
It has been observed that opioids can selectively inhibit certain excitatory inputs to identified neurons. For example, the iontophoretic administration (the induction of an ionized substance through intact skin by the application of a direct current) of morphine into the substantia gelatinosa suppresses the discharge of spinal neurons in lamina IV of the dorsal horn that is evoked by noxious stimuli (e.g. heat) without changing responses to other inputs. While a postsynaptic action at discrete dedritic sites cannot be excluded, these findings suggest that opioids selectively inhibit the release of excitatory transmitters from terminals of nerves carrying pain related stimuli. In other situations, postsynaptic actions of opioids appear to be important. For example, application of opioids to neurons in the locus ceruleus reduces both spontaneous discharge and responses evoked by noxious stimuli. However, excitation of the neurons by antidromic stimulation (i.e. causing the neurons to fire backwards) is also suppressed, and the cells are hyperpolarized by the drugs.
Opioids have been observed to inhibit prostaglandin-induced increases in the accumulation of cyclic AMP in in brain tissue. Of potential relevance to mechanisms that underlie the phenomena of tolerance and withdrawal, the responses to prostaglandins recover in the continued presence of opioids.
Opioid-induced analgesia is due to actions of several sites within the CNS and involves several systems of neurotransmitters. Although opioids do not alter the threshold or responsivity of afferent nerve endings to noxious stimulation or impair the conduction of the nerve impulses along peripheral nerves, they may decrease conduction of impulses of primary afferent fibers when they enter the spinal cord and decrease activity in other sensory endings. There are opioid binding sites (m receptors) on the terminal axons of primary afferents within laminae I and II (substantia gelatinosa) of the spinal cord and in the spinal nucleus of the trigeminal nerve. Morphine-like drugs acting at this site are thought to decrease the release of neurotransmitters, such as substance P, that mediate transmission of pain impulses.
High doses of opioids can produce muscular rigidity in man, and both opioids and endogenous peptides cause catalepsy, circling, and stereotypical behavior in rats and other animals. These effects are probably related to actions at opioid receptors in the substania nigra and striatum, and involve interactions with both dopaminergic and GABA-ergic neurons.
The mechanism by which opioids produce euphoria, tranquility, and other alterations of mood remains unsettled. Microinjections of opioids into the ventral tegmentum activate dopaminergic neurons that project to the nucleus accumbens. Animals will work to receive such injections, and activation (or disinhibition) of these neurons has been postulated to be a critical element in the reinforcing effects of opioids and opioid-induced euphoria. However, the administration of dopaminergic antagonists does not consistently prevent these reinforcing effects. The neural systems that mediate opioid reinforcement in the ventral tegmentum appear to be distinct from those involved in the classical manifestations of physical dependence and analgesia.
Basic Effects of Morphine
CNS. Morphine exerts a narcotic action manifested by analgesia, drowsiness, changes in mood, and mental clouding. The major medical action of morphine sought in the CNS is analgesia, which may usually be induced by doses below those that cause other effects on the CNS, such as sedation or respiratory depression. The relief of pain by morphine-like opioids is relatively selective, in that other sensory modalities (touch, vibration, vision, hearing, etc.) are not inhibited. Patients frequently report that the pain is still present but that they feel more comfortable. Continuous dull pain is relieved more effectively than sharp intermittent pain, but with sufficient amounts of morphine it is possible to relieve even the severe pain associated with renal or biliary colic. In fact, its analgesic action appears to result not from a decrease of pain impulses into the CNS but from an altered perception of the painful stimuli.
Respiration. A second major action of morphine-like drugs is to depress respiration through interaction with m receptors located in the brainstem. At high doses, respiration may become so slow and irregular that life is threatened. In man, death from morphine poisoning is nearly always due to respiratory arrest. The primary mechanism of respiratory depression by morphine involves a reduction in the responsiveness of the brain stem respiratory centers to increases in carbon dioxide tension (PCO2). High concentrations of opioid receptors, as well, as endogenous peptides, are found in the medullary areas believed to be important in ventilatory control. Respiratory depression is mediated by a subpopulation of m receptors (m 1), distinct from those that are involved in the production of analgesia (m 2). Thus, a 'pure' m 1-opioid agonist could theoretically produce analgesia with little respiratory depression.
Cough. Opiates suppress the "cough center" which is also located in the brainstem, the medulla. Such an action is thought to underlie the use of opiate narcotics as cough suppressants. Codeine appears to be particularly effective in this action and is widely used for this purpose.
Gastrointestinal Tract. The opiates have been used for centuries for the relief of diarrhea and for the treatment of dysentery, and these uses were developed long before these agents were used as analgesics or euphoiants. Opiates appear to exert their effect on the gastrointestinal tract primarily in the intestine, where peristaltic movements, which normally propel food down the intestine, are markedly diminished. Also, the tone of the intestine is greatly increased to the point where almost complete spastic paralysis of movement occurs. This combination of decreased propulsion and increased tone leads to a marked decrease in the movement of food through the intestine. This stasis is followed by a dehydration of the feces, which hardens the stool and further retards the advance of material. All these effects contribute to the constipating properties of opiates. Indeed, nothing more effective has yet been developed for treating sever diarrhea.
Opiate Antagonists
Naloxone, when administered to normal individuals, produces no analgesia, euphoria, or respiratory depression. However, it rapidly precipitates withdrawal in narcotic-dependent individuals. Naloxone antagonizes the actions of morphine at all its receptors; however its affinity for m receptors is generally more than ten fold higher than for k or d receptors.
The uses of naloxone include the reversal of the respiratory depression that follows acute narcotic intoxication and the reversal of narcotic-induced respiratory depression in newborns of mothers who have received narcotics. The use of naloxone is limited by a short duration of action and the necessity of parenteral route of administration.
Naltrexone became clinically available in 1985 as a new narcotic antagonist. Its actions resemble those of naloxone, but naltrexone is well is well absorbed orally and is long acting, necessitating only a dose of 50 to 100 mg. Therefore, it is useful in narcotic treatment programs where it is desired to maintain an individual on chronic therapy with a narcotic antagonist. In individuals taking naltrexone, subsequent injection of an opiate will produce little or no effect. naltrexone appears to be particularly effective for the treatment of narcotic dependence in addicts who have more to gain by being drug-free rather than drug dependant. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Medicinal_Chemistry/Analgesics_and_Anti-Inflammatory_Agents.txt |
When fighting cancer, the entire population of neoplastic cells must be eradicated in order to obtain desired results. The concept of "total cell-kill" applies to chemotherapy as it does to other means of treatment: total excision of the tumor is necessary for surgical care, and complete destruction of all cancer cells is required for a cure with radiation therapy. By investigation of a model tumor system, the L1210 leukemia of mice, a number of important principles have been established as follows:
1. A single clonogenic malignant cell can give rise to sufficient progeny to kill the host; to achieve cure it is thus necessary to destroy every such cell. Since the doubling-time of most tumors is relatively constant during logarithmic growth, the life-span of the host is inversely related to the number of malignant cells that are inoculated or that survive therapeutic measures.
2. In contrast to antimicrobial chemotherapy where, in most instances, there are major contributions by the immune mechanisms and other host defenses, these play a negligible role in the therapy of neoplastic disease unless only a small number of malignant cells is present.
3. The cell-kill caused by antineoplastic agents follows first-order kinetics, that is, a constant percentage, rether than a constant number, of cells is killed by a given therapeutic maneuver, this finding has had a profound impact on clinical cancer chemotherapy. For example, a patient with advanced acute lymphocytic leukemia might harbor 1012 or about 1 kg of malignant cells. A drug killing 99.99% of these cells would reduce the tumor mass to about 100mg, and this would be apparent as a complete clinical remission. However, 108malignant cells would remain, any of which could cause a relapse in the disease.
The logical outgrowth of these concepts has been the attempt to achieve total cell-kill by the use of several chemotherapeutic agents concurrently or in rational sequences. The resulting prolonged survival of patients with acute lymphocytic leukemia through the use of such multiple-drug regimens has encouraged the application of these principles the treatment of other neoplasms.
Fundamental advances continue in the chemotherapy of neoplastic diseases. The greatest progress in recent years has not been the discovery of new, useful chemotherapeutic agents but at the conceptual level: the design of more effective regimens for concurrent administration of drugs; the acquisition of knowledge of the mechanisms of action of many antitumor agents, which facilitates the design of new methods to prevent or minimize drug toxicity; the increased use of adjuvant chemotherapy (e,g., the design of chemotherapeutic approaches to destroy micrometastases and prevent the development of secondary neoplasms after removal of destruction of the primary tumor by surgery of irradiation); and increased knowledge about such vital processes as tumor initiation and the dissemination, implantation, and growth of metastases. Of great importance is recognition of the problems imposed by the heterogeneity of tumors, with the realization that individual tumors may contain many subpopulations of neoplastic cells that differ in crucial characteristics, such as karyotype, morphology, immunogenicity, rate of growth, the capacity to metastasize, and , significantly, responsiveness to antineoplastic agents. Information also continues to accumulate in the fields of molecular and cellular biology, resulting in a greater understanding of cellular division and differentiation, tumor immunology, and viral and chemical carcinogenisis. It is hoped that these discoveries will provide new targets for therapy.
A. Alkylating Agents
The chemotherapeutic alylating agents have in common the property of undergoing strongly electrophilic chemical reactions through the formation of carbonium ion intermediates or of transition complexes with the target molecules. These reactions result in the formation of covalent linkages (alkylation) with various nucleophilic substances, including such biologically important moieties as phosphate, amino, sulfydryl, hydroxyl, carbonyl, and imidozole groups. The cytotoxic and other effects of the alkylating agents are directly related to the alkylation of components of DNA. The 7 nitrogen atom of guanine is particularly susceptible to the formation of a covalent bond with both monofunctional and bifunctional alkylators and may well represent the key target that determines the biological effects of these agents. It must be appreciated, however, that other atoms in the purine and pyrimidine bases of DNA-for example, the 1 or 3 nitrogens of adenine, the 3 nitrogen of cytosine, and the 6 oxygen of guanine-may also be alkylated to a lesser degree, as are the phosphate atom of the DNA chains and the proteins associated with DNA.
Nitrogen Mustards
Structure of nitrogen mustards. 3D structure of nitrogen mustard and uracil nitrogen mustard
Alkylating Mechanism of Mechlorethamine With Guanine Base:
Cyclosphosphamide
Efforts to modify the chemical structure of mechlorethamine to achieve greater selectivity for neoplastic tissues led to the development of cyclophosphamide. After studies of the pharmacological activity of cyclophosphamide, clinical investigations by European workers demonstrated its effectiveness in selected malignant neoplasms.
Cyclophosphamide is a classical example of the role of the host metabolism in the activation of an alkylating agent and is one or the most widely used agents of this class. The original rationale that guided its molecular design was twofold. First, if a cyclic phosphamide group replaced the N-methyl of mechlorethamine, the compound might be relatively inert, presumably because the bis-(2-chloroethyl) group of the molecule could not ionize until the cyclic phosphamide was cleaved at the phosphorous-nitrogen linkage. Second, it was hoped that neplastic tissues might posses phosphatase of phosphamidase activity capable of accomplishing this cleavage, thus resulting in the selective production of an activated nitrogen mustard in the malignant cells. In accord with these predictions, cyclophosphamide displays only weak cytotoxic, mutagenic, or alkylating activity and is relatively stable in aqueous solution. However, when administered to experimental animals or patients bearing susceptible tumors, marked chemotherapeutic effects, as well as mutagenicity and cancinogenicity, are seen. Although a definite role for phosphatases of phosphamidases in the mechanism of action of cyclophospamide has not yet been demonstrated, it is clearly established that the drug initially undergoes metabolic activation be the cytochrome P-450 mixed-function oxidase system of the liver, with subsequent transport of the activated intermediate to sites of action. Thus, a crucial factor in the structure-activity relationship of cyclophosphamide concerns its capacity to undergo metabolic activation in the liver, rather than to alkylated malignant cells directly. it also appears that the selectivity of cyclophosphamide against certain malignant tissues may result in part from the capacity of normal tissues, such as liver, to protect themselves against cytotoxicity by further degrading the activated intermediates.
None of the severe acute CNS manifestations reported with the typical nitrogen mustards has been noted with cyclophosphamide. Nosea and vomiting, however, may occur. Although the general cytotoxic action of this drug is similar to that of other alkylating agents, some notable diferences have been observed. When comapred with mechloroethamine, damage to the megakaryocytes and thrombocytopenia are less common. Another unusual manifestation of selectivity consists in more prominent damage to the hair follicles, resulting frequently in alopecia (baldness). The drug is not a vesicant, and local irritaion does occur.
Uracil Mustard
Uracil mustard was synthesized in an unsuccessful attempt to produce an active-site alkylator by linking the bis-(2-chloroethyl) group to the pyrimidine base uracil. Its activity in experimental neoplasms was demonstrated shortly thereafter. No relationship has been demonstrated, however, with the biological function of uracil. Note: side effects of chemotherapy can be treated with marijuana
B. Anti-metabolites - (pyrimidine antagonists)
Cancer is a group of diseases characterized by abnormal and uncontrolled cell division. One important approach to antitumor agents is the design of compounds with structures related to those of pyrimidines and purines that are involved in biosynthesis of DNA. These compounds are known as antimetabolites because they interfere with the formation or utilization of a normal cellular metabolite. This interference generally results from the inhibition of an enzyme in the biosynthetic pathway of the metabolite from the incorporation, as a false building block, into vital macromolecules such as proteins or nucleic acids.
5-Fluorouracile - Pyrimidine Antagonist
Uracil is not a component of DNA. Rather, DNA contains thymine, the methylated analog of uracil. The enzyme thymidylate synthetase is required to catalyze this finishing touch: deoxyuridylate (dUMP) is methylated to deoxythymidylate (dTMP) (see figure below). The methyl donor in this reaction is methylenetetrahydrofolate. Rapidly dividing cells require an abundant supply of deoxythymadylate for the synthesis of DNA. Therefore, the vulnerability of these cells to the inhibition of dTMP synthesis can be exploited in cancer therapy.
The rational for 5-fluorouracil, 5-FU, was to block DNA synthesis by inhibiting the biosynthesis of dTMP, by virtue of its close structural analogy to uracil. Fluorine, being the smallest atom that would substitute for hydrogen at the 5' position, was assumed to create the smallest possible molecular perturbation and thus be converted to the nucleotide and be accepted by the reactive site of thymidylate synthetase as a substrate imposter. In fact, this was the case. The van der Waals radius of the F atom (1.35 A) is only slightly larger than that of the H atom (1.20 A). Therefore, 5-fluorouracil is a fluorinated pyrimidine analogue which stops cell proliferation by blocking DNA synthesis and RNA processing.
Fluorouracil is converted in vivo into fluorodeoxyuridylate (F-dUMP). This analog of dUMP irreversibly inhibits thymidylate synthase after acting as a normal substrate through part of the catalytic cycle. First a sulfhydryl group of the enzyme adds to C-6 of the bound F-dUMP (see figure below). Methylenetetrahydrofolate then adds to C-5 of this intermediate. In the case of dUMP, a hydride ion of the folate is subsequently shifted to the methylene group, and a proton is taken away from C-5 of the bound nucleotide. However, F+ cannot be abstracted from F-dUMP by the enzyme, and so catalysis is blocked at the stage of the covalent complex formed by F-dUMP, methylenetetrahydrofolate, and the sulfhydryl group of the enzyme. We see here and example of suicide inhibition, in which an enzyme converts a substrate into a reactive inhibitor that immediately inactivates its catalytic activity. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Medicinal_Chemistry/Anticancer_Drugs.txt |
A. Histamine
Histamine is 2-(4-imidazolyl)ethylamine and is a hydrophilic molecule comprised of an imadazole ring and an amino group connected by two methylene groups. It arises in vivo by decarboxylation of the amino acid histadine.
Histamine is concentrated in mast cells, cells whose function is essentially to release histamine and immunoglobins when tissue damage occurs. They are especially numerous in parts of the body that are injured often, such as the fingers and toes, or which enjoy frequent contact with the environment, such as the mucosa of the lips, nose, etc. Histamine is also a neurotransmitter in the CNS and a typical problem with some antihistamines is drowsiness. The effort has been to produce compounds that do not enter the brain very well.
There are many drugs with histamine-like properties, and most contain the following fragment:
Histamine contracts many smooth muscles, such as those of the bronchi and gut, but powerfully relaxes others, including those of fine blood vessels. It is also a very potent stimulus to gastric acid production. Effects attributable to these actions dominate the overall response to the drug; however, there are several others, of which edema formation and stimulation of sensory nerve endings are perhaps the most familiar. Some of these effects as bronchoconstriction and contraction of the gut, are mediated by one type of histamine receptor, the H1 receptors, which are readily blocked by pyrilamine and other such classical antihistamines, now more properly described as histamine H1-receptor blocking drugs of simply H1 blockers. Other effects, most notably gastric secretion, are completely refractory to such antagonists, involve activation of H2 receptors, and are susceptible to inhibition by the more recently developed histamine H2-receptor blocking drugs. Still others, such as the hypotension resulting from vascular dilation, are mediated by receptors of both H1 and H2 types, since they are annulled only by a combination of H1 and H2 blockers.
The two classes of histamine receptors also reveal themselves by differential responses to various histamine-like agonists. Thus, 2-methylhistamine preferentially elicits responses mediated by H1 receptors, whereas 4-methylhistamine has corresponingly preferential effect mediated through H2 receptors. These conpounds are representatives of two classes of histtamin-like dtugs, the H1-agonists and H2-agonists.
B. Histamine Antagonists
H1 Antagonists
All of the available antagonists are reversable, competative inhibitors of the actions of histamine. The structure of almost all of the "classic" antihistamines have a tertiary amino group linked by two- or three-atom chain to two aromatic substituens and confrom to the general formula shown below, where Ar is aryl and X is a nitrogen or a carbon aton or a C-O- ether linkage.
Two common examples of H1 antagonists are shown below.
H1-blocking drugs have an established and valued place in the symptomatic treatment of various immediate hypersensitivity reactions, in which their usefulness is attributable to their antagonism of endogenously released histamine, one of several autoacids that elicit allergic response. In addition, the central properties of some of the series are of considerable therapeutic value in suppressing motion sickness.
In treating diseases of allergy, the effect of antihistamines is purely palliative and confined to the suppression in varying degree of symptoms attributable to the pharmacological activity of histamine released by the antigen-antibody reaction. The drugs do not diminish the intensity of this reaction, which is the root cause of the various hypersensitivity diseases. This limitation must be clearly recognized. In bronchial asthma, histamine blockers are singularly ineffectual. The have no role in the therapy of severe attacks in which chief reliance must be placed on epinephrine, isoproterenol, and theophylline. Equally, in the treat5ment of systemic anaphylaxis, in which autoacids other than histamine are again important, the mainstay of therapy is once more epinephrine, with histamine antagonists having only a subordinate and adjuvant role.
Other allergies of the respiratory tract are more amenable to therapy with H1 blockers. The best results are obtained in seasonal rhinitis (hay fever) and conjunctivitis, in which these drugs relieve the sneezing, rhinorrhea, and itching of the eyes, nose and throat.
H2 Antagonists
The H2 blockers are reversible, competitive antagonists of the actions of histamine on H2 receptors. They are highly selective in their action and are highly selective in their action and are virtually without effect on H1 receptors. The most prominent of the effects of histamine that are mediated by H2 receptors is stimulation of gastric acid secretion, and it is the ability of the H2 blockers to inhibit this effect that explains much of their importance. Despite the widespread distribution of H2 receptors in the body, H2 blockers interfere remarkably little with physiological function other than gastric secretion, implying that extragastric H2 receptors are of minor physiological importance
H2 blockers are used in treatment of peptic ulcer disease PUD; a disease in which ulceration occurs in the lower esophagus, stomach, duodenum, or jejunum. The most prominent symptom is gnawing pain that is relieved by food and alkali, but worsened by alcohol and condiments. The proximate cause of PUD is gastric acid hypersecretion.
The synthesis of H2 antagonists was acheived by stepwise modifications of the histamine molecule, which resulted, some 200 compounds later, in the first highly effective drug with potent H2-blocking activity, burimamide. This, like later compounds, retained the imidazole ring of histamine byt possessed a much bulkier side chain. Cimetidine, the first H2 blocker to be introduced for general clinical use, won rapid acceptance for the treatment of ulcers and other gastric hypersecretory conditions and soon became one of the most widely prescribed of all drugs. this success led to the synthesis of numerous congeners. Some of the more popular drugs are shown below.
The synthesis of rantadine is not difficult. The figure below shows the three steps of its synthesis from common starting chemicals.
Proton Pump Inhibitors
The parietal cells secrete acid by means of a membrane pump, identified as an H+, K+-ATPase, that exchanges hydrogen ions for potassium ions. By analogy with the familiar Na+, K+-ATPase, whose function can be inhibited by digitalis, this proton pump can likewise by inhibited by a newly discovered family. Omeprazole is the prototypical "acid pump" inhibitor which was allowed for clinical use in 1989. Its effects on gastric acid reduction are profound, showing a greater decrease of daily acid secretion than is obtained with four cimetidine given four times a day.
It has been demonstrated in vitro that under acid conditions as high as 0.5M, HCl, omeprazole cyclized reversibly to a spiro-dyhydroimidazole intermediate (see figure below), which opens to sulfenic acid (not isolated). Cyclization, by the loss of H2O leads to a cyclic sulfenamide that was isolated and identified. Treatment of the sulfenamide with mercaptoehanol (HSCH2CH2OH) opened the ring to produce the predicted disylfide adduct shown. Since these conditions simulate the gastric environment and H+, K+ATPase was known to have an essential -SH group, it has been proposed that the sulfenamide produced from omeprazole is the chemical species that forms a covalent drug-enzyme complex with H+, K-ATPase in the acid compartment of the parietal cell, thereby blocking \(\ce{H^{+}}\) release.
Cromolyn Sodium
Cromolyn sodium, the disodium salt of 1,3-bis(2-carboxychromone-5-yloxy)-2-hydroxypropane, has the following structure:
Cromolyn does not relax bronchial or other smooth muscle. Nor does it inhibit significantly responses to these muscles to any of a variety of pharmacological spasmogens. It does, however, inhibit the release of histamine and other autocoids (including leukotrienes) from human lung during allergic responses mediated by IgE antibodies and thereby the stimulus for bronchospasm. Inhibition of the liberation of leukotrienes is particularly important in allergic bronchial asthma, where these products appear to be the principal cause of bronchoconstriction. Cromolyn acts on the pulmonary mast cells for the immediate hypersensitivity reaction. Cromolyn does not inhibit the binding of IgE to mast cells nor the interaction between cell-bound IgE and specific antigen; rather, it suppresses the secretory response to this reaction.
Cromolyn sodium is insufflated into the lings by a special device as a micronized powder. The drug is strictly prophylactic; it will not abort an asthmatic attack in progress.
C. Local Anesthetics
The first local anesthetic to be discovered was cocaine, an alkaloid contained in large amounts in the leaves of Erythroxylon coca, a shrub growing in the Andes Mountains. Over 9 million kilograms of these leaves are consumed annually by the 2 million inhabitants of the highlands of Peru, who chew or suck the leaves for the sense of wellbeing it produces.
Local anesthetics are drugs that block nerve conduction when applied locally to nerve conduction when applied locally to nerve tissue in appropriate concentrations. They act on any part of the nervous system and on every type of nerve fiber. For example, when they are applied to the motor cortex impulse transmission from that area stops, and when they are injected into the skin they prevent the initiation and the transmission of sensory impulses. A local anesthetic in contact with a nerve trunk can cause both sensory and motor paralysis in the area innervated. The great practical advantage of the local anesthetic is that their action is reversible: their use is followed by complete recovery in nerve function with no evidence of structural damage to nerve fibers of cells.
The structures some of the typical anesthetics are shown below.
These structures contain hydrophilic and hydrophobic domains that are separated by an intermediate alkyl chain. Linkage of these two domains is of either the ester or amide type. the ester link is important because this bond is readily hydrolyzed during metabolic degradation and inactivation in the body. Procaine, for example, can be divided into three main portions: the aromatic acid (para-aminobenzoic), the alcohol (ethanol), and the tertiary amino group (diethylamino). Changes in any part of the molecule alter the anesthetic potency and the toxicity of the compound. Increasing the length of the alcohol group leads to a greater anesthetic potency. It also leads to an increase in toxicity.
Local anesthetics prevent the generation and the conduction of the nerve impulse. Their site of action is the cell membrane. Local anesthetic and other classes of agents (e.g., alcohols and barbiturates) block conduction by decreasing or preventing the large transient increase in the permeability of the membrane to sodium ions that is produced by a slight depolarization of the membrane. As anesthetic action progressively develops in a nerve, the threshold for electrical excitability gradually increases and the safety factor for conduction decreases; when this action is sufficiently well developed, block of conduction is produced.
The local anesthetics also reduce the permeability of resting nerve to potassium as well as to sodium ions. Since changes in permeability to potassium require higher concentration of local anesthetic, blockade of conduction is not accompanied by any large or consistent change in the resting potential.
All the commonly used local anesthetics contain a tertiary or secondary nitrogen atom and, therefore, can exist either as the uncharged tertiary of secondary amine or as the positively charged substituted ammonium cation, depending on the dissociation content of the compound and the pH of the solutions. The pKa of a typical local anesthetic lies between 8.0 and 9.0, so that only 5 to 20% will be protonated at the pH of the tissues. this fraction, although small, is important because the drug usually has to diffuse through connective tissue and other cellular membranes to reach its site of action, and it is generally agreed that it can do so only in the form of the uncharged amine. Once the anesthetic has reached the nerve, the form of the molecule active in nerve fibers is the cation which combines with some receptor in the membreane to prevent the generationof an action potential. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Medicinal_Chemistry/Antihistamines_and_Local_Anesthetics.txt |
The modern era of the chemotherapy of infection started with the clinical use of sulfanilamide in 1936. The "golden age" of antimicrobial therapy began with the production of penicillin in 1941, when this compound was mass-produced and first made available for limited clinical trial. More than 30% of all hospitalized patients now receive one or more courses of therapy with antibiotics, and millions of potentially fatal infections have been cured. However, at the same time, these pharmaceutical agents have become among the most misused of those available to the practicing physician. One result of widespread use of antimicrobial agents has been the emergence of antibiotic-resistant pathogens, which in turn has created an ever-increasing need for new drugs. Many of these agents have also contributed significantly to the rising costs of medical care.
An antibiotic is any substance produced by a microorganism that is excreted to harm or kill another microorganism. Technically, antibiotics are microbial or fungal products. But these substances can be synthesized and mass produced in the laboratory to use against harmful microorganisms in the environment. Thus, the synthetic chemist has added greatly to our therapeutic armamentarium. Synthetic drugs such as isonaizid and theambutol represent important contributions for the treatment of tuberculosis. While many such antimicrobial agents are not properly termed antibiotics, since they are not produced by living organisms, little distinction should now be made between compounds of natural and synthetic origin.
Antibiotics can further be grouped under the broader heading of chemotherapuetic agents, chemical agents used to treat disease. Good chemotherapuetic agents are able to kill or inhibit the target pathogen without too much damage to the host organism. The basis for this selective toxicity lies in the differences between prokaryotic cells of microorganisms and our own eukaryotic cells. The prokaryotic cells of microorganisms differ in a number of ways from eukaryotic cells, such as absence of cell walls, different size of ribosomes, and details of metabolism. Thus, the goal of antibiotic therapy is to choose or design drugs that target these differences in host and pathogen cells.
A. Cell Wall Synthesis Inhibitors
Penicillin
3-D structure of amoxicillin
History of Penicillin and General Information
Alexander Fleming loved to play, both in the laboratory and out. He always loved snooker and golf and had many whimsical variants on the rules. In the lab he made "germ paintings," in which he would draw with his culture loop using spores of highly pigmented bacteria, which were invisible when he made the painting, but when cultured developed into brightly colored scenes. He followed what Max Delbruck would later call the "principle of limited sloppiness." Fleming abhorred a tidy, meticulous lab; he left culture dishes lying around for weeks and would often discover interesting things in them. Though the story has been told in many sometimes conflicting ways, something like this resulted in the discovery of penicillin. He seems to have left a culture dish lying on the lab bench and then gone away on vacation. When he returned a few spores of an unusual mold had germinated on the plate. When he cultured the bacteria on the plate he found that they grew up to within a few centimeters of the mold, but there were killed. A crude extract of the mold was then shown to have antibacterial properties. Fleming made this discovery in 1928 and by 1929 had named it penicillin (he was told by a colleague that the mold was a type of Penicillium and "penicillozyme" must have seemed cumbersome).
Fleming continued to use penicillin in his lab but not with any great enthusiasm and certainly not to the exclusion of many other
projects. He never developed it into a clinically useful compound, though in 1929 he suggested that it might have important clinical applications. Because he was a bacteriologist and not a chemist, Fleming did not attempt to purify penicillin. He seems to have run into a dead end with penicillin and so during the 1930s, though he kept it in his lab, he did not do much with it. In the late 1930s Australian Howard Florey came to London to work with Charles Sherrington. He worked on lysozyme for a while and then became interested in penicillin. It was Florey, with Chain and other of his group that developed penicillin into a clinical antibiotic. They did this during 1940-41. Fleming, Florey, and Chain shared the 1945 Nobel Prize in Physiology for Medicine.
Fleming became world-famous for penicillin, and was rightly acknowledged as the father of modern antibiotics, but Florey was just as rightly miffed at being denied much of the credit for creating the powerful medical tool we now know. Evidence does not suggest that Fleming deliberately denied Florey his due credit, but Fleming's peculiar, dry sense of humor seems to have caused him not to deny even the wildest attributions to him.
Cephalosporins
Cephalosporins are the second major group of b-lactam antibiotics. They differ from penicillins by having the b-lactam ring fused to a dihydrothiazine ring rather than a thiazolidine. The other difference, which is more significant from a medicinal chemistry stand point, is the existence of a functional group (R) at position 3 of the fused ring system. This now allows for molecular variations to be introduced at the 7-NH2 group, as in the penicillins, as well as to effect changes in properties by diversifying the moieties at position 3.
The first member of the newer series of b-lactams was isolated in 1956 from extracts of Cephalosporium acremonium, a sewer fungus. This species actually produced several antibiotics: cephalosporin C, cephalosporins P1 - P5 and penicillin N. This was also true with Fleming's Penicillium notatum. However, that was not discovered until research on the chemistry of penicillin was worked on. Cephalosporin has also been identified from other fungi such as Emericellopsis and Paecilomyces, two genera that are morphologically similar to Penicillium.
Like penicillin, cephalosporins are valuable because of their low toxicity and their broad spectrum of action against various diseases. In this way, cephalosporin is very similar to penicillin. Cephalosporins are one of the most widely used antibiotics, and economically speaking, has about 29% of the antibiotic market. The cephalosporins are possibly the single most important group of antibiotics today and are equal in importance to penicillin.
The structure and mode of action of the cephalosporins are similar to that of penicillin. They affect bacterial growth by inhibiting cell wall synthesis, in Gram-positive and -negative bacteria.
3-D structure of cefoxitin
Mechanism of Action
Penicillin consists of a thiolidine ring fused to a b-lactam ring, to which a variable R group is attached by a peptide bond. This structure can undergo a variety of rearrangements, which accounts for the instability first encountered by Flemming. In particular, the b-lactam ring is very labile.
In 1957, it was shown that bacteria ordinarily susceptible to penicillin could be grown in its presence if a hypertonic medium were used. The organisms obtained this way are devoid of a cell wall and consequently lyse when transferred to a normal medium. Hence it was inferred that penicillin interferes with the synthesis of the bacterial cell wall. The cell walls of bacteria are essential for their normal growth and development. Peptidoglycan is a heteropolymeric component of the cell wall that provides rigid mechanical stability by virtue of its highly cross-linked lattice work structure, which prevents bacteria from bursting from their high internal osmotic pressure. The peptidoglycan is composed of glycan chains, which are linear strands of alternating pyranoside residues of two amino sugars (N-acetylgucosamine and N-acetylmuramic acid), that are cross-linked by peptide chains. In gram-positive microorganisms, the cell wall is 50 to 100 molecules thick, but it is only 1 or 2 molecules thick in gram-negative bacteria.
In 1965, it was deduced that penicillin blocks the last step in cell wall synthesis, namely the cross-linking of different peptioglycan strands. This cross-linking is accomplished by a transpeptidation reaction that occurs outside the cell membrane. The transpeptidase itself, called glycopeptide transpeptidase, is membrane bound. In the formation of the cell wall of Staphylococcus aureus, the transpeptidase normally forms an acyl-enzyme intermediate with the penultimate D-analine residue of the D-Ala-D-Ala-peptide. This covalent acyl-enzyme intermediate then reacts with the amino group of the terminal glycine in another peptide to form the cross-link (see figure below). Therefore, the end result is that the amino group at one end of a pentaglycine chain attacks the peptide bond between two D-analine residues in another peptide unit, a peptide bond is formed between glycine and one of the D-alanine residues, and the other D-alanine residues is released. It should be noted that bacteria cell walls are unique in containing D amino acids, which form cross-links by a different mechanism from that used to synthesize proteins.
Penicillin mimics the D-Ala-D-Ala moiety of the normal substrate and is welcomed into the active site of the transpeptidase. Bound penicillin then forms a covalent bond with a serine residue at the active site of the enzyme. This penicilloyl-transpeptidase does not further react and the enzyme is irreversibly inhibited (see figure below).
The reason penicillin is so effective in inhibiting glycopeptide transferase is the four-membered b-lactam ring is strained, which makes it highly reactive. Also, the conformation of this part of penicillin is probably very similar to that of the transition state of the normal substrate, a species that interacts strongly with the enzyme.
Because of the high use of penicillin, some bacteria have developed resistance by producing molecules that can disable penicillin. Penicillinase is an enzyme produced by certain penicillin-resistant bacteria which reacts irreversibly with the b-lactam ring. Scientists have responded with other drugs that inturn react and disable penicillinase. One such drug is clavulanic acid. This compound irreversibly binds to penicillinase and prevent the enzyme from working. Therefore, sometimes clavulanic acid is given along with one of the semi-synthetic penicillins.
B. Protein Synthesis Inhibitors.
Erythromycin, Azithromycin
Erythromycin is an orally effective antibiotic discovered in 1952 in the metabolic products of a strain of Streptocyces erythreus, originally obtained from a soil sample collected in the Philippine Archepelago. Erythromycin is one of the macrolide antibiotics, so named because they contain a many-membered lactone ring to which are attached one or more deoxy sugars. It is a white crystalline compound, soluble in water to the extent of 2 mg/ml. the structuras formula of erythromycine is a follows:
Erythromycin-A has a 14-membered macrolide ring, to which two sugars are attached: desosamine on carbon 5, and cladinose on carbon 3.Crystal Image
Erythromycin may be either bacteriostatic or bactericidal, depending on the microorganism and the concentration of the drug. The bactericidal activity is greatest against a small number of rapidly dividing microorganisms and increases markedly as the pH of the medium is raised over the range of 5.5 to 8.5. the antibiotic is most effective in vitro against gram positive cocci such as Strep. pyogens and Strep. Pneumoniae. Resistant strains of these bacteria are rare and are usually isolated from populations of people who have been recently exposed to macrolide antibiotic. For example, in 1979, only 5% of group-A streptococcal strains isolated in Oklahoma were resistant to erythromycin, but 60% of such strains were found to be resistant in Japan. the difference likely reflects the wide use of erythromycin in Japan for respiratory infections.
Mechanism of Action
Erythromycin and other macrolide antibiotics inhibit protein synthesis by binding to 50 S ribosomal subunits of sensitive microorganisms. (Humans do not have 50 S ribosomal subunits, but have ribosomes composed of 40 S and 60 S subunits). Certain resistant microorganisms with mutational changes in components of this subunit of the ribosome fail to bind the drug. The association between erythromycin and the ribosome is reversible and takes place only when the 50 S subunit is free from tRNA molecules bearing nascent peptide chains. The production of small peptides goes on normally in the presence of the antibiotic, but that of highly polymerized homopeptides is suppressed. Gram-positive bacteria accumulate about 100 times more erythromycin than do gram-negative microorganisms. The nonionized from of the drug is considerably more permeable to cells, and this probably explains the increased antimicrobial activity that is observed in alkaline pH.
Tetracycline
The development of the tetracycline antibiotics was the result of a systematic screening of soil specimens collected from many parts of the world for antibiotic-producing microorganisms. The first of these compounds, chlortetracycline, was introduced in 1948. Soon after there initial development, the tetracyclines were found to be highly effective against rickettsiae, a number of gram-positive and gram-negative bacteria, and the agents responsible for conjunctivitis, and psittacosis, and hence became known as "broad spectrum" antibiotics. They are also effective to agents that exert their effects on the bacterial cell wall, such as rickettsiae, Chlamydia, and amebae.
Chlortetracycline
In vitro, tetracycline drugs are primarily bacteriostatic and only multiplying microorganisms are affected. These drugs are closely related bacteriostatic antibiotics, similar in antibacterial spectrum and toxicity. The site of action of tetracyclines is the 30 S subunit of the ribosome, but at least two processes appear to be required for these antibiotics to gain access to the ribosomes of gram-negative bacteria. The first is passive diffusion through hydrophobic pores in the outer cell membrane. The second process involves an active transport system that pumps all tetracyclines through the inner cytoplasmic membrane. Although permeation of these drugs into gram-positive bacteria is less will understood, it too requires an active transport system. Once the tetracyclines gain access to the bacteria cell, they inhibit protein synthesis and bind specifically to 30 S ribosomes. They appear to prevent access of aminoacyl tRNA to the acceptor site on the mRNA-ribosome complex. This prevents the addition of amino acids to the growing peptide chain. Only a small portion of the drug is irreversibly bound, and the inhibitory effects of the tetracyclines can be reversed by washing. Therefore, it is probable that the reversibly bound antibiotic is responsible for the antibacterial action. These compounds also impair protein synthesis in mammalian cells at high concentrations; however the host cells lack the active transport system found in bacteria.
Tetracycline-resistant strains of pneumococci account for about 5% of isolates from pneumococcal pneumonia patients. Infections due to Group A beta-hemolytic streptococci should not be treated with a tetracycline, since as many as 25% of the organisms may be resistant when tested in vitro. Serious staphylococcal disease is also not a primary indication for tetracyclines. Bacterial resistance to one tetracycline is generally accompanied by cross-resistance to the others.
Pharmacology
The tetracyclines are variably absorbed after oral administration. Food interferes with absorption of tetracyclines, with the exception of doxycycline and minocycline. Absorption of tetracyclines is decreased in the presence of antacids containing aluminum, Ca, and Mg, and in preparations containing iron. The half-lives in plasma are about 8 h for oxytetracycline and tetracycline; 13 h for demeclocycline and methacycline; and 16 to 20 h for doxycycline and minocycline.
Tetracyclines penetrate into most tissues and body fluids. However, CSF levels are not reliably therapeutic. Minocycline, because of its high lipid solubility, is the only tetracycline that penetrates into tears and saliva in levels high enough to eradicate the meningococcal carrier state. All tetracyclines, except doxycycline, are excreted primarily in the urine by glomerular filtration, and their blood levels increase in the presence of renal insufficiency. Doxycycline is excreted mainly in the feces. All tetracyclines are partially excreted in bile, resulting in high biliary levels. They are then partially reabsorbed.
C. Synthetic Antibiotics
Sulfonamides
The sulfonamides are synthetic bacteriostatic antimicrobial agents with a wide spectrum encompassing most gram-positive and many gram-negative organisms. These drugs were the first effective chemotherapeutic agents to be employed systematically for the prevention and cure of bacterial infections in man. The considerable medical and public health importance of their discovery and their subsequent widespread use were quickly reflected in the sharp decline in morbidity and mortality figures for the treatable infectious decease. Before penicillin became generally available, the sulfonamides were the mainstay of antibacterial chemotherapy. While the advent of antibiotics has diminished the usefulness of the sulfonamides, they continue to occupy an important, although relatively small, place in the therapeutic armamentarium of the physician.
Sulfonimides are structural analogs and competitive antagonists of para-aminobenzoic acid (PABA), and thus prevent normal bacterial utilization of PABA for the synthesis of the vitamin folic acid. (The role of folic acid in RNA synthesis was already discussed in Anti Cancer Drugs). More specifically, sulfonamides are competitive inhibitors of the bacterial enzyme sulfihydropteroate synthase, which is responsible for the conversion of PABA into dihydrofolic acid, the immediate precursor of folic acid. Sensitive microorganisms are those that must synthesis their own folic acid; bacteria that can utilize preformed folic acid are not affected. Bacteriostasis induced by sulfonamides is counteracted by PABA competitively. Sulfonamides do not affect mamalian cells by this mechanism, since they require preformed folic acid and cannot synthesis it.
Pharmacology
Most sulfonamides are readily absorbed orally, the small intestine being the major site of absorption. Parenteral administration is difficult, since the soluble sulfonamide salts are highly alkaline and irritating to the tissues. The sulfonamides are widely distributed throughout all tissues. High levels are achieved in pleural, peritoneal, synovial, and ocular fluids. CSF levels are effective in meningeal infections, but sulfonamides are rarely used for this indication. When given in pregnancy, high levels are achieved in the fetus. Sulfonamides are loosely and reversibly bound in varying degrees to serum albumin. Since the bound sulfonamide is inactive and nondiffusible, the degree of binding can affect antibacterial effectiveness, distribution, and excretion. The antibacterial action is inhibited by pus.
The sulfonamides are metabolized mainly by the liver to acetylated forms and glucuronides, both therapeutically inactive. Excretion is primarily renal by glomerular filtration with minimal tubular secretion or reabsorption. The relative insolubility of most sulfonamides, especially their acetylated metabolites, may cause precipitation in the renal tubules. The more soluble analogs, such as sulfisoxazole and sulfamethoxazole, should be chosen for systemic therapy, and the patient must be well hydrated. To avoid crystalluria and renal damage, fluid intake should be sufficient to produce a urinary output of 1200 to 1500 mL/day. Sulfonamides should not be used in renal insufficiency.
Antimalarial Drugs - Quinolines
Chloroquine is one of the cheif agents for the chemotherapy of malaria. Although chlorouqine causes a number of effects that singly or in combination may relate to its promary mechanism of plasmodicidal action, this process is not yet shown. From early work, it has been found that chloroquine inhibits the incorporation of phosphate into RNA and DNA. Later it was shown that chloroquine combines strongly with double-stranded DNA. The drug is also reported to inhibit DNA polymerase activity markedly by combing with the DNA primer.
D. Misuses of Antibiotics
A common misuse of these agents is in infections that have been proven to be untreatable. The vast majority of the diseases due to the true viruses will not respond to any of the presently available anti-infective compounds. Thus, the antimicrobial therapy of measles, chickenpox, mumps, and at least 90% of infections of the upper respiratory tract is totally ineffective and, therefore, worse than useless
Fever of undetermined etiology may be of two types: one that is present for only a few days to a week and another that persists for an extended period. Both of these are frequently treated with antimicrobial agents. Most instances of pyrexia of short duration, in the absence of localizing signs, are probably associated with undefined viral infections, often of the upper respiratory tract, and do not respond to antibiotics. In the bulk of these cases, defervescence takes place spontaneously within a week or less. Studies of prolonged fever have shown that three common infectious causes are tuberculosis, often of the disseminated variety, hidden pyogenic intraabdominal abscess and infectious endocarditis. Also, the so-called collagen disorders and various neoplasms, especially lymphoma are frequently responsible for prolonged and significant degrees of fever. Various types of cancer, metabolic disorders, hepatitis, asymptomatic regional enteritis, atypical rheumatoid arthritis, and a number of other noninfectious disorders may present themselves as cases of fever of unknown etiology. The most rational approach to the problem of fever of unknown origin is not one that concentrates of the elevated temperature alone but one that involves a thorough search for its cause. The patient should not be unnecessarily exposed to chemotherapy in the hope that, if an agent is not effective, another one or combination of drugs will be helpful.
Because of misuse of antibiotics many strains of bacteria have become resistant to the effects of these drugs. Here are some examples of resistance that has occurred in staphylococcus species:
1. In 1952, almost 100% of Staphylococcus infections were susceptible to penicillin. By 1982, fewer than 10% of staph cases could be cured with penicillin. The resistance initially was due to one type of resistance mechanism, and alternative drugs were available. For example, in the late 1960’s, physicians switched to methicillin for staph infections.
2. In the early 1980’s, Staphylococcus strains were found that were resistant to penicillins, methicillin, naficillin, and cephalosporins. The source of these bacteria was ahospital. That is, the toughest cases of bacterial infections will be treated in hospitals, so that is where these highly resistant bacteria are most likely to be found. Approximately 19,000 hospital patients die each year due to nosocomial (hospital-acquired) bacterial infections.
3. In 1992, roughly 15% of all Staphylococcus strains in the U.S. were methicillin-resistant. By 1993, only one surefire Staphylococcus killer remained: vancomycin. However, vancomycin-resistant strains of other bacteria have been found, and now Staphylococcus has even become resistant to this antibiotic. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Medicinal_Chemistry/Antimicrobial_Drugs.txt |
A. Pharmacodynamics: interaction of drugs with their sites of action
While there are several types of exceptions, the effects of most drugs result from their interaction with functional macromolecular components of the organism. Such interaction alters the function of the pertinent cellular component and thereby initiates the series of biochemical and physiological changes that are characteristic of the response to the drug. The term receptor is used to denote the component of the organism with which the chemical agent interacts. By virtue of interactions with such receptors, drugs do not create effects but merely modulate ongoing function. Thus, drugs cannot impart a new function to a cell.
Figure. - hypothetical drug in receptor site.
Structure-Activity
The affinity of a drug for a specific macromolecular component of the cell and its intrinsic activity are intimately related to its chemical structure. The relationship is frequently quite stringent, and relatively minor modifications in the drug molecule, particularly such subtle changes as stereochemistry, may result in major changes in pharmacological properties. Exploitation of structure-activity relationships has on many occasions led to the synthesis of valuable therapeutic agents. Since changes in molecular configuration need to alter all actions and effects of a drug equally, it is sometimes possible to develop a congener with a more favorable ratio of therapeutic to toxic effects, enhanced selectivity among different cells or tissues, or more acceptable secondary characteristics than those of the parent drug. In addition, effective therapeutic agents have been fashioned by developing structurally related competitive antagonists of other drugs or of endogenous substances known to be important in biochemical or physiological function. Minor modifications of structure can also have profound effects on the pharmacokinetic properties of drugs.
Stereochemistry
Enantemerism can be produced by sp3 hybridized carbon atoms. Because free rotation about the chiral carbon is not possible, two stable forms of the molecule can exist. A molecule with two nonidentical asymmetric centers can exist as (22 = 4) four stereo isomers. Interaction with biological receptors can differ greatly between two enantomers, even to the point of no binding. There are numerous examples among drug molecules where only one isomer exhibits the desired pharmacology. Some isomers may even cause side effects or entirely different effects than its mirror image.
Ephedrine has two chiral centers and four isomers:
Different isomers can be used in different cases depending on the desired effect. Clinically, D(-) ephedrine is used to a large extent as an anti-asthmatic and, formerly, as a presser amine to restore low blood pressure as a result of trauma. L(+) pseudo-ephedrine is used primarily as a nasal decongestant.
Potencies of the Ephedrines
Isomer Relative activity
D(-) Pseudoephedrine 1
L(+) Pseudoephedrine 7
L(+) Ephedrine 11
D(-) Ephedrine 36
If the biological receptor has at least three binding sites, the receptor easily can differentiate enantomers (see figures below). The R(-)isomer has three points of interaction and is held in the conformation shown to maximize binding energy, whereas, the S(+)isomer can have only two sites of interaction.
It should be noted that the structure of alpha and beta adrenergic receptors are not entirely known. Also we should not forget that there is also enantioselectivity with respect to pharmacokinetics, such as, absorption, distribution, metabolism, and excretion.
Drug-Receptor Interactions
Cell signaling is the method in which cells communicate between each other in order to coordinate their activities and react to changes in their environments. Cell signaling involves a signal molecule (an agonist) and a specific signal receptor. Agonistic drugs are those which mimick natural signaling molecules and couse similar effects, while antagonists compete or inhibit agonists and hamper their effects.
Agonist Drugs. Agonist drugs mimic cell signaling molecules by activating the same receptor sites and causing similar effects. Both are described quantitatively by the same methods. If one assumes that an agonist drug interacts reversibly with its receptor and that the resultant effect is proportional to the number of receptors occupied, the following reaction can be written:
$\text{Drug (D)} + \text{Receptor (R)} \ce{ <=>[k_1][k_2]} DR \rightarrow \text{Effect}$
This reaction sequence is analogous to the interaction of substrate with enzyme, and the magnitude of effect can be analyzed in a manner similar to that for enzymatic product formation. The application equation is identical in form with the Michaelis-Menten equation:
$\text{Effect} = \dfrac{(\text{Maximal Effect}) [D]}{K_d+[D]} \label{2}$
where[D] is the concentration of free drug and KD (equal to K2 / K1) is the dissociation constant for the drug-receptor complex. This equation describes a simple rectangular hyperbola . There is no effect at [D] = 0; the effect is half-maximal when [D] = KD, that is when half of the receptors are occupied; the maximal effect is approached asymptotically as [D] increases above KD (Figure A below). Therefore, doubling the dose does not double the drug effect, but creates a less than two-fold consequence. It is frequently convenient to plot the magnitude of effect versus log [D], since a wide range of drug concentrations is easily displayed and a portion of the curve is more linear. In this case, the result is the familiar sigmoidal log dose-effect curve (Figure B below).
Equation \ref{2} can be rearranged by taking the reciprocal of both sides. The graph of the resulting equation is called the Lineweaver-Burk plot:
$\dfrac{1}{\text{Effect}} = \dfrac{K_D}{(\text{Max Effect} [D])} + \dfrac{1}{\text{Max Effect}} \label{3}$
A plot of 1/Effect versus 1/[D] yields a straight line that intersects the Y-axis at 1/(Max. Effect) and that has a slope equal to KD/(Max. Effect). Extrapolation of this line to the X-axis yields the value of -1/KD (Figure C below). Thus, values of $K_D$ and Max. Effect can be readily calculated from such a plot. This representation is especially useful for analyzing drug antagonism.
Figures A, B, and C. These were produced using Lotus 123r5. To download the spreadsheet, click here.
Antagonists. Certain drugs, called antagonists, interact with receptor sites to inhibit the actions of an agonists, while initiating no effect themselves. In this type of interaction, called competitive inhibition, the 1/Effect intercept of the plot of 1/[D] versus 1/[S] (Figure C) is the same in the presence and absence of an inhibitor, although the slope is different. This reflects the fact that the Max. Effect is not altered by a competitive inhibitor. The identification of competitive inhibition is that it can be overcome by a sufficiently high concentration of substrate.
A noncompetitive antagonist prevents the agonist from producing any effect at a given receptor site. This could result from irreversible interaction of the antagonist at any site to prevent binding of agonist. It could also follow reversible interaction with any component of the system so as to decrease the effect binding of agonist. These results may be conceptualized as removal of receptor from the system. The Max. Effect is reduced but the agonist's ability to still act normally at the receptor site is unaltered. The affinity of the agonist for the receptor and its potency are thus unaltered. On a Lineweaver-Burk plot (Fig. C), the intercept on the 1/Effect axis is increased and the new slope, is steeper. In contrast with the Max. Effect, KD is not affected by this kind of inhibition and so the x-intercept is unaltered. Noncompetitive inhibition cannot be overcome by increasing the agonist concentration.
Antagonists may thus be classified as acting reversibly or irreversibly. If the antagonist binds at the active site for the agonist, reversible antagonists will be competitive and irreversible antagonists will be noncompetitive. If binding is elsewhere, however, these simple rules do not hold, and any combination is possible.
Down load the spreadsheet used to make Figures A, B, and C and change KD and Max Effect to see the resulting changes in the graphs. (This spreadsheet can be also used in Microsoft Excel).
Interactions Between Drugs
There are two primary factors contributing to drug interactions. First, many drugs are bound to plasma proteins and this binding serves as a reservoir of inactive drugs. If a second drug displaces a drug already bound to the protein (by competing for the same protein), more of the previously bound drug will be able to pass out of the bloodstream and be available to the receptor and thus a more intense effect may be produced: the displacing drug would increase the effects or the toxicity of the displaced drug.
Second, a drug that is metabolized by the liver may induce new enzymes, which can then metabolize any of a variety of new drugs. Thus, an enzyme-inducing drug such as pentobarbital will decrease the activity of other drugs metabolized by the liver by increasing their rates of metabolism. Therefore, a wide variety of factors influence a drug's action in the body, making the use of more than one drug at a time risky, whether they are used separately or mixed in a concoction.
B. Pharmacokinetics
Dynamics of drug absorption, distribution and elimination.
Pharmacokinetics deals with the absorption, distribution, biotransformation, and excretion of drugs. These factors, coupled with dosage, determine the concentration of a drug at its sites of action and, hence, the intensity of its effects as a function of time. Many basic principles of biochemistry and enzymology and the physical and chemical principles that govern the active and passive transfer and the distribution of substances across biological membranes are readily applied to the understanding of this important aspect of medicinal chemistry.
Drug Elimination
Drugs are eliminated from the body either unchanged or as metabolites. Excretory organs, the lung excluded, eliminate polar compounds more efficiently than substances with high lipid solubility. Lipid-soluble drugs are thus not readily eliminated until they are metabolized to more polar compounds.
The kidney is the most important organ for elimination of drugs and their metabolites. Substances excreted in the feces are mainly unabsorbed orally ingested drugs or metabolites excreted in the bile and not reabsorbed from the intestinal tract. Excretion of drugs in milk is important not because of the amounts eliminated but because the excreted drugs are potential sources of unwanted pharmacological effects in the nursing infant. Pulmonary excretion is important mainly for the elimination of anesthetic gases and vapors: occasionally, small quantities of other drugs of metabolites are excreted by this route.
Drug elimination follows first-order kinetics. To illustrate first order kinetics we might consider what would happen if we were instantly inject (with an IV) a person with a drug, collect blood samples at various times and measure the plasma concentrations Cp of the drug. We might see a steady decrease in concentration as the drug is eliminated, as shown in the figure below.
If we measure the slope of this curve at a number of times we are actually measuring the rate of change of concentration at each time point. This can be written mathematically as Equation \ref{4}:
$\dfrac{dC_p}{dt} = - k_{el}C_p \label{4}$
where $k_{el}$ is an elimination constant.
Exercise $1$
Can you make a plot from this equation to calculate Kel? How can this equation be rearranged to determine Kel?
If we integrate, we find that the plasma concentration at a given time is Equation \ref{5}:
$C_p = C_P^o e^{-k_{el}t} \label{5}$
where Cpo is the initial plasma concentration. In the process of deriving this equation we can calculate the half life to be Equation 6:
$t_{1/2} = \dfrac{0.693}{k_{el}}\label{6}$
Table 1: Some Typical kel and t1/2 Values
Drug kel, hr-1 t1/2, hr
Acetaminophen 0.28 2.5
Diazepam 0.021 33
Digoxin 0.017 40
Gentamicin 0.35 2.1
Lidocaine 0.43 1.6
Theophylline 0.063 11
Metabolism and Excretion
So far we have considered elimination by excretion into urine only. Usually drugs are eliminated by excretion AND metabolism. Schematically this can be represented as:
where ke is the excretion rate constant and km is the metabolism rate constant. Here we have two parallel pathways for elimination although there could be more pathways, ie. excretion by exhalation, in sweat, or as is commonly the case, more than one metabolite. The differential equations for the two components shown in this diagram could be written just as we did above.
Apparent Volume of Distribution, V
We can also use the equations above to calculate the plasma concentration at any time when we know kel and Cpo. However, usually we don't know Cpo ahead of time, but we do know the dose. To calculate Cpo we need to know the volume that the drug is distributed into. That is, the apparent volume of the mixing container, the body. This apparent volume of distribution is not a physiological volume. It won't be lower than blood or plasma volume but it can may be much larger than body volume for some drugs. It is a mathematical 'fudge' factor relating the amount of drug in the body and the concentration of drug in the measured compartment, usually plasma. This can be expressed as Equation 7:
or
Immediately after an intravenous dose is administered, the amount of drug in the body is the dose. Thus we get equation 8:
or
Some example values for apparent volume of distribution are listed in the table below.
Drug V (l/kg) V (l, 70 kg)
Sulfisoxazole 0.16 11.2
Phenytoin 0.63 44.1
Phenobarbital 0.55 38.5
Diazepam 2.4 168
Digoxin 7 490
The last figure, for digoxin, is much larger than body volume. The drug must be extensively distributed into tissue, leaving low concentrations in the plasma, thus the body as a whole appears to have a large volume, of distribution. Remember, this is not a physiological volume.
From Equations 8 and 5 we can produce Equation 9:
Here are some Example Calculations* to try.
Clearance, CL
Clearance is the most important concept to be considered when a rational regiment for drug administration is to be designed. The clinician usually wants to maintain steady- state concentrations of a drug within a known therapeutic range. Assuming complete bioavailability, the steady state will be achieved when the rate of drug elimination equals the rate of drug administration in equation 10:
Dosing rate = CL*Css
where CL is clearance and Css is the steady-state concentration of the drug.
Clearance can be defined as the volume of plasma which is completely cleared of drug per unit time. The symbol is CL and the units are ml/min, L/hr, i.e. volume per time. Another way of looking at Clearance is to consider the drug being eliminated from the body ONLY via the kidneys. (If we were to also assume that all of the drug that reaches the kidneys is removed from the plasma then we have a situation where the clearance of the drug is equal to the plasma flow rate to the kidneys. All of the plasma reaching the kidneys would be cleared of drug.)
The amount cleared by the body per unit time is dU/dt, the rate of elimination (also the rate of excretion in this example). To calculate the volume which contains that amount we can divide by Cp. That is the volume = amount/concentration. Thus equation 11:
equation12 is
As we have defined the term here it is the total body clearance. We have considered that the drug is cleared totally by excretion in urine. Below we will see that the total body clearance can be divided into a clearance due to renal excretion and that due to metabolism.
Clearance is a useful term when talking of drug elimination since it can be related to the efficiency of the organs of elimination and blood flow to the organ of elimination. It is useful in investigating mechanisms of elimination and renal or hepatic function in cases of reduced clearance of test substances. Also the units of clearance, volume/time (e.g. ml/min) are easier to visualize, compared with elimination rate constant (units 1/time, e.g. 1/hr).
Total body clearance, CL, can be separated into clearance due to renal elimination, CLr and clearance due to metabolism, CLm.
CLr = ke * V (renal clearance) and CLm = km * V (metabolic clearance)
where these are combined to give equation 13:
CL = CLr + CLm
Equation 11 can be rearranged to get:
thus a plot of dU/dt versus Cp will give a straight line through the origin with a slope equal to the clearance, CL.
Pharmacokinetics of Oral Administration
Most commonly the absorption process of oral administration follows first order kinetics. Even though many oral dosage forms are solids which must dissolve before being absorbed the overall absorption process can often be considered to be a single first order process. At least that's the assumption we will use for now.
Schematically this model can be represented as:
Where Xg is the amount of drug to be absorbed, Xp is the amount of drug in the body, and ka is the first order absorption rate constant.
Differential Equation
The differential equation for Xg is Equation 14:
This is similar to the equation for after an IV injection.
Integrating this we get Equation 15:
Xg = Xg0 * e-ka * t = F * Dose * e-ka * t
where F is the fraction of the dose which is absorbed, the bioavailability.
For the amount of drug in the body Xp ( = V * Cp), the differential equation is Equation 16:
The first term of this equation is ka * Xg which is absorption, and the second term is kel * V * Cp which is elimination.
Even without integrating this equation we can get an idea of the plasma concentration time curve.
At the start Xg >> V * Cp therefore the value of is positive, the slope will be positive and Cp will increase. With increasing time Xg will decrease, while initially Cp is increasing, therefore there will be a time when ka * Xg = kel * V * Cp. At this time will be zero and there will be a peak in the plasma concentration. At even later times Xg --> 0, and will become negative and Cp will decrease.
Integrated equation
Now we will integrate the equation. Starting with the differential equation we can substitute Xg = Xg0 *e-ka * t.
If we use F * DOSE for Xg0 where F is the fraction of the dose absorbed, the integrated equation for Cp versus time is Equation 17:
Notice that the right hand side of this equation (Equation 17) is a constant multiplied by the difference of two exponential terms. A biexponential equation.
We can plot Cp as a constant times the difference between two exponential curves. If we plot each exponential separately we get the following:
Notice that the difference starts at zero, increases, and finally decreases again. By adding the two lines in the second plot we get the actual plasma concentration. Plotting this difference we get:.
We can calculate the plasma concentration at anytime if we know the values of all the parameters of Equation 17.
We can also calculate the time of peak concentration using the equation:
As an example we could calculate the peak plasma concentration given that F = 0.9, DOSE = 600 mg, ka = 1.0 hr-1, kel = 0.15 hr-1, and V = 30 liter.
= 2.23 hour
= 21.18 x [ 0.7157 - 0.1075] = 12.9 mg/L
As another example we could consider what would happen with ka = 0.2 hr-1 instead of 1.0 hr-1
= 5.75 hour
= 72 x (0.4221 - 0.3166) = 7.6 mg/L lower and slower than before.
Multiple doses
Using Equation 17 above we can create a spreadsheet that will describe the effects of multiple doses given at different time intervals (see plot below). Using a graph such as this, we can coordinate drug doses with proper time intervals in order to will keep drug concentrations at there optimum levels..
Plot of plasma concentration due to multiple oral doses of a hypothetical drug. The doses are 200 mg every 8 hours; the fraction absorbed was 1.00; the elimination constant is 0.2/hr; the absorption constant is 1.0/hr; and the volume concentration is 30 L. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Medicinal_Chemistry/Basic_Aspects_of_Drug_Activity.txt |
Cardiovascular disease constitutes the largest single cause of death in the industrialized countries. As with cancer, which is a distant second in terms of mortality, cardiovascular disease morbidity increases with age, accounting for about two-thirds of all deaths in persons over 75. Even though some diseases affect primarily the heart and other diseases effect the vascular system, they cannot be divorced from each other. This obvious interdependence makes a unified imperative. One of the major diseases, atherosclerosis, affects and ultimately damages the heart, kidneys, and other organs.
A. Antihypertensive Agents
Hypertension is generally defined as an elevation of systolic and/or arterial blood pressure and a value of 140/90 torr is generally accepted as the upper limit of normotension. Certain risk factors (e.g., hypercholesterolemia, diabetes, smoking, and a family history of vascular disease) in conjunction with hypertension predispose to arteriosclerosis and consequent cardiovascular morbidity. Patient populations with sustained diastolic blood pressures in the range of 105 to 129 torr are unequivocally benefited by effective reduction of blood pressure. The benefits of antihypertensive treatment are the avoidance of accelerated or malignant hypertension, a lower incidence of hypertensive renal failure, and a decrease in the incidence of hemorrhagic stroke and cardiac failure. Only recently has it been demonstrated that aggressive care of patients with mild diastolic hypertension can apparently reduce the incidence of myocardial infarction.
Inhibition of Sympathetic Function
b-blockers
Since they where introduced in the 1960s, b-adrenergic blocking agents have become the most commonly used drugs for cardiovascular diseases. Propranolol was the first b-blocker to come into wide clinical use, and it remains the most important of these compounds. It is a highly potent, nonselective adrenergic blocking agent with not intrinsic sympathomimetic activity. However, because of its ability to block b receptors in bronchial smooth muscle and skeletal muscle, propranolol interferes with bronchodiation produced by epinephrine and with glycogenolysis, which ordinarily occurs during hypoglycemia. Thus, the drug is usually not used in individuals with asthma and must be used cautiously in diabetics who are receiving insulin. As a consequence, there has been a search for b-blockers that are cardioselective and a number of drugs now have been developed that exhibit some degree of specificity for b1-adrenergic receptors. Metaprolol is a prototype for these more specific drugs.
Much of the pharmacology of b-blockers can be deduced from a knowledge of the functions subserved by the involved receptors and the physiological conditions under which they are activated. Thus, b-receptor blockade has little effect on the normal heart with the subject at complete rest, but may have profound effects when sympathetic control of the heart is high, as during exercise. b-blockers decrease heart rate and cardiac output, prolongs mechanical systole, and slightly decreases blood pressure in resting subjects. Peripheral resistance is increased as a result of compensatory sympathetic reflexes, and blood flow to all tissues except the brain is reduced. Some of these drugs also have direct actions on cell membranes, which are commonly described as membrane stabilizing and local anesthetic. The local anesthetic potency of propranolol is about equal to that of lidocaine.
b-blockers are effective antihypertensive agents. Chronic treatment of hypertensive patients with b-adrenergic blocking agents results in a slowly developing reduction in blood pressure. Several mechanisms have been proposed for the efficacy of b-blockers in the management of hypertension. Reduction in cardiac output occurs rather promptly after administration of b-blockers. However, the hypotensive effects of b-blockers does not appear as rapidly. The release of renin from the juxtaglumarular apparatus is stimulated by adrenergic agonists, and this effect is blocked by drugs such as propranolol. Also, b-adrenergic agonists are known to increase modestly the release of norepinephrine from adrenergic nerve terminals, which causes vasoconstriction. b-blockers block this effect, and such impairment of the release of norepinephrine following sympathetic nerve stimulation might contribute to the antihypertensive effects of the drugs.
a-blockers
a-Adrenergic blocking agents exist in most blood vessels, particularly in coetaneous resistance vessels. Since their stimulation leads to constriction and therefore blood pressure elevation, it stands to reason that blocking such stimulation would lead to a diminution of blood pressure. Phenoxybenzamine binds covalently to the a receptor and produces an irreversible type of blockade. Phentolamine and tolazoline bind reversibly and antagonize the actions of sympathomimetic amines competitively.
Reserpine
Rauwofia serpentina is a climbing shrub indigenous of India and neighboring countries. In 1954, it was reported that rauwolfia or reserpine was helpful in the treatment of psychotic patients because it acts centrally to produce characteristic sedation and a state of indifference to environmental stimuli; effects which resemble phenothiazines (discussed in Psychoactive Drugs). Subsequent discovery of the ability of rauwolfia alkaloids and related compounds to deplete biogenic amines from storage sites in the body initiated a great number of investigations directed at elucidating the interaction between these amines and reserpine. There are a number of rauwolfia alkaloids with complex structures. The structure of reserpine is as follows:
It is clear that reserpine interferes with intracellular storage of catecholamines, but the amounts of reserpine in tissues are much too small to assume a stoichiometric displacement. Reserpine antagonizes the uptake of norepinephrine by isolated chromaffin granules by inhibiting the ATP-Mg2+-dependent uptake mechanism of the granule membrane. The drug also binds to the vesicular membranes for days, accounting for the irreversibility of the process.
Reserpine depletes stores of catecholamines in many organs, including the brain and adrenal medulla, and most of its pharmacological effects have been attributed to this action. Since it is the reuptake and not the release of catecholamines that is inhibited, the existing pool must be fully depleted before antihypertensive effects become apparent. Also,, after administration of relatively large doses, reserpine causes a transient sympathomimetic effect followed by a slowly developing fall in blood pressure frequently associated with bradycardia. For normal doses, reduced concentrations of catecholamines can be measured within a hour after administration of reserpine, and depletion is maximal at 24 hours. Most of the catecholamine is deaminated intraneuronally, and pharmacological effects of the released mediator are minimal unless MAO has been inhibited.
The decrease in norepinephrine synthesis induced by reserpine is the block of dopamine uptake into storage granules that contain the enzyme dopamine b-hydroxylase (see norepinephrine synthesis). Furthermore, the increased concentration of free catecholamine presumably feeds back to inhibit tyrosine hydroxylase, since norepinephrine competes with the pterin cofactor for the enzyme.
Supersensitivity to catecholamines is observed following chronic administration of reserpine. The site of change is presumably postjunctional and may by due to alterations of the adrenergic receptors. Such adaptive change is usual following chronic deprivation of transmitter.
Reserpine is used as an effective antihypertensive agent, particularly when used with other agents such as diuretics. Its low cost, once-daily administration, and minimal change in effect when compliance is erratic make it useful as an agent for long-term treatment of patients with uncomplicated mild hypertension. However, reserpine causes mental depression in 25% of patients.
Diuretics
A diuretic is a substance that increases the rate of urine volume output, as the name implies. Most diuretics also increase urinary excretion of solutes, especially sodium and chloride. In fact, most diuretics that are used clinically act by decreasing the rate of sodium reabsorbtion from the nephron tubules, which in turn causes naturesis and this in turn causes diuresis. The most common clinical use of diuretics is to reduce extracellular fluid volume, especially in diseases associated with edema and hypertension.
Carbonic Anhydrase Inhibitors
Soon after the introduction of the sulfonamides as antibacterial agents in the 1930s, changes in the electrolyte balance of patients were noted as was systemic acidosis accompanied by an alkalization of the urine due to increased rate of HCO3- excretion. Proposals by several researchers established that inhibition of the enzyme carbonic anhydrase (CA) accounted for the electrolytic imbalances produced. Since the antibacterial sulfonamides were relatively weak inhibitors, a successful search for more potent CA inhibition ensued. Acetazolamide became the first successful drug introduced into clinical use.
Among the enormous number of sulfonamides that have been synthesized and tested, acetazolamide has been studied the most extensively as an inhibitor of carbonic anhydrase. The other drugs of this class that are available in the U.S. are dichlorphenamide and methazolamide. There structural formulas are as follows:
Mechanism of Action. The kidneys control acid-base balance of the body by excreting either an acidic or a basic urine. The overall mechanism by which the kidneys accomplish this is as follows: Large numbers of bicarbonate ions are filtered continuously into the nephron tubules of the kidneys, and if they are excreted into the urine, this removes base from the blood. On the other hand, large numbers of hydrogen ions are also secreted into the tubular lumen by the tubular epithelial cells, thus removing acid from the blood.
Bicarbonate ions enter the tubular lumen of the kidney nephron with the glumerular filtrate. Bicarbonate ions do not readily permeate the luminal membranes of the renal tubular cells; therefore, bicarbonate ions that are filtered by the glomerulus cannot be directly reabsorbed. Instead, bicarbonate is reabsorbed by a special process in which it first combines with hydrogen ions to from H2CO3, which eventually becomes CO2 and H2O.
\[\ce{ H^{+} + HCO3 <=> H2CO3 <=> CO2 + H2O}\]
This reabsorbption of bicarbonate ions is initiated by a reaction in the tubules between bicarbonate ions filtered at the glomerulus and hydrogen ions secreted by the tubular cells. The H2CO3 formed then dissociates into CO2 and H2O. The CO2 can move easily across the tubular membrane; therefore, it instantly diffuses into the tubular cell, where it recombines with H2O, under the influence of carbonic anhydrase, to generate new a H2CO3 molecule. This H2CO3 in turn dissociates to form bicarbonate ion and hydrogen ion. The hydrogen ions are secreted from the cell into the tubular lumen, by sodium-hydrogen pump, in exchange for sodium. The bicarbonate ions together with the exchanged Na+, then enter the peritubular blood supply. The hydrogen ion, now in the tubular lumen, combines with another bicarbonate ion to form H2CO3, which then again dehydrates to CO2, which reenters the tubular cell. The net result is reabsorbtion of most of the bicarbonate. Some of these concepts can be seen here in an animated nephron (you must have the shockwave plugin to view this). animated
A hydrogen-bonding mechanism that acts competitively explains the action of acetazolamide-like carbonic anhydrase inhibitors that have diuretic properties. Carbonic acid is the normal substrate that fits into a cavity of and complexes with the enzyme carbonic anhydrase. This complex is strongly stabilized by four hydrogen bonds (see figure below).
The acetazolamide-like drugs fit into the cavity of the enzyme also effectively bond, presumably to the same four areas by hydrogen bonds (see figure below).
Thus these sulfonamide agents competitively prevent the carbonic acid from binding at this site which inhibits the reabsorption of NaHCO3 and H20. More than 99% of the enzyme activity in the kidney must be inhibited before physiological effects become apparent.
Following the administration of acetazolamide, the urine volume promptly increases. The urinary excretion of bicarbonate and fixed cation, mostly sodium (also potassium) and the normally acidic pH becomes alkaline. As a result, the concentration of bicarbonate in the extracellular fluid decreases and metabolic acidosis results. The urinary concentration of chloride falls.
The presence of carbonic anhydrase in a number of intraocular structure, including the cilliary processes, and the high concentration of bicarbonate in the aqueous humor have focused attention on the role that the enzyme might play in the secretion of aqueous humor. acetazolamide reduces the rate of aqueous humor formation; intaocular pressure in patients with glaucoma is correspondingly reduced.
Benzothiadiazines
Thiazines and related compounds comprise the most frequently used antihypertensive agents in the United States. They were synthesized as an out growth of studies on carbonic anhydrase inhibitors. Thiazides act directly of the kidney to increase the excretion of sodium chloride and an accompanying volume of water. Also many of these drugs act as corbonic anhydrase inhibitors. At the molecular level, it is unknown how benzothiadiazines inhibit the reuptake of sodium chloride.
B. Antianginal Drugs: Coronary Vasodilators
Angina pectoris, or ischemic heart disease, is the name given to the symptomatic oppressive pain resulting from myocardial ischemia. In simplest terms it results when the oxygen demand of myocardial tissues exceeds the circulatory supply. Once a local anemia due to an obstruction exists, biochemical changes are inevitable. Metabolic products will accumulate in the area, contractility declines, and NE release occurs from sympathetic neurons. The end result is pain. The underlying pathology of typical angina is usually advanced atherosclerosis and stenosis of the coronary vasculature which causes localized oxygen starvation. Episodes are be precipitated by emotional stress or exercise, but they usually cease rapidly with rest or nitroglycerin. In contrast, variant angina is caused by vasospasm of the coronary vessel and may or may not be associated with severe atherosclerosis. Patients with variant angina develop chest pain while at rest.
Until recently, many of the drugs used to prevent anginal attacks were no more effective than a placebo. In fact. the use of placebos has been reported to relieve symptoms in as many as 50% of patients with angina pectoris. For over a century, however, nitroglycerin has been known to be useful to prevent or relieve acute anginal attacks. More recently, the efficacy of b-adrenergic antagonists has been established for the long-term prophylaxis of typical angina. In addition, the calcium channel blockers appear to be effective for the treatment of vasospastic angina. While antianginal agents provide only symptomatic treatment, administration of these drugs do appear to decrease the incidence of sudden death associated with myocardial ischemia and infarction.
Organic Nitrates
The organic nitrates and nitrites are dilators of arterial and venous smooth muscle. The venodilation results in decreased left and right ventricular end-diastolic pressure, which are greater on a percentage basis than is the decrease in systemic arterial pressure. Net systemic vascular resistance is usually relatively unaffected; heart rate is unchanged or slightly increased; and pulmonary vascular resistance is consistently reduced. These drugs correct the inadequacy of myocardial oxygenation by increasing the supply of oxygen to ischemic myocardium by direct dilatation of the coronary vasculature and by decreasing the oxygen demand for oxygen by a reduction in cardiac work. The latter results from the decrease in vascular pressure enabling the heart to pump blood easier.
Glyceryl Trinitrate
Glyceryl trinitrate, or nitroglycerine, is a dense sweet-smelling oil that is highly explosive. The most utilized dosage form of nitroglycerin has been the sublingual tablet. Buccal absorption is rapid, offering almost instantaneous relief of sufficient duration (<30 min) for the emergency. Nevertheless, because of the valuable properties of nitroglycerin is now known to have in angina pectoralis, continuous blood levels of the drug are desirable. Therefore, different and innovative dosage forms are being developed. Because nitroglycerin is efficiently absorbed through the skin, this has led to the introduction of nitroglycerin skin patches. These patches contain the drug in a form which results in its continuous release.
3-D
Mechanism of Action
Nitric oxide (NO) has been shown to be an important messenger in many signal transduction processes. This free radical gas is naturally produced endogenously from arginine in a complete reaction that is catalyzed by nitric oxide synthetase (NOS).
Nitroglycerin, nitrites, and organic nitrates also lead to the formation of the reactive free radical NO and is the basis of their mechanism of action. However, it is not known the exact enzyme that converts these drugs into NO.
Nitric oxide diffuses freely across membranes but has a short life, less than a few seconds, because it is highly reactive. Nitric oxide then activates a heme prosthetic group on the enzyme guanylate cyclase in the cell membrane. The heme molecule is, in effect, functioning as an extremely sensitive detector of NO. A portion of the enzyme protrudes to the interior of the cell and causes the formation of cyclic guanosine monophosphate (cGMP), a so-called second messenger. The cGMP in turn has many effects, one of which is to change the degree of phosphorylation of several enzymes that indirectly inhibit contraction. Especially, the pump that pumps calcium ions from the sarcoplasm into the sarcoplasmic reticulum in activated as well as the cell membrane pump that pumps calcium ions out of the cell itself; these effects reduce the intracellular calcium ion concentration, thereby inhibiting contraction.
Viagra
Viagra, an experimental heart medication with a great side effect.More on the Chemistry of Viagra
C. Digitalis
Digitalis is the dried leaf of the foxglove plant, Digitalis purpurea. Seeds and leaves of a number of other species also contain active cardiac principles. The two molecules in digitalis responsible for its pharmacological activity are digoxigenin and digitoxigenin. As you can see from the structures below, these genins are chemically related to sex and adrenocortical hormones.
Digitalis has a powerful action of the myocardium that is unrivaled in value for the treatment of heart failure. It is also used to slow the ventricular rate in the presence of atrial fibrillation and flutter. The main pharmacodynamic property of digitalis is its ability to increase the force of myocardial contraction. The beneficial effects of the drug in patients with heart failure - increased cardiac output; decreased heart size. venous pressure, and blood volume; diuresis and relief of edema - are all explained on the basis of increased contractile force, a positive inotropic action.
The most important component to the positive inotropic effect of digitalis direct inhibition of the membrane-bound Na+, K+-activated ATPase, which leads to an increase in intracellular [Ca+]. It seems clear that digitalis, in therapeutic concentrations, exerts no direct effect on the contractile proteins or on the interactions between them. Also, it seems most unlikely that the positive inotropic effect of digitalis is due to any action of the cellular mechanisms that provide the chemical energy for contraction. The hydrolysis of ATP by the Na+, K+-ATPase provides the energy for the so-called sodium-potassium pump - the system in the sarcolemma of cardiac fibers that actively extrudes sodium and transports potassium into the fibers. Digitalis drugs bind specifically to the Na+, K+-ATPase, inhibit its enzymatic activity, and impair the active transport of these two monovalent cations. As a result, there is a gradual increase in intracellular [Na+] and a gradual decrease in internal [K+]. These changes are small at therapeutic concentrations of the drug. It is judged to be crucially related to the positive inotropic effect of digitalis. This is so because in cardiac fibers, intracellular Ca2+ is exchanged for extracellular Na+ by a different transmembrane pump. When internal [Na+] is increased because of inhibition of the Na+, K+ pump by digitalis, the exchange of extracellular Na+ for intracellular Ca2+ is diminished, and internal [Ca2+] increases. The probable consequence of this is an increased store of Ca2+ in the sarcoplasmic reticulum and, with each action potential, a greater release of Ca2+ to activate the contractible apparatus.
Interactions of Na+, K+-ATPase with its various substrates are complex. Thus binding affinities with ATP, cofactor MG2+, Na+, K+, and a digitalis glycoside are all important to the overall effect. It is now accepted that the digitalis receptor is one or more of the conformations of Na+, K+-ATPase that occur during the ion pump's operation, possibly during a state in which the drug helps to stabilize one of the intermediate states of the enzyme.
Evidence suggests that the entire glycoside molecule participates in the proposed drug-receptor interaction. The steric relationship of the lactone ring to the steroid nucleus is absolute. The double bond is also critical since saturation results in an almost total loss of activity. The required stereochemical positioning of rings C and D in relation to each other (cis) and of A and B (cis), and the configuration of the OH at C-14 (between rings C and D) have all been established. The figure below is a highly simplified version of a proposed interaction of the lactone side chain with such a digitalis receptor.
It is believed that the polarized carbonyl group, with its electron-rich oxygen, hydrogen bonds to a hydroxyl group on the enzyme's surface, probably to a serine residue, and the carbon atom bonded to the steroid nucleus is attracted to an electron dense site at a secondary location.More Info on Digitalis
D. Anticoagulants
From any viewpoint blood is chemically the most complex tissue in the body. In addition to the multitude of cells and platelets, it contains inorganic ions, various plasma proteins, hormones, lipids, vitamins, a large variety of enzymes, nucleic acid breakdown products, a large number of unknown types of environmentally ingested chemical at varying stages of metabolic conversion, gases, and water. Among these is a group of more than a dozen chemical factors that will cause the blood to coagulate when properly triggered.
Hemostasis is the spontaneous arrest of bleeding from damaged vessels. Precapillary vessels contract immediately when cut. Within seconds, thrombocytes are bound to the exposed collagen of the injured vessel. Platelets also stick to each other and a viscous mass is formed. This platelet plug can stop bleeding quickly, but it must be reinforced by fibrin for long-term effectiveness. This reinforcement is initiated by the local stimulation of the coagulation process by the expose collagen of the cut vessel and the released contents and membranes of platelets. The two pathways of blood coagulation are shown in the figure below. The circled clotting factors are dependent on vitamin K for their activation.
Thrombogenesis is an altered state of hemostasis. An intravascular thrombus results from a pathological disturbance of hemostasis. The arterial thrombus is initiated by the adhesion and the release of circulating platelets to a vessel wall. This initial adhesion and the release of adenosine diphosphate (ADP) from platelets is followed by platelet-platelet aggregation. The thrombus grows to occlusive proportions in the areas of slower arterial blood flow. A platelet plug formed solely by platelet interaction is unstable. After the initial aggregation and viscous metamorphosis of platelets, fibrin becomes an important constituent of a thrombus. Production of thrombin occurs by activation of the reactions of blood coagulation at the site of the platelet mass. This thrombin stimulates further platelet aggregation not only by inducting the release of mote ADP from the platelets but also by stimulating the synthesis of prostaglandins, which are more powerful than ADP.
Dicumarol
The first orally active anticoagulant, dicoumerol, which is a molecule consisting of two 4-hydroxycoumarin moieties bonded at their 3-position via a CH2 bridge, was isolated from decomposed yellow sweet clover. It was discovered and identified because of hemorrhagic death of cattle ingesting this improperly stored feed in the early 1920s (sweet clover disease). This followed demonstration that oral use of this compound increased clotting time and decreased the incidence of post surgical intravascular thrombus formation.
The oral anticoagulants are antagonists of vitamin K. Their administration to man or other animals leads to the appearance of precursors of the four vitamin K-dependent clotting factors in plasma and liver. These precursor proteins are biologically inactive in tests of coagulation. The precursor protein to prothrombin can be activated to thrombin nonphysiologically by snake venoms, demonstrating that the portion of the molecule necessary for this activity is intact. However, the prothrombin and other precursor proteins cannot bind divalent cations such as calcium and thus cannot interact with phospholipid-containing membranes, which are their normal sites of activation. This was puzzling for some time because abnormal prothrombin has the same number to amino acid residues and gives the same amino acid analysis after acid hydrolysis as does normal prothrombin. NMR studies revealed that normal prothrombin contains g-carboxyglutamate (see figure below), a previously unknown residue. The vitamin K-sensitive step in the synthesis of clotting factors is the carboxylation of ten or more glutamic acid residues at the amino-terminal end of the precursor protein to form g-carboxyglutamate. These amino acid residues are much stronger chelators of calcium than glutamate. The binding of Ca2+ by prothrombin anchors it to phospholipid membranes derived from blood platelets following injury. Th functional significance of the binding of prothrombin to phospholipid surfaces is that it brings prothrombin into close proximity with two proteins that mediate its conversion into thrombin-Factor Xa and Factor V. The amino-terminal fragment of prothrombin, which contains the Ca2+-binding sites, is released in this activation step. Thrombin free in this way from the phospholid surface can then activate fibrinogen in the plasma.
In hepatic microsomes, the reduction of vitamin K to its hydroquinone form (vitamin KH2) precedes the bicarbonate-dependent carboxylation of precursor prothrombin, descarboxyprothrombin, to prothrombin (see figure below). This carboxylase activity for the synthesis of prothrombin is linked to an epoxidase activity for vitamin KH2, which oxidizes the vitamin to vitamin K epoxide (KO). An epoxide reductase, which requires NADH, converts vitamin K epoxide back to vitamin KH2. This reaction is the site of action of the coumarins and the site of genetic resistance to the coumarins, which is also characterized by an increase requirement for vitamin K. Thus it is the recycling of the vitamin to its reduced active cofactor that results ultimately in decreased thrombin levels. The vitamin K analog chloro-K1 (in which the 2-methyl group of vitamin K1 is replaced by a chloro group) directly inhibits the carboxylase and epoxidase reaction, which are not sensitive to coumarins.
Vitamin K is reduced to the hydroquinone form (KH2). Stepwise oxidation to vitamin K epoxide (KO) is coupled to protein carboxylation, wherein descarboxyprothrombin is converted to prothrombin by carboxylation of glutamate residues to g-carboxyglutamate. Enzymatic reduction of the epoxide with NADH as a cofactor regenerates vitamin KH2. The oxidation of vitamin KH2. The oxidation of vitamin K is inhibited by the chloro analog of vitamin K epoxide is the coumarin-sensitive step.
Dicoumerol, the prototype of coumarin drugs, is of relatively low potency, with a slow onset of up to 5 days for peak activity and hypoprothrombinemia. The anticoagulant effect may persist for more than 1 week after stopping the drug. Even though over doses can be antidoted with vitamin K1, clinical adjustment of anticoagulation, particularly downward, is difficult. Warfarin has become the most widely used of the coumarin drugs. It is the most potent, with many patient being maintained on only 5 mg/day
Warfarin was initially introduced as a rodenticide because it was thought too dangerous for human use. It is still used in pest control. The drug, as sweetened pellets, causes rats to die from internal bleeding. In the early 1960s rats resistant to warfarin were noted in London. They were nicknamed "super rats." Several years later this phenomenon appeared in humans. It has been shown to be an inherited autosomal dominant trait. Person with this trait require a 20-fold increase in the drug to achieve anticoagulation-easily fatal to normal patients. Explanations for this unusual phenomenon have been proposed. One is that a tissue protein regulating the synthesis of one or more of the clotting factors became genetically altered. Another is a mutation in the enzyme daphorase that makes it less susceptible to coumarin drug inhibition.
Questions
1. What factors affect cardiac O2 supply and demand?
2. What are the symptoms and etiology of angina pectoris?
3. Both the nitrates and beta-adrenergic blocking agents have antianginal properties. Compare and contrast the pharmacologic effects of these two classes of compounds.
4. What are the sites of action of the nitrates?
5. When prescribing nitrates what would you advise a patient concerning adverse effects?
6. What advantages do the calcium-channel blockers have in the treatment of angina? What is their mechanism of action? What arecontraindications to their use?
7. What are the two major objectives of drug therapy in angina pectoris?
8. What is the mechanism of action of nitroglycerine? | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Medicinal_Chemistry/Cardiovascular_Drugs.txt |
Neurotransmitters released from nerve terminals bind to specific receptors, which are specialized macromolecules embedded in the cell membrane. The binding action initiates a series of specific biochemical reactions in the target cell that produce a physiological response. These effects can be modified by various drugs that act as agonists or antagonists.
The autonomic system consists of two major divisions: the Sympathetic Nervous System and the Parasympathetic Nervous System. These often function in antagonistic ways. A signal is transmitted from the spinal cord to peripheral areas through two successive neurons. The first neuron (preganglionic), which originates in the spinal cord, will synapse with the second neuron (postganglionic) in a ganglion. Parasympathetic ganglia tend to lie close to or within the organs or tissues that their neurons innervate, whereas sympathetic ganglia lie at a more distant site from their target organs. Both systems have associated sensory fibers that send feedback information into the central nervous system regarding the functional condition of target tissues.
The significant difference between the two systems is that their postganglionic fibers secrete different neurotransmitters. Those of the parasympathetic system secrete acetylcholine (ACh), hence the name cholinergic, whereas the postganglionic fibers secrete norepinephrine (NE), hence the name adrenergic. The preganglionic fibers of both systems secrete ACh; therefore, both preganglionic fibers are cholinergic. Motor neurons which are not part of the autonomic nervous system also release acetylcholine (see Figure 1).
Figure 1. (a) Preganglionic neurons (solid line) of the sympathitic division of the autonomic nervous system release acetycholine at their synapses with postganglionic neurons (dashed line). Although exceptions occur, the postganglionic neurons release mainly norepinephrine at their function with effectors. (b) Pregangionic neurons (solid line) of the parasympathetic division of the autonomic nervous system release acetycholine at their synapses with postganglionic neurons (dashed line), and the postgangionic neurons also release acetycholine at their effectors. (c) Somatic efferent neurons release acetylcholine at their junctions with skeletal muscles.S.K.Anderson
Acetylcholine acts on more than one type of receptor. Henry Dale, a British physiologist working in London in 1914, found that two foreign substances, nicotine and muscarine, could each mimic some, but not all, of the parasympathetic effects of acetylcholine. It was found that Nicotine stimulates receptors on skeletal muscle and sympathetic and parasympathetic postganglionic neurons, however, muscarine stimulates receptor sites located only at the junction between postganglionic parasympathetic neurons and the target organ. Dale therefore classified the many actions of acetylcholine into nicotinic effects and muscarinic effects. It has subsequently become clear that there are two distinct types of acetylcholine receptors affected by either muscarine or nicotine. To restate this again, nicotinic receptors cause sympathetic postganglionic neurons and parasympathetic postganglionic neurons to fire and release their chemicals and skeletal muscle to contract. Muscarinic receptors are associated mainly with parasympathetic functions and stimulates receptors located in peripheral tissues (e.g., glands, smooth muscle). Acetylcholine activates all of these sites.
Advanced biochemical techniques have now shown a more fundamental difference in the two types of cholinergic receptors. The nicotinic receptor is a channel protein that, upon binding by acetylcholine, opens to allow diffusion of cations. The muscarinic receptor, on the other hand, is a membrane protein; upon stimulation by neurotransmitter, it causes the opening of ion channels indirectly, through a second messenger. For this reason, the action of a muscarinic synapse is relatively slow. Muscarinic receptors predominate at higher levels of the central nervous system, while nicotinic receptors, which are much faster acting, are more prevalent at neurons of the spinal cord and at neuromuscular junctions in skeletal muscle.
A cholinergic drug is any of various drugs that inhibit, enhance, or mimic the action of the neurotransmitter acetylcholine within the body. Acetylcholine stimulation of the parasympathetic nervous system helps contract smooth muscles, dilate blood vessels, increase secretions, and slow the heart rate. Some cholinergic drugs, such as muscarine, pilocarpine, and arecoline, mimic the activity of acetylcholine in stimulating the parasympathetic nervous system. These drugs, however, have few therapeutic uses. Other cholinergic drugs, such as atropine and scopolamine, inhibit the action of acetylcholine and thus suppress all the actions of the parasympathetic nervous system. These drugs help dry up such bodily secretions as saliva and mucus and relax smooth-muscle walls. They are used therapeutically to relieve spasms of the smooth-muscle walls of the intestines, to relieve bronchial spasms, to diminish salivation and bronchial secretions during anesthesia, and to dilate the pupil during ophthalmological procedures.
Nicotine
Nicotine is an organic compound that is the principal alkaloid of tobacco. Nicotine occurs throughout the tobacco plant and especially in the leaves. The compound constitutes about 5 percent of the plant by weight. Both the tobacco plant (Nicotiana tabacum) and the compound are named for Jean Nicot, a French ambassador to Portugal, who sent tobacco seeds to Paris in 1550.
Crude nicotine was known by 1571, and the compound was obtained in purified form in 1828; the correct molecular formula was established in 1843, and the first laboratory synthesis was reported in 1904. Nicotine is one of the few liquid alkaloids. In its pure state it is a colorless, volatile base (pKa -8.5) with an oily consistency, but when exposed to light or air, it acquires a brown color and gives off a strong odor of tobacco.
The complex and often unpredictable changes that occur in the body after administration of nicotine are due not only to its actions on a variety of neuroeffector and chemosensitive sites but also to the fact that the alkaloid has both stimulant and depressant phases of action. The ultimate response of any one system represents the summation of the several different and opposing effects of nicotine. For example, the drug can increase the heart rate by excitation of sympathetic cardiac ganglia, and it can slow down the heart rate by stimulation of parasympathetic cardiac ganglia. In addition, the effects of the drug on the chemoreceptors of the carotid and aortic bodies and on medullary centers influence heart rate, as do also the cardiovascular compensatory reflexes resulting from changes in blood pressure caused by nicotine. Finally, nicotine causes a discharge of epinephrine from the adrenal medulla, and this hormone accelerates cardiac rate and raises blood pressure.
Nicotine is unique in its biphasic effects. In the medulla, small doses of nicotine evoke the discharge of catacholamines, and in larger doses prevent their release in response to splanic nerve stimulation. Its biphasic effect causes a stimulant effect when inhaled in short puffs, but when smoked in deep drags it can have a tranquilizing effect. This is why smoking can feel invigorating at some times and can seem to block stressful stimuli at others.
Nicotine markedly stimulates the central nervous system (CNS). Appropriate doses produce tremors in both man and laboratory animals; with somewhat larger dose, the tremor is followed by convulsions. The excitation of respiration is a prominent action of nicotine; although large doses act directly on the medulla oblongata, smaller doses augment respiration reflexly by excitation of the chemoreceptors of the carotid and aortic bodies. Stimulation of the CNA is followed by depression, and death usually results from failure of respiration due to both central analysis and peripheral blockade of muscles of respiration. Nicotine also causes vomiting by central and peripheral actions. The central component of the vomiting response is due to stimulation of the chemoreceptor trigger zone is in the medulla.oblongata. In addition, nicotine activates vagal and spinal afferent nerves that from the sensory input of the reflex pathways involved in the act of vomiting.
Although acetylcholine causes vasodilation and a decrease in heart rate, when administered intravenously to the dog, nicotine characteristically produces an increase in heart rate and blood pressure. This is because in general, the cardiovascular responses to nicotine are due to stimulation of the sympathetic ganglia and the adrenal medulla, together with the discharge of catacholamines from sympathetic nerve endings.
Nicotine is commercially obtained from tobacco scraps and is used as an insecticide and as a veterinary vermifuge (wormer). Nitric acid or other oxidizing agents convert nicotine to nicotinic acid, or niacin, which is used as a food supplement.
Medicinal Uses
Tabacco
Tabacco Info
Tabacco Death
Cigarettes - Tar, Nicotine, and Carbon Monoxide Content
Nicotine Addiction
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How deadly is nicotine? (don't take this site too seriously)
Nicotine Dosages
Muscarine
Muscarine, and alkaloid obtained from the poisonous mushroom Amanita Muscaria, produces the effects predictable from stimulation of postgangiolinc parasympathetic fibers. The symptoms usually occur within 15-30 minutes of ingestion or injection, and are focused on the involuntary nervous system. The muscarinic alkaloids stimulate the smooth muscle and therby increase motility; large doses cause spasm and severe diarrhea. The bronchial musculature is also stimulated, causing asmatic-like attacks. Excessive salivation, sweating, tears, lactation (in pregnant women), plus severe vomiting also occur. The most prominent cardiovascular effects are the a marked fall in the blood pressure and a slowing or temporarily cessation of the heart. Victims normally recover within 24 hours, but severe cases may result in death due to respiratory failure. All effects of muscarine-like drugs are prevented by the alkaloid atropine. Furthermore, neither atropine-like nor muscarine-like drugs show effects at the neuromuscular junction.
Although muscarine and muscarine like alkaloids are of great value as pharmacological tools, present clinical use is largely restricted. Since evidence is beginning to accumulate that there are distinct subtypes of muscarinic receptors, there has been a renewed interest in synthetic analogs that may enhance the tissue selectivity of muscarinic agonists. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Medicinal_Chemistry/Cholinergic_Drugs_I_-_Nicotinic_and_Muscarinic_Receptors.txt |
Anticholinesterase Agents
Acetylcholine is inactivated by the enzyme acetylcholinesterase (enlarged), which is located at cholinergic synapses and breaks down the acetylcholine molecule into choline and acetate. Three particularly well-known drugs, neostigmine, physostigmine, and diisopropyl fluorophosphate, inactivate acetylcholinesterase so that it cannot hydrolyze the acetylcholine released at the nerve ending. As a result, acetylcholine increases in quantity with successive nerve impulses so that large amounts of acetylcholine can accumulate and repetitively stimulate receptors. In view of the widespread distribution of cholinergic neurons, it is not surprising that the anticholinesterase agents as a group have received more extensive application as toxic agents, in the form of agricultural insecticides and potential chemical-warfare "nerve gases," than as therapeutic agents. Nevertheless, several members of this class of compounds are clinically useful.
The active center of acetylcholinesterase consists of a negative subsite, which attracts the quaternary group of choline through both coulombic and hydrophobic forces, and an esteratic subsite, where nucleophilic attack occurs on the acyl carbon of the substrate. The catalytic mechanism resembles that of other serine esterases, where a serine hydroxyl group is rendered highly nucleophilic through a charge-relay system involving the close apposition of an imidazole group and , presumably, a carboxyl group on the enzyme. During enzymatic attack on the ester, a tetrahedral intermediate between enzyme and ester is formed that collapses to an acetyl enzyme conjugate with the concomitant release of choline. The acetyl enzyme is labile to hydrolysis, which results in the formation of acetate and active enzyme. Acetylcholinesterase is one of the most efficient enzymes known and has the capacity to hydrolyze $3 \times 10^5$ acetylcholine molecules per molecule of enzyme per minute: this is equivalent to a turnover time of 150 microseconds.
Drugs such as neostigmine and pydrostigmine that have a carbamyl ester linkage are hydrolyzed by acetylcholinesterase, but much more slowly than acetylcholine because they from more stable intermediates. Therefore, these drugs inactivate acetylcholinesterase for up to several hours, after which they are displaced so that acetylcholinesterase. These drugs are used for their effects on skeletal muscle and the eye (pupillary constriction and decreased intraocular pressure) and for the treatment of atropine poisoning. On the other hand, diisopropyl fluorophosphate, which has military potential as a powerful nerve gas poison, inactivates acetylcholinesterase for weeks, which makes this a particularly lethal poison.
Generally the pharmacological properties of anticholinesterase agents can be predicted merely by knowing those loci where acetylcholine is released physiologically by nerve impulses. and the responses of the corresponding effector organs to acetylcholine. While this is true of the main, the diverse locations of cholinergic synapses increase the complexity of the response. Potentially, the antcholiesterase agents can produce all of the following effects: 1) stimulation of muscarinic receptor responses at autonomic effector organs; 2) stimulation, followed by paralysis, of all autonomic ganglia and skeletal muscle; and 3) stimulation, with occasional subsequent depression, of cholinergic receptor sites in the CNS. However, with smaller doses, particularly those employed therapeutically, several modifying factors are significant.
Neostigmine and Pydrostigmine
Neostigmine and pydrostigmine are among the principal anticholinesterases. These drugs have only a few clinical uses, mainly in augmenting gastric and intestinal contractions (in treatment of obstructions of the digestive tract), in generally augmenting muscular contractions (in the treatment of myasthenia gravis), and in constricting the eye pupils (in the treatment of glaucoma). Other anticholinesterases in larger doses, however, are widely used as toxins that achieve their effects by causing a continual stimulation of the parasympathetic nervous system. Parathion and malathion are thus highly effective agricultural insecticides, while tabun and serin are nerve gases used in chemical warfare to induce nausea, vomiting, convulsions, and death in humans. This action produces a decrease in the rate of destruction of acetylcholine in the synaptic cleft and hence an increase in the amount of transmitter available to interact with the receptors.
Serin Nerve Gas
Acetylcholine Antagonists
Atropine, a naturally occurring alkaloid of "Atropa belladonna", the deadly nightshade, inhibits the actions of acetylcholine on autonomic effectors innervated by postganglionic cholinergic nerves as well as on smooth muscles that lack cholinergic innervation. Since atropine antagonizes the muscarinic actions of acetylcholine, it is known as an antimuscarinic agent. Evidence shows that atropine and related compounds compete with muscarinic agonists for identical binding sites on muscarinic receptors.
In general, antimuscarinic agents have only a moderate effect on the actions of acetylcholine at nicotinic receptor sites. Thus, at autonomic ganglia, where transmission primarily involves an action of acetylcholine on nicotinic receptors, atropine produces only partial block. At the neuomuscular junction, where the receptors are exclusively nicotinic, extremely high doses of atropine or related drugs are required to cause any degree of blockade. In the central nervous system, cholinergic transmission appears to be predominantly nicotinic in the spinal chord and both nicotinic and muscarinic in the brain. Many of the CNS effects of atropine-like drugs are probably attributable to their central antimuscarinic actions.
Atropine
When used as premedication for anaesthesia, atropine decreases bronchial and salivary secretions, blocks the bradycardia associated with some drugs used in anaesthesia such as halothane, suxamethonium and neostigmine, and also helps prevent bradycardia from excessive vagal stimulation.
There is usually an increase in heart rate and sometimes a tachycardia as well as inhibition of secretions (causing a dry mouth) and relaxation of smooth muscle in the gut, urinary tract and biliary tree. Since atropine crosses the blood brain barrier CNS effects in the elderly may include amnesia, confusion and excitation. Pupillary dilatation and paralysis of accommodation occur, with an increase in intraocular pressure especially in patients with glaucoma. Occasionally small intravenous doses may be accompanied by slowing of the heart rate due to a central effect - this resolves with an extra increment of intravenous atropine Being a sympathetic cholinergic blocking agent, signs of parasympathetic block may occur such as dryness of the mouth, blurred vision, increased intraocular tension and urinary retention.
Atropine Nerve Gas Treatment
Sarin is a nerve agent in the organophosphate family. It is dispersed in a droplet or mist form. Upon inhalation, for instance, the symptoms (in order of occurrence) include: runny nose, bronchial secretions, tightness in the chest, dimming of vision, pin-point pupils, drooling, excessive perspiration, nausea, vomiting, involuntary defecationand urination, muscle tremors, convulsions, coma, and death. Primary treatment for nerve agents is atropine sulfate. It is commonly carried in auto-injectors by military personnel in dosages of 1-2 mgs. However, in many cases, massive doses may be necessary to reverse the effects of the anticholinesterase agents. Frequently, 20-40 mgs. of atropine may be necessary.
Picture of Auto-Injector
Curare
In 1799 the famous Prussian explorer and scientist Baron Von Humboldt discovered a potent drug called curare. On an expedition into the jungles of Venezuela, he watched an Indian hunter bring down a large animal with a single shot from his bow and arrow. The arrow had been poisoned with curare, a potion with two curious properties, derived from the jungle plants. Curare injected into the bloodstream, as it was when hunting animals, was deadly. It immobilized the body, attacked the vital organs, and caused death almost instantaneously. Humboldt discovered the second property of curare in a more dramatic fashion. He became sick, and a native witch doctor forced Humboldt to drink some curare that had been diluted with water. Terrified that he was going to die, Humboldt was surprised to find that after drinking the curare, he felt significantly better. Curare, when it was diluted and taken orally, he discovered, could have a positive medicinal value without causing any damage to vital organs.
Curare is a generic term for various South American arrow poisons. The main active ingredient in curare is d-tubocurarine, which has the chemical structure shown below.
In brief, d-tubocurarine is an antagonist of the cholinergic receptor sites at the post junctional membrane and thereby blocks competitively the transmitter action of acetylcholine. When the drug is applied directly to the end-plate of a single isolated muscle fiber under microscopic control, the muscle cell becomes insensitive to motor-nerve impulses and to direct applied acetylcholine; however, the end -plate region and the remainder of the muscle fiber membrane retain their normal sensitivity to the application of potassium ions, and the muscle fiber still responds to direct electrical stimulation. Because acetylcholine release into the neuromuscular junction muscle is what initiates contraction, curare causes muscle relaxation and paralysis.
There are several clinical application for neuromuscular blockage. The most important by far is the induction of muscle relaxation during anesthesia for effective surgery. Without such drugs deeper anesthesia, requiring more anesthetic, would be needed to achieve the same degree of muscle relaxation: tracheal intubation would also be impossible because of strong reflex response to tube insertion. It is the decreased need for anesthetics, however, that represents increased surgical safety.
Neuromuscular blockers also find limited utility in convulsive situations such as those precipitated by tetanus infections and to minimize injury to patients undergoing electroconvulsive therapy. Manipulation of fractured or dislocated bones may also be aided by such drugs.
Botulus Toxin
Botulus toxin* is a bacterial poison that prevents the release of acetylcholine by all types of cholinergic nerve terminals. Apparently, the toxin blocks release of vesicular acetylcholine at the preterminal portion of the axon, but why this is confined to cholinergic fibers is not known.
GABA
In most instances of natural neuron inhibition GABA is the inhibitory transmitter. GABA has the specific effect of opening anion channels in nerves, allowing large numbers of chloride ions to diffuse into the terminal fibril. the negative charges of these ions cancel much of the excitatory effect of the positively charged sodium ions that enter the terminal fibril when as action potential arrives. The cell is thus become hyperpolarized and the action potential in these neuron fibrils becomes greatly reduced. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Medicinal_Chemistry/Colinergic_Drugs_II_-_Anticholinesterase_Agents_and_Acetylcholine_Antagonists.txt |
A. Sedatives and Hypnotics
A sedative drug decreases activity, moderates excitement, and calms the recipient. A hypnotic drug produces drowsiness and facilitates the onset and maintenance of a state of sleep that resembles natural sleep in its electrocephalographic characteristics and from which the recipient may be easily aroused; the effect is sometimes called hypnosis. Sedation, pharmacological hypnosis, and general anesthesia are usually regarded as only increasing depths of a continuum of central nervous system (CNS) depression. Indeed, most sedative or hypnotic drugs, when used in high doses, can induce general anesthesia. One important exception is the benzodiazepines
Benzodiazepines
The term benzodiazepine refers to the portion of the structure composed of a benzene ring (A) fused to a seven-membered diazepine ring (B). However, since all of the important benzodiazepines contain a aryl substituent (ring C) and a 1, 4-diazepine ring, the term has come to mean the aryl-1,4-benzodiazepines. There are several useful benzodiazepines available. The skeletal structure and two examples are shown below.
.3-D
The effects of the benzodiazepines virtually all result from action of these drugs on the CNS, even when lethal doses are used. The most prominent of these effects are sedation, hypnosis, decreased anxiety, muscle relaxation, and anticonvulsant activity. As the dose of a benzodiazepine is increased, sedation progresses to hypnosis and hypnosis to stupor. They are used as sedatives, hypnotics, antianxiety agents (in panic disorder), anticonvulsants, muscle relaxants, in anesthesia and in alcoholism.
The actions of benzodiazepines are a result of potentiation of neural inhibition that is mediated by gamma-aminobutyric acid (GABA). This view is supported by behavioral and electrophysiological evidence that the effects of benzodiazepines are reduced or prevented by prior treatment with antagonists of GABA or inhibitors of the synthesis of the transmitter. Benzodiazepine receptors are located on the alpha subunit of the GABA receptor (see figure below) located almost exclusively on postsynaptic nerve endings in the CNS (especially cerebral cortex). Benzodiazepines enhance the GABA transmitter in the opening of postsynaptic chloride channels which leads to hyperpolarization of cell membranes. That is, they "bend" the receptor slightly so that GABA molecules attach to and activate their receptors more effectively and more often.
The remarkable safety of the benodiazepines can be accounted for by the self-limited nature of neuronal depression that requires the release of an endogenous inhibitory neurotransmitter to be expressed. That is, they do not directly act to open chloride ion channels, and therefore, are not lethal in over dose as are barbiturates.
Benzodiazepines are highly lipid soluble. There is rapid diffusion into the CNS followed by redistribution to inactive tissue sites. Benzodiazepines have a high volume of distribution and rapidly cross the placenta. The duration of action is determined by rate of metabolism and elimination. Diazepam is not water soluble and is dissolved in propylene glycol; therefore it may cause venous irritation and thrombophlebitis. Diazepam also has unpredictable absorption after IM injection. Benzodiazepines are extensively bound to albumin.
Valium Ads 1, 2, 3
Barbiturates
The barbiturates once enjoyed a long period of extensive use as sedative-hypnotic drugs; however, except for a few specialized uses, they have been largely replaced by the much safer benzodiazepines.
Barbiturates are CNS depressants and are similar, in many ways, to the depressant effects of alcohol. To date, there are about 2,500 derivatives of barbituric acid of which only 15 are used medically. The first barbiturate was synthesized from barbituric acid in 1864. The original use of barbiturates was to replace drugs such as opiates, bromides, and alcohol to induce sleep. Barbiturates are broken down chemically within the liver and eliminated via the kidneys at different rates according to their type. With regular use, the body develops a tolerance to barbiturates that translates into a need for larger and more frequent doses to attain the desired affect. However, while the tolerance increases in terms of realizing a desired effect, tolerance to the lethal level does not.
In general, structural changes that increase lipid solubility decrease duration of action, decrease latency to onset of activity, accelerate metabolic degradation, and increase hypnotic potency. Thus, large aliphatic groups at R2 confer greater activity than do methyl groups, but the compounds have a shorter duration of action; however, groups longer than seven carbons tend to have convulsive activity. Introduction of polar groups, such as ether, keto, hydroxyl, amino, or carboxyl groups, into alkyl side chains decreases lipid solubility and abolishes hypnotic activity.
Barbiturates facilitate GABA-ergic inhibition in a way that resembles some of the actions of the benzodiazepines, discussed above. However, barbiturates do not displace benzodiazepines from their binding sites. Instead, they enhance such binding by increasing the affinity for benzodiazepines; they also enhance the binding of GABA and its agonist analogs to specific sites in neural membranes. These effects are almost completely dependent upon the presence of chloride or other anions that are known to permeate through chloride channels and they are completely inhibited by picritoxin. (The picrotoxin group of toxins are naturally-occurring GABA antagonists which can cause death due to convulsions.)
While both barbiturates and benzodiazepines are capable of potentiating GABA-induced increases in chloride conductance, significant differences in their modes of action can be detected. Pentobarbital appears to increase the lifetime of the open state of the chloride channels that are regulated by GABA-ergic receptors; the magnitude of this effect more than offsets a barbiturate-induced decrease in the frequency of channel openings. By contrast, high concentrations of diazepam increase the frequency of channel openings with little effect on the lifetime of the open state. It is thought that barbiturates prolong the activation of the channel by decreasing the rate of dissociation of GABA from its receptor.
B. Antipsychotic Drugs
Schizophrenia comes in many varieties. One of the most common types is seen in the person who hears voices and has delusions of grandeur, intense fear, or other types of feelings that are unreal. Many schizophrenics are highly paranoid, with a sense of persecution from outside sources.
Schizophrenia appears to result from excessive excitement of a group of neurons that secrete dopamine in the behavioral centers of the brain, including in the frontal lobes. An alternative possibility is either hypersensitive or excess D2 dopamine receptors. Therefore, drugs used to treat this disorder decrease the level of dopamine excreted from these neurons or antagonize dopamine.
Dopamine has been implicated as a cause of schizophrenia because many patients with Parkinson's disease develop schizophrenic-like symptoms when they are treated with the drug L-DOPA. Also, drugs known to enhance central dopamine activity can worsen symptoms and even produce psychotic-like signs in normal individuals. It has been suggested that in schizophrenia, excess dopamine is secreted by a group of dopamine-secreting neurons whose cell bodies lie in the mesencephalon, medial to the substantia nigra. These neurons give rise to the so-called mesolinic dopaminergic system that projects nerve fibers to the medial and anterior portions of the limbic system, especially to the hippocampus, amygdala, anterior caudate nucleus, and portions of the prefrontal lobes. All of these are powerful behavioral control centers. An even more compelling reason for believing that schizophrenia is caused by excess production of dopamine is that many drugs that are effective in treating schizophrenia-such as chlorpromazine, haloperidol, and thiothizene-all decrease the secretion of dopamine at the dopaminergic nerve endings or decrease the effect of dopamine on subsequent neurons.
Phenothiazines
The phenothiazines as a class, and especially chlorpromazine, the prototype, are among the most widely used drugs in medical practice and are primarily employed in the management of patients with serious psychiatric illnesses. In addition, many members of the group have other clinically useful properties, including antiemetic, antinausea, and antihistaminic effects and the ability to potentiate analgesics, sedatives and general anesthetics.
Phenothiazine compounds were synthesized in Europe in the late nineteenth century as part of the development of aniline dyes such as methylene blue. In the late 1930s a derivative of phenothiazine was found to have antihistamine and a strong sedative effect and so the drug was introduced as into clinical anesthesia. It was noted that chlorpromazine by itself did not cause a loss of consciousness but produced only a tendency to sleep and a lack of interest in what was going on. These central actions became known as neuroleptic soon after.
Phenothiazine has a tricyclic structure in which two benzene rings are linked by a sulfur and a nitrogen atom (see figures below). Substitution of an electron-withdrawing group at R2 (but not at position 3 or 4) increases the efficacy of phenothiazines and other tricyclic congeners.
Neuroleptic drugs reduce initiative and interest in the environment, and they reduce displays of emotion or affect. Initially there may be some slowness in response to external stimuli and drowsiness. However subject are easily aroused, capable of giving appropriate answers to direct questions, and seem to have intact intellectual functions; there is no ataxia, incoordination, or dysathria at ordinary doses. Psychotic patients become less agitated and restless, and withdrawn or autistic patients sometimes become more responsive and communicative. Aggressive and impulsive behavior diminishes. Gradually (over a period of days). psychotic symptoms of hallucinations, delusions, and disorganized or incoherent thinking tend to disappear.
The most prominent observable effects of typical neuroleptic agents are strikingly similar. In low doses, operant behavior is reduced but spinal reflexes are unchanged. Exploratory behavior is diminished, and responses to a a variety of stimuli are fewer, slower, and smaller, although the ability to discriminate stimuli is retained. Conditioned avoidance behaviors are selectively inhibited, while unconditioned excape or avoidance responses are not.
Behavioral activation, stimulated environmentally of pharmacologically, is blocked. Feeding is inhibited. Most neuroleptics block the emesis and aggression induced by apomorphine--a dopaminergic agonist. In high doses, most neuroleptic agents induce characteristic cataleptic immobility that allows the animal to be placed in abnormal postures that persist. Muscle tone is altered, and ptosis (drooping of the eyelids) is typical. Even very high doses of most neuroleptics do not induce comma, and the lethal dose is extraordinarily high.
Mechanism. It is well established that benzodiazepines block dopaminergic receptors in the brain. There are three major central dopamine pathways: the nigrostriatal, which is affiliated with motor effects produced by antipsychotic drugs; the tuberoinfudibular, which is associated with the endocrine effects of neuroleptics; and the mesolimbic, which is the most likely to relate to the symptoms of schizophrenia. Of the three central dopamine receptor subtypes D1, D2 and D3, the D2 receptor, is believed to be most relevant to antipsychotic drug action. Most interesting, however, is that both D1 and D2 are altered in drug-naive schizophrenics. Though neuroleptic drugs are D1 and D2 antagonists, in vitro D2 effects are achieved at 103 lower concentrations.
D2 receptors are also located outside the blood brain barrier. One area is in the the chemoreceptor trigger zone of the medulla which is the reason that many of the phenothiazine drugs work as antiemetics and stop nausea.
Side Effects. There are several resulting syndromes which can occur from using antipsychotic drugs. A parkinsonian syndrome that may be indistinguishable from idiopathic parkinsonism my develop during administration of antipsychotic drugs. The most noticeable signs are rigidity and tremor at rest, especially involving the upper extremities. Tardive dyskenesia is a late-appearing neurological syndrome also associated with antipsychotic drug use. It is characterized by stereotypical involuntary movements consisting in sucking and smacking of the lips, lateral jaw movements, and fly-catching dartings of the tongue. There may be purposeless, quick movements of the extremities and slower, more dystonic movements and postures of the extremities, trunk, and neck may also be seen. All of these movements disappear during sleep as they do in parkinsonism. Symptoms of these conditions my persists indefinitely after discontinuation of the medication, although sometimes they disappear with time.
Dibenzodiazepine - Risperidone
In 1994 an addition tot he antipsychotic drugs is risperidone (Risperdal). This drug antagonises D2 and serotonin type 2 receptors. The drug also antagonizes for other receptors such as a adrenergic and histaminergic H1 receptors.
C. Anti Depressants
Major depression is the most common of the major mental illnesses, and it must be distinguished from normal grief, sadness, and disappointment. Major depression is characterized by feelings of intense sadness and despair, mental slowing and loss of concentration, pessimistic worry, agitation, and self-depreciation. Physical changes also occur, such as weight loss, decreased libido, and disruption of hormonal circadian rhythms.
Before the advent of psychotherapy in the 1950s, treatment of depression consisted of stimulants such as caffeine and amphetamines to ameliorate the depressive phases and barbiturates to allay agitation, anxiety, and insomnia. At best, such attempts at therapy may have offered transient relief to some patients. Suffering usually decreased little.
Monoamine Oxidase Inhibitors
Monoamine oxidase inhibitors were the first effective antidepressants used. These were discussed in detail in the section on Adrenergic Mechanisms and therefore will not be further discussed here.
Serotonin
Serotonin (5-hydroxytryptamine or 5-HT) is a monoamine neurotransmitter found in cardiovascular tissue, in endothelial cells, in blood cells, and in the central nervous system. The role of serotonin in neurological function is diverse, and there is little doubt that serotonin is an important CNS neurotransmitter. The cell bodies for serotonergic neurons are found in the raphe region in the brainstem/pons region. Lesions of this area can be made using 5,6 or 5,7-dihydroxytryptamine in a similar manner to 6-hydroxydopamine and have helped define the CNS pathways for 5-HT.
The monoamine serotonin is itself a precursor for melatonin production in the pineal gland. The biosynthesis of serotonin from the amino acid tryptophan is similar to that found for the catecholamines, and 5-hydroxytryptophan can cross the BBB to increase central levels of 5-HT. Although some of the serotonin is metabolized by monoamine oxidase to 5-hydroxyindole acetic acid, most of the serotonin released into the post-synaptic space is removed by the neuron through a reuptake mechanism inhibited by the tricyclic antidepressants and the newer, more selective antidepressants such as fluoxetine and sertraline.
Serotonin receptors are diverse and numerous. Over the past several years, over fourteen different serotonin receptors have been cloned and sequenced through molecular biological techniques. Overall, there are seven distinct families of 5-HT receptors, with as many as five within a particular family. Only one of the 5-HT receptors is a ligand-gated ion channel (the 5-HT3 receptor), and the other six families are all G protein-coupled receptors.
Tricyclic Anti-depressants
Imipramine, amitriptylin, and other closely related drugs are among the drugs currently most widely used for the treatment of major depression. Because of there structure ( see below). They are often referred to as the tricyclic antidepressants. Although these compounds seem to be similar to the phenothiazines chemically, the ethylene group of imiprimine's middle ring imparts dissimilar stereochemical properties and prevents conjegation of the rings, as occurs with the phenothiazines.
One might expect an effective antidepressant drug to have a stimulating or mood-elevating effect when given to a normal subject. Although this may occur with the MAOIs, it is not true of the tricyclic antidepressants. If a dose of imiprimine given to a normal subject, he feels sleepy and tends to be quieter, his blood pressure falls slightly, and he feels light headed. These drug effects are usually perceived to be unpleasant, and cause a feeling of unhappiness and increased anxiety. Repeated administration for several days may lead to accentuation of these symptoms and, in addition, to difficulty in concentrating and thinking. In contrast, if the drug is given over a period of time ( two to three weeks) to depressed patients an elevated mood occurs. For this reason, the tricyclics are not prescribed on an "as-needed" basis.
Mechanism. All tricyclic antidepressants in current use in the U.S. potentiate the actions of biogenic amines in the CNS by blocking its re-uptake at nerve terminals. However, the potency and selectivity for the inhibition of the uptake of norepinephrine, serotonin, and dopamine vary greatly among the agents. The tertiary amine tricyclics seem to inhibit the serotonin uptake pump, whereas the secondary amine ones seem better in switching off the NE pump (see table below). For instance, imipramine and amitriptyline are potent and selective blockers of serotonin transport with small effects on NE uptake, while desipramine and nortriptyline inhibit the uptake of norepinephrine and exert smaller effects on serotonin inhibition. None of these agents is very effective as an inhibitor of dopamine transport; this contrasts with the rather nonselective inhibitory actions of cocaine and amphetamine on the uptake of both norepinephrine and dopamine. These are poor antidepressants, despite the fact that it has a stimulant and even euphoriant effect in some people.
Drug Type of Amine Serotonin Norepinephrine Dopamine
Amitriptyline tertiary ++++ + 0
Imiprimine tertiary +++ ++ 0
Doxepine tertiary ++ + 0
Desipramine secondary 0 ++++ 0
Maprotiline secondary 0 +++ 0
Nortriptyline secondary + ++ 0
Protriptyline secondary ? +++ 0
D-Amphetamine - 0 +++ ++++
Selective Serotonin Reuptake Inhibitors
In recent years, selective serotonin reuptake inhibitors (SSRIs) have been introduced for the treatment of depression. Prozac is the most famous drug in this class. Lilly's sales of Prozac in 1993 exceeded 1 billion US dollars. Clomiprimine, fluoxetine (Prozac), sertraline and paroxetine selectively block the reuptake of serotonin, thereby increasing the levels of serotonin in the central nervous system. Note the similarities and differences between the tricyclic antidepressants and the selective serotonin reuptake inhibitors. The SSRIs generally have fewer anticholinergic side effects, but caution is still necessary when co-administering any drugs that affect serotonergic systems (e.g., monoamine oxidase inhibitors). Some of the newer, SSRIs (e.g., clomipramine) have been useful in the treatment of obsessive-compulsive disorders.
Here are some data to give you an idea of what transport systems are likely to be altered by the different antidepressants:
Drug Selectivity for 5-HT (Norepinephrine / Serotonin)
clomiprimine 14
fluoxetine 54
fluvoxamine 160
paroxetine 280
sertraline 840
From: Hyttel J. Pharmacological characterization of selective serotonin reuptake inhibitors (SSRIs). International Clinical Psychopharmacology. 9 Suppl 1: 19-26, 1994 Mar.
Lithium Salts
Lithium is widely used as \(\ce{Li2CO3}\) to control manic behavior in manic-depressive patients. No totally acceptable mechanism for its action exists. Postulations involve action that would likely adjust overactive catecholaminergic activity, which is the accepted occurrence in mania. Recent research has shown that \(\ce{Li^{+}}\) is a glutamate reuptake inhibitor. Other research has shown that \(\ce{Li^{+}}\) may inhibit glutamate stimulation of nerve cells. Several studies showing that \(\ce{Li^{+}}\) has some antidepressant effects are known. Some weak biphasic alterations of norepinephrine and serotonin turnover in the brain were established.
D. Stimulants
Amphetamines
Amphetamines were discussed under the topic of Adrenergic Mechanisms and therefore will not be further discussed here.
Methylxanthines
Caffeine, theophylline and theobromine share in common several pharmacological actions of therapeutic interest. They stimulate the central nervous system, act on the kidney to produce diuresis, stimulate cardiac muscle, and relax smooth muscle, notably bronchial muscle. Because the various xanthines differ markedly in the intensity of their action on various structures, one particular xanthine has been used more than another for a particular therapeutic effect. Since theobromine displays low potency in these pharmacological actions, it has all but disappeared from the therapeutic scene.
Caffeine, theophyline, and theobromine occur naturally in plants widely distributed geographically. Caffeine is found in the coffee bean, tea leaves, guarana, and other plants. It is probably the most-used of all psychoactive drugs. From the figure below, we can see that the methylxanthines have a structure which is very similar to adenine.
We have already discussed the role of the second messenger, cAMP, in the response to norepinephrine and epinephrine in the section on Adrenergic Mechanisms. The mechanism of action of caffeine and other methylxanthines is inhibiting the degradation of cAMP. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Medicinal_Chemistry/Psychoactive_Drugs.txt |
Adenosine-5'-triphosphate (ATP) is comprised of an adenine ring, a ribose sugar, and three phosphate groups. ATP is often used for energy transfer in the cell. The enzyme ATP synthase produces ATP from ADP or AMP + Pi using energy produced from metabolism in the mitochondria. ATP has many uses. It is used as a coenzyme, in glycolysis, for example. ATP is also found in nucleic acids in the processes of DNA replication and transcription. In a neutral solution, ATP has negatively charged groups that allow it to chelate metals. Usually, Mg2+ stabilizes it.
Introduction
ATP is a molecule which can hydrolyze to ADP and inorganic phosphate when it is in water. The formation of solvated ADP and hydrogen phosphate from solvated ATP and water has a ΔG of -30.5 kJ/mol. The negative ∆G means that the reaction is spontaneous (given an infinite amount of time it will proceed) and produces a net release of energy. However, because it requires energy to rupture the P-O bond connecting the phosphate that leaves ATP, ATP molecules do not instantly fall apart and can be used to transport useful energy around the cell. The energy required to rupture the bond contributes to the activation barrier that prevents the reaction from happening instantly.
Hydrolysis of ATP
At pH 7 the balanced reaction for hydrolysis is:
\[ATP ^{4-} + H_2O \rightleftharpoons ADP^{3-} + HPO_4^{2-} + H^+\] ∆G = -30.5 kJ
Why is ATP hydrolysis an exergonic reaction?
1. The entropy, which is the level of disorder, of solvated ADP + solvated HPO42- is greater than that of solvated ATP (∆SRXN > 0). Additionally, the reaction is exothermic (∆HRXN ≈ -20 kJ/mol).2 Therefore the thermodynamics implies the reaction is spontaneous (∆G = ∆H - T∆S < 0).
2. Electrostatic repulsion of the four negative charges on the oxygens of the ATP molecule. Naturally, like charges repel and opposite charges attract. Therefore, if there are four negative charges in close proximity to one another, they will naturally repel each other. This makes ATP a relatively unstable molecule because it will want to give away its phosphate groups, when given the chance, in order to become a more stable molecule.
3. Resonance stabilization of ADP and of HPO42- is greater than that of ATP. The oxygen molecules of the ADP are sharing electrons. Those electrons are constantly being passed back and forth between the oxygens, creating an effect called resonance. This stables the ADP. Resonance does not occur in ATP; therefore, it is a more unstable molecule.
4. There is a greater degree of solvation of HPO42-, H+, and ADP, relative to ATP. This means that it is easier for ATP to lose one of its phosphate groups. But, it takes a large amount of water to force ADP to lose one of its phosphates.
ATP in the Cell FIX ME
ATP is the primary energy transporter for most energy-requiring reactions that occur in the cell. The continual synthesis of ATP and the immediate usage of it results in ATP having a very fast turnover rate. This means that ADP is synthesized into ATP very quickly and vice versa. For example, it takes only a few seconds for half of the ATP molecules in a cell to be converted into ADP to be used in driving endergonic (non-spontaneous) reactions and then converted back into ATP using exergonic (spontaneous) reactions.
ATP is useful in many cell processes such as glycolysis, photosynthesis, beta oxidation, anaerobic respiration, active transport across cell membranes (as in the electron transport chain), and synthesis of macromolecules such as DNA.
Problems FIX ME
1. In cellular respiration, which process produces the most ATP?
2. True or false: ATP may be used to regulate certain enzymes.
3. From one molecule of glucose, how many molecules of ATP will be produced?
4. Where is ATP synthase located?
5. True or false: ATP is generated through substrate level phosphorylation.
Answers
1. Electron transport chain
2. True
3. 32 - 34 molecules of ATP
4. It is located in the inner mitochondrial membrane.
5. True
Contributors and Attributions
• Tiffany Lui, University of California, Davis.
• Jonathan Gutow, University of Wisconsin Oshkosh. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Metabolism/ATP_ADP-Gutow_Draft.txt |
Adenosine-5'-triphosphate (ATP) is comprised of an adenine ring, a ribose sugar, and three phosphate groups. ATP is often used for energy transfer in the cell. ATP synthase produces ATP from ADP or AMP + Pi. ATP has many uses. It is used as a coenzyme, in glycolysis, for example. ATP is also found in nucleic acids in the processes of DNA replication and transcription. In a neutral solution, ATP has negatively charged groups that allow it to chelate metals. Usually, Mg2+ stabilizes it.
Introduction
ATP is an unstable molecule which hydrolyzes to ADP and inorganic phosphate when it is in equilibrium with water. The high energy of this molecule comes from the two high-energy phosphate bonds. The bonds between phosphate molecules are called phosphoanhydride bonds. They are energy-rich and contain a ΔG of -30.5 kJ/mol.
Hydrolysis of ATP
Removing or adding one phosphate group interconverts ATP to ADP or ADP to AMP. Breaking one phosphoanhydride bond releases 7.3 kcal/mol of energy.
$\ce{ATP + H_2O \rightarrow ADP + P_{i}} \tag{ΔG = -30.5 kJ/mol}$
$\ce{ATP + H_2O \rightarrow AMP + 2 P_{i} } \tag{ΔG = -61 kJ/mol}$
$\ce{2 ADP + H_2O \rightarrow 2 AMP + 2 P_{i}} \tag{ΔG = -61 kJ/mol}$
At pH 7,
$\ce{ATP ^{4-} + H_2O \rightleftharpoons ADP^{3-} + HPO_4^{2-} + H^{+}} \nonumber$
Why is ATP hydrolysis an exergonic reaction?
1. The entropy, which is the level of disorder, of ADP is greater than that of ATP. Therefore, due to thermodynamics, the reaction spontaneously occurs because it wants to be at a higher entropy level. Also, the Gibbs' free energy of ATP is higher than that of ADP. Naturally, molecules want to be at a lower energy state, so equilibrium is shifted towards ADP.
2. Electrostatic repulsion of the four negative charges on the oxygens of the ATP molecule. Naturally, like charges repel and opposite charges attract. Therefore, if there are four negative charges in close proximity to one another, they will naturally repel each other. This makes ATP a relatively unstable molecule because it will want to give away its phosphate groups, when given the chance, in order to become a more stable molecule.
3. Resonance stabilization of ADP and of Pi is greater than that of ATP. The oxygen molecules of the ADP are sharing electrons. Those electrons are constantly being passed back and forth between the oxygens, creating an effect called resonance. This stables the ADP. Resonance does not occur in ATP; therefore, it is a more unstable molecule.
4. There is a greater degree of solvation of Pi, H+, and ADP, relative to ATP. This means that it is easier for ATP to lose one of its phosphate groups. But, it takes a large amount of water to force ADP to lose one of its phosphates.
ATP in the Cell
ATP is the primary energy transporter for most energy-requiring reactions that occur in the cell. The continual synthesis of ATP and the immediate usage of it results in ATP having a very fast turnover rate. This means that ADP is synthesized into ATP very quickly and vice versa. For example, it takes only a few seconds for half of the ATP molecules in a cell to be converted into ADP to be used in driving endergonic (non-spontaneous) reactions and then converted back into ATP using exergonic (spontaneous) reactions.
ATP is useful in many cell processes such as glycolysis, photosynthesis, beta oxidation, anaerobic respiration, active transport across cell membranes (as in the electron transport chain), and synthesis of macromolecules such as DNA.
Problems
1. In cellular respiration, which process produces the most ATP?
2. True or false: ATP may be used to regulate certain enzymes.
3. From one molecule of glucose, how many molecules of ATP will be produced?
4. Where is ATP synthase located?
5. True or false: ATP is generated through substrate level phosphorylation.
Answers
1. Electron transport chain
2. True
3. 32 - 34 molecules of ATP
4. It is located in the inner mitochondrial membrane.
5. True
Contributors and Attributions
• Tiffany Lui, University of California, Davis. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Metabolism/ATP_ADP.txt |
The biochemical processes of metabolism by which molecules are synthesized or built up. Often anabolism is a reductive process in which carbons have hydrogen atoms added. Fully saturated fatty acids are the most reduced form of carbon. NADPH is the primary electron donator for anabolic reactions.
Anabolism
Gluconeogenesis is the metabolic process by which organisms produce sugars (namely glucose) for catabolic reactions from non-carbohydrate precursors. Glucose is the only energy source used by the brain (with the exception of ketone bodies during times of fasting), testes, erythrocytes, and kidney medulla. In mammals this process occurs in the liver and kidneys.
Introduction
The need for energy is important to sustain life. Organisms have evolved ways of producing substrates required for the catabolic reactions necessary to sustain life when desired substrates are unavailable. The main source of energy for eukaryotes is glucose. When glucose is unavailable, organisms are capable of metabolizing glucose from other non-carbohydrate precursors. The process that coverts pyruvate into glucose is called gluconeogenesis. Another way organisms derive glucose is from energy stores like glycogen and starch.
Overview
Gluconeogenesis is much like glycolysis only the process occurs in reverse. However, there are exceptions. In glycolysis there are three highly exergonic steps (steps 1,3,10). These are also regulatory steps which include the enzymes hexokinase, phosphofructokinase, and pyruvate kinase. Biological reactions can occur in both the forward and reverse direction. If the reaction occurs in the reverse direction the energy normally released in that reaction is now required. If gluconeogenesis were to simply occur in reverse the reaction would require too much energy to be profitable to that particular organism. In order to overcome this problem, nature has evolved three other enzymes to replace the glycolysis enzymes hexokinase, phosphofructokinase, and pyruvate kinase when going through the process of gluconeogenesis:
1. The first step in gluconeogenesis is the conversion of pyruvate to phosphoenolpyruvic acid (PEP). In order to convert pyruvate to PEP there are several steps and several enzymes required. Pyruvate carboxylase, PEP carboxykinase and malate dehydrogenase are the three enzymes responsible for this conversion. Pyruvate carboxylase is found on the mitochondria and converts pyruvate into oxaloacetate. Because oxaloacetate cannot pass through the mitochondria membranes it must be first converted into malate by malate dehydrogenase. Malate can then cross the mitochondria membrane into the cytoplasm where it is then converted back into oxaloacetate with another malate dehydrogenase. Lastly, oxaloacetate is converted into PEP via PEP carboxykinase. The next several steps are exactly the same as glycolysis only the process is in reverse.
2. The second step that differs from glycolysis is the conversion of fructose-1,6-bP to fructose-6-P with the use of the enzyme fructose-1,6-phosphatase. The conversion of fructose-6-P to glucose-6-P uses the same enzyme as glycolysis, phosphoglucoisomerase.
3. The last step that differs from glycolysis is the conversion of glucose-6-P to glucose with the enzyme glucose-6-phosphatase. This enzyme is located in the endoplasmic reticulum.
Glycolysis
Regulation
Because it is important for organisms to conserve energy, they have derived ways to regulate those metabolic pathways that require and release the most energy. In glycolysis and gluconeogenesis seven of the ten steps occur at or near equilibrium. In gluconeogenesis the conversion of pyruvate to PEP, the conversion of fructose-1,6-bP, and the conversion of glucose-6-P to glucose all occur very spontaneously which is why these processes are highly regulated. It is important for the organism to conserve as much energy as possible. When there is an excess of energy available, gluconeogenesis is inhibited. When energy is required, gluconeogenesis is activated.
1. The conversion of pyruvate to PEP is regulated by acetyl-CoA. More specifically pyruvate carboxylase is activated by acetyl-CoA. Because acetyl-CoA is an important metabolite in the TCA cycle which produces a lot of energy, when concentrations of acetyl-CoA are high organisms use pyruvate carboxylase to channel pyruvate away from the TCA cycle. If the organism does not need more energy, then it is best to divert those metabolites towards storage or other necessary processes.
2. The conversion of fructose-1,6-bP to fructose-6-P with the use of fructose-1,6-phosphatase is negatively regulated and inhibited by the molecules AMP and fructose-2,6-bP. These are reciprocal regulators to glycolysis' phosphofructokinase. Phosphofructosekinase is positively regulated by AMP and fructose-2,6-bP. Once again, when the energy levels produced are higher than needed, i.e. a large ATP to AMP ratio, the organism increases gluconeogenesis and decreases glycolysis. The opposite also applies when energy levels are lower than needed, i.e. a low ATP to AMP ratio, the organism increases glycolysis and decreases gluconeogenesis.
3. The conversion of glucose-6-P to glucose with use of glucose-6-phosphatase is controlled by substrate level regulation. The metabolite responsible for this type of regulation is glucose-6-P. As levels of glucose-6-P increase, glucose-6-phosphatase increases activity and more glucose is produced. Thus glycolysis is unable to proceed.
Problems
1. How many enzymes are unique to Gluconeogenesis?
2. What is reciprocal regulation and why is it important to Glycolysis and Gluconeogenesis?
3. Where does the activity of glucose-6-phosphatase occur?
4. Why is it necessary for gluconeogenesis to incorporate other enzymes in its pathway that are different from glycolysis?
5. Draw glycolysis and Gluconeogenesis side by side with the products, reactants and enzymes for each step.
Contributors
• Darik Benson, Undergraduate University California Davis | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Metabolism/Anabolism/Gluconeogenesis.txt |
The pentose phosphate pathway is the major source for the NADPH required for anabolic processes. There are three distinct phases each of which has a distinct outcome. Depending on the needs of the organism the metabolites of that outcome can be fed into many other pathways. Gluconeogenesis is directly connected to the pentose phosphate pathway. As the need for glucose-6-phosphate (the beginning metabolite in the pentose phosphate pathway) increases so does the activity of gluconeogenesis.
Introduction
The main molecule in the body that makes anabolic processes possible is NADPH. Because of the structure of this molecule it readily donates hydrogen ions to metabolites thus reducing them and making them available for energy harvest at a later time. The PPP is the main source of synthesis for NADPH. The pentose phosphate pathway (PPP) is also responsible for the production of Ribose-5-phosphate which is an important part of nucleic acids. Finally the PPP can also be used to produce glyceraldehyde-3-phosphate which can then be fed into the TCA and ETC cycles allowing for the harvest of energy. Depending on the needs of the cell certain enzymes can be regulated and thus increasing or decreasing the production of desired metabolites. The enzymes reasonable for catalyzing the steps of the PPP are found most abundantly in the liver (the major site of gluconeogenesis) more specifically in the cytosol. The cytosol is where fatty acid synthesis takes place which is a NADPH dependent process.
Oxidation Phase
• The beginning molecule for the PPP is glucose-6-P which is the second intermediate metabolite in glycolysis. Glucose-6-P is oxidized in the presence of glucose-6-P dehydrogenase and NADP+. This step is irreversible and is highly regulated. NADPH and fatty acyl-CoA are strong negative inhibitors to this enzyme. The purpose of this is to decrease production of NADPH when concentrations are high or the synthesis of fatty acids is no longer necessary.
• The metabolic product of this step is gluconolactone which is hydrolytrically unstable. Gluconolactonase causes gluconolactone to undergo a ring opening hydrolysis. The product of this reaction is the more stable sugar acid, 6-phospho-D-gluconate.
• 6-phospho-D-gluconate is oxidized by NADP+ in the presence of 6-phosphogluconate dehydrogenase which yields ribulose-5-phosphate.
• The oxidation phase of the PPP is solely responsible for the production of the NADPH to be used in anabolic processes.
Isomerization Phase
• Ribulose-5-phosphate can then be isomerized by phosphopentose isomerase to produce ribose-5-phosphate. Ribose-5-phosphate is one of the main building blocks of nucleic acids and the PPP is the primary source of production of ribose-5-phosphate.
• If production of ribose-5-phosphate exceeds the needs of required ribose-5-phosphate in the organism, then phosphopentose epimerase catalyzes a chiralty rearrangement about the center carbon creating xylulose-5-phosphate.
• The products of these two reactions can then be rearranged to produce many different length carbon chains. These different length carbon chains have a variety of metabolic fates.
Rearrangement Phase
• There are two main classes of enzymes responsible for the rearrangement and synthesis of the different length carbon chain molecules. These are transketolase and transaldolase.
• Transketolase is responsible for the cleaving of a two carbon unit from xylulose-5-P and adding that two carbon unit to ribose-5-P thus resulting in glyceraldehyde-3-P and sedoheptulose-7-P.
• Transketolase is also responsible for the cleaving of a two carbon unit from xylulose-5-P and adding that two carbon unit to erythrose-4-P resulting in glyceraldehyde-3-P and fructose-6-P.
• Transaldolase is responsible for cleaving the three carbon unit from sedoheptulose-7-P and adding that three carbon unit to glyceraldehyde-3-P thus resulting in erythrose-4-P and fructose-6-P.
• The end results of the rearrangement phase is a variety of different length sugars which can be fed into many other metabolic processes. For example, fructose-6-P is a key intermediate of glycolysis as well as glyceraldehyde-3-P.
Problems
1. Where does the PPP take place and why there?
2. The PPP has three main phases, what are the outcomes of those phases?
3. What is Ribose-5-phosphate and why is it important?
4. In the isomerization phase of the PPP there are two enzymes that catalyze two different reactions. How are the out comes of these two reactions different? (Hint: What is being changed in these reactions?)
5. Name all eight enzymes in the PPP and briefly describe the processes.
Contributors
• Darik Benson, University California Davis | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Metabolism/Anabolism/Pentose_Phosphate_Pathway.txt |
Catabolism is the biochemical processes of metabolism by which molecules are broken down. Often catabolism is an oxidative process in which carbons have hydrogen atoms removed. CO2 is the most oxidized form of carbon. NADH is the primary electron acceptor for catabolic reactions.
Catabolism
The best source of energy for eukaryotic organisms are fats. Glucose offers a ratio 6.3 moles of ATP per carbon while saturated fatty acids offer 8.1 ATP per carbon. Also the complete oxidation of fats yields enormous amounts of water for those organisms that do not have adequate access to drinkable water. Camels and killer whales are good example of this, they obtain their water requirements from the complete oxidation of fats.
Introduction
There are four distinct stages in the oxidation of fatty acids. Fatty acid degradation takes place within the mitochondria and requires the help of several different enzymes. In order for fatty acids to enter the mitochondria the assistance of two carrier proteins is required, Carnitine acyltransferase I and II. It is also interesting to note the similarities between the four steps of beta-oxidation and the later four steps of the TCA cycle.
Entry into Beta-oxidation
Most fats stored in eukaryotic organisms are stored as triglycerides as seen below. In order to enter into beta-oxidation bonds must be broken usually with the use of a Lipase. The end result of these broken bonds are a glycerol molecule and three fatty acids in the case of triglycerides. Other lipids are capable of being degraded as well.
Key molecules in beta-oxidation: (left) A triglyceride molecule, (middle) Glycerol, (right) Fatty Acids (unsaturated)
Steps of Beta-oxidation
Activation
• Once the triglycerides are broken down into glycerol and fatty acids they must be activated before they can enter into the mitochondria and proceed on with beta-oxidation. This is done by Acyl-CoA synthetase to yield fatty acyl-CoA.
• After the fatty acid has been acylated it is now ready to enter into the mitochondria.
• There are two carrier proteins (Carnitine acyltransferase I and II), one located on the outer membrane and one on the inner membrane of the mitochondria. Both are required for entry of the Acyl-CoA into the mitochondria.
• Once inside the mitochondria the fatty acyl-CoA can enter into beta-oxidation.
Oxidation
A fatty acyl-CoA is oxidized by Acyl-CoA dehydrogenase to yield a trans alkene. This is done with the aid of an [FAD] prosthetic group.
Hydration
The trans alkene is then hydrated with the help of Enoyl-CoA hydratase
Oxidation
The alcohol of the hydroxyacly-CoA is then oxidized by NAD+ to a carbonyl with the help of Hydroxyacyl-CoA dehydrogenase. NAD+ is used to oxidize the alcohol rather then [FAD] because NAD+ is capable of the alcohol while [FAD] is not.
Cleavage
Finally acetyl-CoA is cleaved off with the help of Thiolase to yield an Acyl-CoA that is two carbons shorter than before. The cleaved acetyl-CoA can then enter into the TCA and ETC because it is already within the mitochondria.
Problems
1. Where does beta-oxidation occur?
2. What is the average net yield of ATP per carbon?
3. Where exactly is water formed during the process of fatty acid degradation? (Hint: H2O is formed when when the one of the products of beta-oxidation is passed through another of the metabolic pathways)
4. During the process of beta-oxidation, why is it that [FAD] is used to oxidize an alkane to an alkene while NAD+ is used to oxidize an alchol to a carbonyl
5. Draw out the entire process of the degradation of a triglyceride, include enzymes and products and reactants for each step.
Contributors
• Darik Benson, Undergraduate University California Davis | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Metabolism/Catabolism/Beta-Oxidation.txt |
Before the concept of biological oxidation can be understood and explored, the fundamental chemical process through which oxidation and reduction take place should be first established.
Foundation
All reactions which involve electron flow are considered oxidation-reduction reactions. The basic definition can be defined as: One reactant is oxidized (loses electrons), while another is reduced (gains electrons). A couple of basic oxidation-reduction or "redox" example's are given here.
Example 1
The reaction of magnesium metal with oxygen, involves the oxidation of magnesium
$2Mg(s) + O_2(g)→ 2MgO(s) \label{1}$
Since the magnesium solid is oxidized, we expect to see a loss of electrons. Similarly, since oxygen must therefore be reduced, we should see a gain of electrons.
As the magnesium is oxidized there is a loss of 2 electrons while simultaneously, oxygen gains those two electrons. Another example of a redox reaction is with the two gasses CO2 and H2. This redox reaction also demonstrates the importance of implementing "oxidation numbers" in the methodology of redox reactions, allowing for the determination of which reactant is being reduced and which reactant is being oxidized.
Example 2
The reaction of carbon dioxide gas with hydrogen gas, involving the oxidation of hydrogen
$CO_2 (g) + H_2 (g) → 2CO (g) + H_2O (g) \label{3}$
Since the hydrogen gas is being oxidized (reductant), we expect to see an overall loss of electrons for the resulting molecule. Similarly, we expect to see a gain in the overall number of electrons for the resulting molecule of the oxidant (CO2).
Here it is possible to infer that the carbon of CO2 is being reduced by review of its unique oxidation number. Such that, C (of CO2) goes from an oxidation number of +4 to C (of CO) having an oxidation number of +2, representing a loss of two electrons. Similarly, H2 is noted as going from an oxidation number of 0 to +1, or gaining one electron in a reduction process. For more information on oxidation numbers, review the following link: Oxidation-Reduction Reactions
A Basic Biological Model
The flow of electrons is a vital process that provides the necessary energy for the survival of all organisms. The primary source of energy that drives the electron flow in nearly all of these organisms is the radiant energy of the sun, in the form of electromagnetic radiation or Light. Through a series of nuclear reactions, the sun is able to generate thermal energy (which we can feel as warmth) from electromagnetic radiation (which we perceive as light). However, the particular wavelength of the electromagnetic spectrum we are able to detect with the human eye is only between 400 and 700 nm in wavelength. It should therefore be noted that the visible part of the electromagnetic spectrum is actually a small percentage of the whole; where a much greater percentage remains undetectable for the human eye.
In physics, the use of the term "light" refers to electromagnetic radiation of any wavelength, independent of its detectability for the human eye. For plants, the upper and lower ends of the visible spectrum are the wavelengths that help drive the process of splitting water (H2O) during photosynthesis, to release its electrons for the biological reduction of carbon dioxide (CO2) and the release of diatomic oxygen (O2) to the atmosphere. It is through the process of photosynthesis that plants are able to use the energy from light to convert carbon dioxide and water into the chemical energy storage form called glucose.
Plants represent one of the most basic examples of biological oxidation and reduction. The chemical conversion of carbon dioxide and water into sugar (glucose) and oxygen is a light-driven reduction process:
$6CO_2 + 6H_2O \rightarrow C_6H_{12}O_6 + 6O_2 \label{5}$
The process by which non photosynthetic organisms and cells obtain energy, is through the consumption of the energy rich products of photosynthesis. By oxidizing these products, electrons are passed along to make the products carbon dioxide, and water, in an environmental recycling process. The process of oxidizing glucose and atmospheric oxygen allowed energy to be captured for use by the organism that consumes these products of the plant. The following reaction represents this process:
$C_6H_{12}O_6 + O_2 \rightarrow 6CO_2 + 6H_2O +Energy \label{6}$
It is therefore through this process that heterotrophs (most generally "animals" which consume other organisms obtain energy) and autotrophs (plants which are able to produce their own energy) participate in an environmental cycle of exchanging carbon dioxide and water to produce energy containing glucose for organismal oxidation and energy production, and subsequently allowing the regeneration of the byproducts carbon dioxide and water, to begin the cycle again. Therefore, these two groups of organisms have been allowed to diverge interdependently through this natural life cycle.
Physical Chemistry's Understanding
Biological oxidation-reduction reactions, or simply biological oxidations utilize multiple stages or processes of oxidation to produce large amounts of Gibbs energy, which is used to synthesize the energy unit called adenosine triphosphate or ATP. To efficiently produce ATP, the process of glycolysis must be near an abundance of oxygen. Since glycolysis by nature is not an efficient process, if it lacks sufficient amounts of oxygen the end product pyruvate, is reduced to lactate with NADH as the reducing agent. However, in a more favorable aerobic process, the degradation of glucose through glycolysis proceeds with two additional processes known as the citric acid cycle and the terminal respiratory chain; yielding the end products carbon dioxide and water, which we exhale with each breath.
The products NADH and FADH2 formed during glycolysis and the citric acid cycle are able to reduce molecular oxygen (O2) thereby releasing large amounts of Gibbs energy used to make ATP. The process by which electrons are transferred from NADH or FADH2 to O2 by a series of electron transfer carriers, is known as oxidative phosphorylation. It is through this process that ATP is able to form as a result of the transfer of electrons.
Thee specific examples of redox reactions that are used in biological processes, involving the transfer of electrons and hydrogen ions as follows. During some biological oxidation reactions, there is a simultaneous transfer of hydrogen ions with electrons (1). In other instances, hydrogen ions may be lost by the substance being oxidized while transferring only its electrons to the substance being reduced (2). A third type of biological oxidation might involve only a transfer of electrons (3). It should be noted that biological oxidation rarely proceeds in a direct manner, and generally involves complex mechanisms of several enzymes. The outline below recaps the three processes of biological oxidation stated above, in descending order.
Table 1: Transfer of hydrogen ions and electrons for the general reaction scheme of A + B with intermediate stage shown
Reactants Intermediate Stage Products
AH2 + B [A + 2H+ + 2e- + B] A + BH2
AH2 + B [A + 2H+ + 2e- + B] A + B2- + 2H+
A2- + B [A + 2e- + B] A + B2-
In the last stage of the metabolic process (the terminal respiratory chain), the sequence by which electrons are carried is determined by relative redox potentials. The carrier molecules used to transfer electrons in this stage are called cytochromes, which are an electron-carrying protein containing a heme group. The iron atom of each cytochrome molecule can exist either in the oxidized (Fe3+) or reduced (Fe2+) form. Within the terminal respiratory chain, each carrier molecule alternates between the reduced state and the oxidized state, with molecular oxygen as the final electron acceptor at the end.
It is through the knowledge of redox potentials, that the knowledge of biological processes can be further expanded. The standard reduction potential is denoted as Eo' and is often based on the hydrogen electrode scale of pH 7, rather than pH 0, a common reference point for listed values. Moreover, the superscript symbol ( o ) denotes standard-state conditions, while the adjacent superscript symbol ( ' ) denotes the pH scale of 7 for biochemical processes.
It therefore becomes possible to trace the energy transfer in cells back to the fundamental flow of electrons from one particular molecule to another. Where this electron flow occurs via the physics principle of higher potential to lower potential; similar to a ball rolling down a hill, as opposed to the opposite direction. All of these reactions involving electron flow can be attributed to the basic definition of the oxidation-reduction pathway stated above.
Contributors and Attributions
• Brent Younglove (Hope) | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Metabolism/Catabolism/Biological_Oxidation.txt |
Photosynthesis is responsible for creating NADPH and ATP and the Calvin-Benson-Bassham cycle (CBB) uses those high energy molecules to drive the production of glyceraldehyde-3-phosphate (G-3-P). G-3-P can then be used to synthesize hexose sugars which are the primary source of nutrients for heterotrophs.
Introduction
Because ATP and NADPH are required for the CBB to proceed it is necessary for photosynthesis to occur prior. Photosynthesis (a light dependent reaction) uses light energy to produce ATP and NADPH which can then be used to drive synthesis of of carbohydrate molecules in the CBB, namely glyceraldehyde-3-phosphate. Although the CBB cycle has been given the nick name the "dark reaction" the enzymes involved are activated by light. Light stimulates changes in pH in the different regions of the plant cell which then in turn create a better environment for the CBB enzymes. The enzymes in the CBB cycle are very similar to other enzymes found in other metabolic path ways with the exception that they are found in the stoma instead of in the cytoplasm like in glycolysis.
Steps of the Calvin Cycle
The diagram directly below is an extremely abbreviated version of the CBB cycle.
Phase 1
This stage is very similar to the isomerization phase of PPP. Enzymes for these reactions are in red
1. The enzyme Rubisco (ribulose bisphosphate carboxylase) catalyses the carboxilation of ribulose-1,5-bisphosphate in a two step reaction. Ribulose-1,5-bisphosphate must first be phosphorolated by the enzyme Phosphoribulose kinase. The outcome of this carboxylation are two molecules of 3-Phosphoglycerate.
Phase 2
This phase of CBB very closely resembles part of gluconeogenesis. Enzymes for these reactions are in red
1. 3-Phosphoglycerate is then phosphorolated with the aid of Phosphoglycerate kinase to yield 1,3-Bisphosphoglycerate.
2. Next 1,3-Bisphosphoglycerate is reduced by NADPH to yield NADP+ and Glyceraldehyde-3-phosphate with the aid of Glyceraldehyde-3-phosphate dehydrogenase. One of every six Glyceraldehyde-3-phosphate molecules is exported into the cytoplasm to be use in the synthesis of Glucose and other metabolic processes.
Phase 3
This phase of CBB very closely resembles the rearrangement phase of PPP. Enzymes for these reactions are in red
1. Glyceraldehyde-3-phosphate is then reversibly converted to Dihydroxyacetone phosphate by Triose phosphate isomerase.
2. Next Dihydroxyacetone is converted into fructose-6-phosphate (F-6-P) by Aldolase and Fructose bisphosphatase. Aldolase condenses the two DHAP molecules to form Fructose-1,6-bisphosphate. Because of its high (-)delta G the transformation of Fructose-1,6-bisphosphate to Fructose-6-phosphate is thought to be the rate limiting step of the CBB cycle. F-6-P can then be converted into glucose via two enzymatic steps with the help of Phosphoglucoisomerase and glucose-6-Phosphatase.
3. Dihydroxyacetone can also go on to condense with Erythrose-4-phosphate to form Sedoheptulose-1,7-bisphosphate(SBP). This reaction is also catalyzed by Aldolase.
4. SBP is then de-phosphorolated by Sedoheptulose bisphosphatase to yield Sedoheptulose-7-phosphate (S7P).
5. After several rearrangement reactions utilizing Transketolase and Transaldolase enzymes, Xylulose-5-Phosphate (X5P) and Ribose-5-phosphate (R5P) are synthesized.
6. Lastly X5P and R5P are isomerised by Phosphopentose epimerase and Phosphopentose isomerase to yield Ribulose-5-phosphate which can then be put back into the cycle.
Problems
1. What are the three phases of the CBB cycle?
2. What is considered the rate determining step of the CBB cycle?
3. Where does the ATP and NADPH come from that is used in the CBB cycle?
4. Examine the CBB and identify the steps that are similar to the PPP and also the steps similar to gluconeogenesis.
5. After watching the animation write up balanced reactions for each phase of CBB cycle.
Contributors and Attributions
• Darik Benson (University of California Davis) | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Metabolism/Catabolism/Calvin-Benson-Bassham_Cycle.txt |
The electron transport chain (aka ETC) is a process in which the NADH and [FADH2] produced during glycolysis, β-oxidation, and other catabolic processes are oxidized thus releasing energy in the form of ATP. The mechanism by which ATP is formed in the ETC is called chemiosmotic phosphorolation.
Introduction
The byproducts of most catabolic processes are NADH and [FADH2] which are the reduced forms. Metabolic processes use NADH and [FADH2] to transport electrons in the form of hydride ions (H-). These electrons are passed from NADH or [FADH2] to membrane bound electron carriers which are then passed on to other electron carriers until they are finally given to oxygen resulting in the production of water. As electrons are passed from one electron carrier to another hydrogen ions are transported into the intermembrane space at three specific points in the chain. The transportation of hydrogen ions creates a greater concentration of hydrogen ions in the intermembrane space than in the matrix which can then be used to drive ATP Synthase and produce ATP (a high energy molecule).
Overview
In the diagram located below there are the major electron transporters responsible for making energy in the ETC.
The Electron Carriers
• I (NADH-ubiquinone oxidioreductase): An integral protein that receives electrons in the form of hydride ions from NADH and passes them on to ubiquinone
• II (Succinate-ubiquinone oxidioreductase aka succinate dehydrogenase from the TCA cycle): A peripheral protein that receives electrons from succinate (an intermediate metabolite of the TCA cycle) to yield fumarate and [FADH2]. From succinate the electrons are received by [FAD] (a prosthetic group of the protein) which then become [FADH2]. The electrons are then passed off to ubiquinone.
• Q (Ubiquinone/ ubiquinol): Ubiquinone (the oxidized form of the molecule) receives electrons from several different carriers; from I, II, Glycerol-3-phosphate dehydrogenase, and ETF. It is now the reduced form (ubiquinol) which passes its electron off to III.
• III (Ubiquinol-cytochrome c oxidioreductase): An integral protein that receives electrons from ubiquinol which are then passed on to Cytochrome c
• IV (Cytochrome c oxidase):An integral protein that that receives electrons from Cytochrome c and transfers them to oxygen to produce water within the mitochondria matrix.
• ATP Synthas: An integral protein consisting of several different subunits. This protein is directly responsible for the production of ATP via chemiosmotic phosphorolation. It uses the proton gradient created by several of the other carriers in the ETC to drive a mechanical rotor. The energy from that rotor is then used to phosphorolate ADT to ATP.
Not Shown
• ETF (Electron-transferring flavoprotein) Dehydrogenase: This peripheral protein located on the matrix side of the inner membrane is a part the B-oxidation cycle. Electrons from acyl-CoA are donated to an electron-transfer flavoprotien which are then transferred to ETF (Electron-transferring flavoprotein) Dehydrogenase in the form of [FADH2]. ETF dehydrogenase then passes those electrons from [FADH2] to ubiquinone and on through the ETC.
• Glycerol-3-phosphate dehydrogenas:This peripheral protein located on the intermembrane space side of the inner membrane is a part of the glycerol-3-phosphate transport system. It accepts a proton from glycerol-3-phosphate to a prosthetic [FAD] group which yields [FADH2]. From [FADH2] the electrons are then given to ubiquinone and on through the ETC.
Electron Flow
It should be noted from the diagram below that ubiquinone (a hydrophobic carrier that resides within the membrane) receives electrons from several different electron carriers. Cytochrome c (a hydrophilic carrier found with in the intermembrane space) on the other hand only transfers electrons from III to IV. The driving force of the ETC is the fact that each electron carrier has a higher standard reduction potential than the one that it accepts electrons from. Standard reduction potential is a measure of the ability to accept or donate electrons. Oxygen has the highest (most positive) standard reduction potential which means that is is most likely to accept electrons from other carriers. That is precisely why it is found at the end of the ETC.
Proton Motive Force
Proton motive force refers to the energy obtained from the proton gradient created by several of the electron carriers. Only three of the four mentioned electron carriers are capable of transporting protons from the matrix to the intermembrane space: I, III, and IV. It is this proton gradient that drives phosphorolation of ADP to ATP as well as several other important transport systems. As proton concentration builds up in the intermembrane space a gradient is created and protons are transported from high to low concentration. The energy from the transfer of protons is used to change ADP into ATP though phosphorolation. ATP synthase is the protein responsible for ADP phosphorolation.
It is also important for proper concentrations of substrates to be maintained within and without the mitochondria to allow for chemiosmotic phosphorolation. The two main types of proteins responsible for maintaining proper substrate concentrations are pyruvate and phosphate symporters and ADP/ATP antiporters. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Metabolism/Catabolism/Electron_Transport_Chain.txt |
The majority of the energy conserved during catabolism reactions occurs near the end of the metabolic series of reactions in the electron transport chain. The electron transport or respiratory chain gets its name from the fact electrons are transported to meet up with oxygen from respiration at the end of the chain.
Introduction
The overall electron chain transport reaction is:
2 H+ + 2 e- + 1/2 O2 → H2O + energy
Notice that 2 hydrogen ions, 2 electrons, and an oxygen molecule react to form as a product water with energy released is an exothermic reaction. This relatively straight forward reaction actually requires eight or more steps. The energy released is coupled with the formation of three ATP molecules per every use of the electron transport chain.
Pre-Initiation of Electron Transport Chain
The electron transport chain is initiated by the reaction of an organic metabolite (intermediate in metabolic reactions) with the coenzyme NAD+ (nicotinamide adenine dinucleotide). This is an oxidation reaction where 2 hydrogen atoms (or 2 hydrogen ions and 2 electrons) are removed from the organic metabolite. (The organic metabolites are usually from the citric acid cycle and the oxidation of fatty acids--details in following pages.) The reaction can be represented simply where M = any metabolite.
MH2 + NAD+ → NADH + H+ + M: + energy
One hydrogen is removed with 2 electrons as a hydride ion (H-) while the other is removed as the positive ion (H+). Usually the metabolite is some type of alcohol which is oxidized to a ketone. NAD+ is a coenzyme containing the B-vitamin, nicotinamide, shown on a previous page.
The purpose of the other seven steps in the electron transport chain is threefold:
1. to pass along 2H+ ions and 2e- to eventually react with oxygen;
2. to conserve energy by forming three ATP's; and
3. to regenerate the coenzymes back to their original form as oxidizing agents.
Initiation of Electron Transport Chain
Once the NADH has been made from a metabolite in the citric acid cycle inside of the mitochondria, it interacts with the first complex 1 enzyme, known as NADH reductase. This complex 1 contains a coenzyme flavin mononucleotide (FMN) which is similar to FAD.
The sequence of events is that the NADH, plus another hydrogen ion enter the enzyme complex and pass along the 2 hydrogen ions, ultimately to an interspace in the mitochondria. These hydrogen ions, acting as a pump, are utilized by ATP synthetase to produce an ATP for every two hydrogen ions produced. Three complexes (1, 3, 4) act in this manner to produce 2 hydrogen ions each, and thus will produce 3 ATP for every use of the complete electron transport chain.
In addition, NADH passes along 2 electrons to first FMN, then to an iron-sulfur protein (FeS), and finally to coenzyme Q. The net effect of these reactions are to regenerate coenzyme NAD+. This regeneration of reactants occurs in many of the reactions so that a cycling effect occurs. The NAD+ is ready to react further with metabolites in the citric acid cycle. Coenzyme Q, which also picks up an additional 2 hydrogen ions to make CoQH2, is soluble in the lipid membrane and can move through the membrane to come into contact with enzyme complex 3.
In summary, the very first enzyme complex in the electron transport chain is coupled with the formation of ATP. The coupled reaction may be written as:
MH2 + NAD+ → NADH + H+ + M + energy
ADP + P + energy → ATP + H2O
Electron Transport - Enzyme Complex 3
Coenzyme QH2 carrying an extra 2 electrons and 2 hydrogen ions now starts a cascade of events through enzyme complex 3, also known as cytochrome reductase bc.
Cytochromes are very similar to the structure of myoglobin or hemoglobin. The significant feature is the heme structure containing the iron ions, initially in the +3 state and changed to the +2 state by the addition of an electron. The CoQH2 (yellow)passes along the 2 electrons first to cytochrome (blue) b1 heme (magenta), then b2 heme , then to an iron-sulfur protein (green), then to cytochrome c1 (red with black heme), and finally to cytochrome c (not shown). Co Q is represented by the inhibitor antimycin (yellow) in the graphic.
In the meantime the 2 hydrogen ions are channeled to the interspace of the mitochondria for ultimate conversion into ATP.
Complex 4
Refer to the middle graphic: Cytochrome c is a small molecule which is also able to move in the lipid membrane layer and diffuses toward cytochrome a complex 4. At this time it continues the transport of the electrons, and provides the third and final time that 2 hydrogen ions are channeled to the interspace of the mitochondria for ultimate conversion into ATP.
ATP synthetase is also found at numerous locations in the bilayer membrane of the mitochondria. Three ATP are produced by the pumping action of the re-entry of the hydrogen ions through the ATP synthetase.
Finally, oxygen has diffused into the cell and the mitochondria for the finally reaction of metabolism. Oxygen atom reacts with the 2 electrons and 2 hydrogens to produce a water molecule.
Problems
1. What are the initial reactants which start the electron transport chain?
2. What are the final products of the chain?
3. What is the definition of a coupled reaction?
4. When NADH converts back to NAD+, is this oxidation or reduction?
5. When CoQ converts to CoQH2, is this oxidation or reduction?
6. When iron +3 ions add an electron to become +2 ion, is this oxidation or reduction?
Outside Links
• Link to a complete animated version of Electron Transport - Brooks-Cole
• Link to: Rodney Boyer Animation of Electron Transport
• Animation of Electron Transport - Thomas M. Terry, The University of Connecticut | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Metabolism/Catabolism/Electron_Transport_Chain_II.txt |
Fermentation is the process by which living organisms recycle $NADH \rightarrow NAD^+$. $NAD^+$ is a required molecule necessary for the oxidation of Glyceraldehyde-3-phosphate to produce the high energy molecule 1,3-bisphosphoglycerate (Step 6 of Glycolysis). Fermentation occurs in the cytosol of cells.
Introduction
Because $NAD^+$ is used in Glycolysis it is important that living cells have a way of recycling $NAD^+$ from $NADH$. One way that a cell recycles $NAD^+$ is through the process of respiration, a set of sequential electron transfers involving an electron transport chain to a terminal electron acceptor. In aerobic organisms, the terminal electron acceptor is oxygen. In anaerobic organisms, the terminal electron acceptor can vary from species to species and include but are not limited to various metals like Fe(III), Mn(IV) and Co(III), CO2, nitrate, sulfur This process reduces NADH back to $NAD^+$ which can then be used again in step 6 of Glycolysis or other red/ox reactions in the cell. Another way that $NAD^+$ is recycled from $NADH$ is by a process called fermentation.
Example: Lactic acid fermentation in contracting muscle
Lactic acid fermentation occurs by converting pyruvate into lactate using the enzyme Lactate dehydrogenase and producing $NAD^+$ in the process. This process takes place in oxygen depleted muscle and some bacteria. It is responsible for the sour taste of sauerkraut and yogurt. $NAD^+$ is required for the oxidation of glyceraldehyde-3-P to produce 1,3-Bisphosphoglycerate (Step 6 of Gycolysis). If the supply of $NAD^+$ is not replenished by the ETC or fermentation, glycolysis is unable to proceed. Fermentation is a necessary process for anaerobic organisms to produce energy. The yield of energy is much less than if the organism were to continue on through the TCA cycle and ETC, but energy is produce nonetheless.
Example: Alcoholic fermentation in yeast
The purpose of fermentation in yeast is the same as that in muscle and bacteria, to replenish the supply of NAD+ for glycolysis, but this process occurs in two steps:
1. Alcoholic fermentation consists of pyruvate being first converted into acetaldehyde by the enzyme pyruvate decarboxylase and releasing $CO_2$.
2. In the second step acetaldehyde is converted into ethanol using alcohol dehydrogenase and producing $NAD^+$ in the process. It is this recycled $NAD^+$ that can be used to continue on with glycolysis.
Problems
1. Draw the chemical structures of pyruvate, ethanol and lactate (the reactant and products of fermentation)
2. Why is fermentation necessary? (Hint: see step 6 of Glycolysis)
3. What type of environment is necessary for fermentation to occur?
4. Where does fermentation occur? What part of the cell?
5. Explain the alternative to fermentation and why it is able to proceed. (Hint: Final electron acceptor)
Contributors and Attributions
• Darik Benson (Undergraduate University California Davis)
Glycolysis
Glycolysis is the catabolic process in which glucose is converted into pyruvate via ten enzymatic steps. There are three regulatory steps, each of which is highly regulated.
Introduction
There are two phases of Glycolysis:
1. the "priming phase" because it requires an input of energy in the form of 2 ATPs per glucose molecule and
2. the "pay off phase" because energy is released in the form of 4 ATPs, 2 per glyceraldehyde molecule.
The end result of Glycolysis is two new pyruvate molecules which can then be fed into the Citric Acid cycle (also known as the Kreb's Cycle) if oxygen is present, or can be reduced to lactate or ethanol in the absence of of oxygen using a process known as Fermentation. Glycolysis occurs within almost all living cells and is the primary source of Acetyl-CoA, which is the molecule responsible for the majority of energy output under aerobic conditions. The structures of Glycolysis intermediates can be found in the following diagram:
Phase 1: The "Priming Step"
The first phase of Glycolysis requires an input of energy in the form of ATP (adenosine triphosphate).
1. alpha-D-Glucose is phosphorolated at the 6 carbon by ATP via the enzyme Hexokinase (Class: Transferase) to yield alpha-D-Glucose-6-phosphate (G-6-P). This is a regulatory step which is negatively regulated by the presence of glucose-6-phosphate.
2. alpha-D-Glucose-6-phosphate is then converted into D-Fructose-6-phosphate (F-6-P) by Phosphoglucoisomerase (Class: Isomerase)
3. D-Fructose-6-phosphate is once again phosphorolated this time at the 1 carbon position by ATP via the enzyme Phosphofructokinase (Class: Transferase) to yield D-Fructose-1,6-bisphosphate (FBP). This is the committed step of glycolysis because of its large \(\Delta G\) value.
4. D-Fructose-1,6-bisphosphate is then cleaved into two, three carbon molecules; Dihydroxyacetone phosphate (DHAP) and D-Glyceraldehyde-3-phosphate (G-3-P) by the enzyme Fructose bisphosphate aldolase (Class: Lyase)
5. Because the next portion of Glycolysis requires the molecule D-Glyceraldehyde-3-phosphate to continue Dihydroxyacetone phosphate is converted into D-Glyceraldehyde-3-phosphate by the enzyme Triose phosphate isomerase (Class: Isomerase)
Phase 2: The "Pay Off Step"
The second phase of Glycolysis where 4 molecules of ATP are produced per molecule of glucose. Enzymes appear in red:
1. D-Glyceraldehyde-3-phosphate is phosphorolated at the 1 carbon by the enzyme Glyceraldehyde-3-phosphate dehodrogenase to yield the high energy molecule 1,3-Bisphosphoglycerate (BPG)
2. ADP is then phosphorolated at the expense of 1,3-Bisphosphoglycerate by the enzyme Phosphoglycerate kinase (Class: Transferase) to yield ATP and 3-Phosphoglycerate (3-PG)
3. 3-Phosphoglycerate is then converted into 2-Phosphoglycerate by Phosphoglycerate mutase in preparation to yield another high energy molecule
4. 2-Phosphoglycerate is then converted to phosphoenolpyruvate (PEP) by Enolase. H2O, potassium, and magnesium are all released as a result.
5. ADP is once again phosphorolated, this time at the expense of PEP by the enzyme pyruvate kinase to yield another molecule of ATP and and pyruvate. This step is regulated by the energy in the cell. The higher the energy of the cell the more inhibited pyruvate kinase becomes. Indicators of high energy levels within the cell are high concentrations of ATP, Acetyl-CoA, Alanine, and cAMP.
Because Glucose is split to yield two molecules of D-Glyceraldehyde-3-phosphate, each step in the "Pay Off" phase occurs twice per molecule of glucose.
Problems
1. What is the net yield of Glycolysis as far as ATP?
2. Name the enzymes that are key regulatory sites in Glycolysis.
3. Why are the enzymes in the previous question targets for regulation?
4. Why is the priming phase necessary?
5. Draw the entire pathway for glycolysis including enzymes, reactants and products for each step.
Kreb's Cycle
Organisms derive the majority of their energy from the Kreb's Cycle, also known as the TCA cycle. The Kreb's Cycle is an aerobic process consisting of eight definite steps. In order to enter the Kreb's Cycle pyruvate must first be converted into Acetyl-CoA by pyruvate dehydrogenase complex found in the mitochondria.
Introduction
In the presence of oxygen organisms are capable of using the Kreb's Cycle. The reason oxygen is required is because the NADH and [FADH2] produced in the Kreb's Cycle are able to be oxydized in the electron transport chain (ETC) thus replenishing the supply of NAD+ and [FAD].
Steps
In order for pyruvate from glycolysis to enter the Kreb's Cycle it must first be converted into acetyl-CoA by the pyruvate dehydrogenase complex which is an oxidative process wherein NADH and CO2 are formed. Another source of acetyl-CoA is beta oxidation of fatty acids.
1. Acetyl-CoA enters teh Kreb Cycle when it is joined to oxaloacetate by citrate synthase to produce citrate. This process requires the input of water. Oxaloacetate is the final metabolite of the Kreb Cycle and it joins again to start the cycle over again, hence the name Kreb's Cycle. This is known as the committed step
2. Citrate is then converted into isocitrate by the enzyme aconitase. This is accomplished by the removal and addition of water to yield an isomer.
3. Isocitrate is converted into alpha-ketogluterate by isocitrate dehydrogenase. The byproducts of which are NADH and CO2.
4. Apha-ketogluterate is then converted into succynl-CoA by alpha-ketogluterate dehydrogenase. NADH and CO2 are once again produced.
5. Succynl-CoA is then converted into succinate by succynl-CoA synthetase which yields one ATP per succynl-CoA.
6. Succinate coverts into fumerate by way of the enzyme succinate dehydrogenase and [FAD] is reduced to [FADH2] which is a prosthetic group of succinate dehydrogenase. Succinate dehydrogenase is a direct part of the ETC. It is also known as electron carrier II.
7. Fumerate is then converted to malate by hydration with the use of fumerase.
8. Malate is converted into oxaloacetate by malate dehydrogenase the byproducts of which are NADH.
Contributors and Attributions
• Darik Benson, Undergraduate University California Davis | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Metabolism/Catabolism/Fermentation.txt |
The complicated processes of metabolism wouldn't be possible without the help of certain high-energy molecules. The main purpose of these molecules is to transfer either inorganic phosphate groups (Pi) or hydride (H-) ions. The inorganic phosphate groups are used to make high energy bonds with many of the intermediates of metabolism. These bonds can then be broken to yield energy, thus driving the metabolic processes of life. Hydride ions can be transferred from one intermediate to another resulting in a net oxidation or reduction of the intermediate. Oxidation corresponds to a loss of hydride and reduction to the gaining of hydride. Certain reduced forms of high energy molecules such as NADH and [FADH2] can donate their electrons to the electron carriers of the electron transport chain (ETC) which results in the production of ATP (only under aerobic conditions).
ATP
ATP (Adenosine Triphosphate) contains high energy bonds located between each phosphate group. These bonds are known as phosphoric anhydride bonds.
There are three reasons these bonds are high energy:
1. The electrostatic repulsion of the positively charged phosphates and negatively charged oxygen stabilizes the products (ADP + Pi) of breaking these bonds.
2. The stabilization of products by ionization and resonance. As the bonds are broken there is an increased stability due to the resonance of that product's structure.
3. The entropy increases. There is a greater stability in the products because there exists a greater entropy; i.e. more randomness. 1 mole of reactants has a higher energy than 2 moles of products. Disorder is favored over order according to the 2nd law of thermodynamics.
ADP
ADP (Adenosine Diphosphate) also contains high energy bonds located between each phosphate group. It has the same structure as ATP, with one less phosphate group. The same three reasons that ATP bonds are high energy apply to ADP's bonds.
NAD+
NAD+ (Nicotinamide adenine dinucleotide (oxidized form)) is the major electron acceptor for catabolic reactions. It is strong enough to oxidize alcohol groups to carbonyl groups, while other electron acceptors (like [FAD]) are only able to oxidize saturated carbon chains from alkanes to alkenes. It is an important molecule in many metabolic processes like beta-oxidation, glycolysis, and TCA cycle. With out NAD+ the aforementioned processes would be unable to occur.
NADH
NADH (reduced form) is an NAD+ that has accepted electrons in the form of hydride ions. NADH is also one of the molecules responsible for donating electrons to the ETC to drive oxidative phosphorolation and also pyruvate during fermentation processes.
NADP+
NADP+ (Nicotinamide adenine dinucleotide phosphate (oxidized form)) is the major electron donator for anabolic reactions.
NADPH
Nicotinamide adenine dinucleotide phosphate (reduced form)
Problems
1. What is the name of the high energy bond in ATP and ADP?
2. What is the major electron donator in anabolic reactions.
3. Without looking draw the structures of ATP, NAD+, NADH, NADP+, NADPH.
4. What properties make the phosphoric anhydride bond a high energy bond? (Hint: There are three reasons)
5. Think about all of the metabolic pathways, find similarities and differences between the steps that use NADH and the ones that use NADPH.
Contributors and Attributions
• Darik Benson (UCD) | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Metabolism/Important_High_Energy_Molecules_in_Metabolism.txt |
Feeder Pathways: Other catabolic pathways produce products that can enter glycolysis or the TCA cycle. Two examples are given below.
MP2. Overview of Metabolic Pathways: Anabolism
Anabolic reactions are those that lead to the synthesis of biomolecules. In contrast to the catabolic reactions just discussed (glycolysis, TCA cycle and electron transport/oxidative phosphorylation) which lead to the oxidative degradation of carbohydrates and fatty acids and energy release, anabolic reactions lead to the synthesis of more complex biomolecules including biopolymers (glycogen, proteins, nucleic acids) and complex lipids. Many biosynthetic reactions, including those for fatty acid synthesis, are reductive and hence require reducing agents. Reductive biosynthesis and complex polymer formation require energy input, usually in the form of ATP whose exergonic cleavage is coupled to endergonic biosynthesis.
Cells have evolved interesting mechanism so as not to have oxidative degradation reactions (which release energy) proceed at the same time and in the same cell as reductive biosynthesis (which requires energy input). Consider this scenario. You dive into a liver cell and find palmitic acid, a 16C fatty acid. From where did it come? Was it just synthesized by the liver cell or did it just enter the cell from a distant location such as adipocytes (fat cells). Should it be oxidized, which should happen if there is a demand for energy production by the cell, or should the liver cell export it, perhaps to adipocytes, which might happen if there is an excess of energy storage molecules? Cells have devised many ways to distinguish these opposing needs. One is by using a slightly different pool of redox reagents for anabolic and catabolic reactions. Oxidative degradation reactions typically use the redox pair NAD+/NADH (or FAD/FADH2) while reductive biosynthesis often uses phosphorylated variants of NAD+, NADP+/NADPH. In addition, cells often carry out competing reactions in different cellular compartments. Fatty acid oxidation of our example molecule (palmitic acid) occurs in the mitochondrial matrix, while reductive fatty acid synthesis occurs in the cytoplasm of the cell. Fatty acids entering the cell destined for oxidative degradation are transported into the mitochondria by the carnitine transport system. This transport system is inhibited under conditions when fatty acid synthesis is favored. We will discuss the regulation of metabolic pathways in a subsequent section. One of the main methods, as we will see, is to activate or inhibit key enzymes in the pathways under a given set of cellular conditions. The key enzyme in fatty acid synthesis, acetyl-CoA carboxylase, is inhibited when cellular conditions require fatty acid oxidation.
The following examples give short descriptions of anabolic pathways. Compare them to the catabolic pathways from the previous section.
• Glucose synthesis, better known as Gluconeogenesis: In glycolysis, glucose (C6H12O6), a 6C molecule, is converted to two, 3C molecules (pyruvate) in an oxidative process that requires NAD+ and makes two net ATP molecules. In a few organs, most predominately in the liver, the reverse pathway can take place. The liver does this to provide glucose to the brain when the body is deficient in circulating glucose, for example, under fasting and starving conditions. (The liver under these conditions can get its energy from oxidation of fatty acids). The reactions in gluconeogenesis are the same reactions in glycolysis but run in reverse, with the exception of three glycolytic steps which are essentially irreversible. These three steps have bypass enzymes in the gluconeogenesis pathway. Although the synthesis of glucose is a reductive pathway, it uses NADH instead of NADPH as the redundant as the same enzyme used in glycolysis is simply run in reverse. Gluconeogenesis, which also occurs in the cortex of the kidney, is more than just a simple reversal of glycolysis, however. It can be thought of as the net synthesis of glucose from non-carbohydrate precursors. Pyruvate, as seen in the section on catabolism, can be formed from protein degradation to glucogenic amino acids which can be converted to pyruvate. It can also be formed from triacylglycerides from the 3C molecule glycerol formed and released from adipocytes after hydrolysis of three fatty acids from triacylglycerides. However, in humans, glucose can not be made in net fashion from fatty acids. Fatty acids can be converted to acetyl-CoA by fatty acid oxidation. The resulting acetyl-CoA can not form pyruvate since the enzyme that catalyzes the formation for acetyl-CoA from pyruvate, pyruvate dehydrogenase, is irreversible and there is no bypass reaction known. The acetyl-CoA can enter the TCA cycle but since the pathway is cyclic and proceeds in one direction, it can not form in net fashion oxaloacetate. Although oxaloacetate can be remove from the TCA cycle and be use to form phosphoenolpyuvate, a glycolytic intermediate, one acetyl-CoA condenses with one oxaloacetate to form citrate which leads back to one oxaloacetate. Hence fatty acids can not be converted to glucose and other sugars in a net fashion.
• Pentose Phosphate Shunt: This two-part pathway doesn't appear to start as a reductive biosynthetic pathway as the first part is the oxidative conversion of a glycolytic intermediate, glucose-6-phosphate, to ribulose-5-phosphate. The next, nonoxidative branch leads to the formation of ribose-5-phosphate, a key biosynthetic intermediate in nucleic acid synthesis as well as erthyrose-4-phosphate used for biosynthesis of aromatic amino acids . The oxidative branch is important in reductive biosynthesis as it is a major source of the reductant NADPH used in biosynthetic reactions.
• Fatty acid and isoprenoid/sterol biosynthesis: Acetyl-CoA is the source of carbon atoms for the synthesis of more complex lipids such as fatty acids, isoprenoids, and sterols. When energy needs in a cell are not high, citrate, the condensation product of oxaloacetate and acetyl-CoA in the TCA cycle, builds up in the mitochondrial matrix. It is then transported by the citrate transporter (an inner mitochondrial membrane protein) to the cytoplasm, where it is cleaved back to oxaloacetate and acetyl-CoA by the cytoplasmic enzyme citrate lyase. The oxaloacetate is returned to the mitochondria by conversion first to malate (reduction reaction using NADH), which can move back into the mitochondria through the malate transporter, or further conversion to pyruate, using the cytosolic malic enzyme, which uses NADP+ to oxidize malate to pyruvate which then enters the mitochondria. The acetyl-CoA formed in the cytoplasm can then be used in reductive biosynthesis using NADPH as the reductant to form fatty acids, isoprenoids, and sterols. The NADPH for the reduction comes from the oxidative branch of the pentose phosphate pathway and from the reaction catalyzed by malic enzyme. The liver cells can still run the glycolytic pathway as the NADH/NAD+ ratio is low in the cytoplasm while NADPH/NADP+ ratio is high.
Now its time to see how the various pathways fit together to form an integrated set of pathways.
MP3. Metabolic Maps
Now its time to see how the various pathways fit together to form an integrated set of pathways. Metabolic map pathways are by nature very messy and complex. I've created a series of maps below which display some important anabolic and catabolic pathways and how they connect. Many pathways have been omitted. These maps will evolve with time as more relevant information is added. The first maps show the interconnected pathways without much detail. Subsequent maps give increasingly amount of detail. Eventually, the most detailed maps will contain web links to show how given reactions are regulated and will display interactive Jmol molecular models of key enzymes.
These maps are tailored to support foundational courses in chemical reactivity that highlight specific metabolic pathways to illustrate how enzyme-catalyzed reactions can be explain using the language of organic and inorganic chemistry. Of course they are also useful for foundational level biochemistry courses which seek to give an overview of metabolic pathways and their connections. More detailed and comprehensive sites are available on the web. Once such site is the Kyoto Encylopedia of Gene and Genomes (KEGG) Pathway Data Base.
INTEGRATED METABOLIC PATHWAYS MAPS (pdf version)
• INDIVIDUAL METABOLIC PATHWAY MAPS (pdf version)
Best viewed in Firefox as Safari. Some links don't show in Chrome or IE!
• Anabolic
MP4. Regulation of Metabolic Pathways: How Is It Regulated
Exquisite mechanisms have evolved that control the flux of metabolites through metabolic pathways to insure that the output of the pathways meets biological demand and that energy in the form of ATP is not wasted by having opposing pathways run concomitantly in the same cell.
Enzymes can be regulated by changing the activity of a preexisting enzyme or changing the amount of an enzyme.
A. Changing the activity of a pre-existing enzyme: The quickest way to modulate the activity of an enzyme is to alter the activity of an enzyme that already exists in the cell. The list below, illustrated in the following figure, gives common ways to regulate enzyme activity
1. Figure: Regulation of the Activity of Pre-existing Enzymes
B. Changing the amount of an enzyme: Another and less immediate but longer duration method to modulate the activity of an enzyme is to alter the activity of an enzyme that already exists in the cell. The list below, illustrated in the following figure, shows way in which enzyme concentration is regulated. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Metabolism/Metabolic_Pathways/MP1._Overview_of_Metabolic_Pathways%3A_Catabolism.txt |
The use of food by organisms is termed nutrition. Vitamins and minerals necessary for biochemical processes. There are three general categories of food: (1) Essential fiber which are non-digestible polysaccharide material, essential for normal functioning of animal digestive systems (i.e. colon), (2) Energy-yielding nutrients which are protein, carbohydrate and lipid and (3) Micronutrients.
Protein
Animals are unable to synthesize certain amino acids (humans can only make 10 of the 20 common amino acids). The amino acids that an animal is unable to synthesize must be obtained from the diet (i.e. by consuming plants or microorganisms), and these amino acids are termed "essential amino acids".
Excess dietary protein becomes a source of metabolic energy
• Glucogenic amino acids: can be converted into glucose
• Ketogenic amino acids: can be converted into fatty acids or keto acids
• If plenty of fats and carbohydrates are available, then the glucogenic and ketogenic amino acids from excess dietary protein are converted to triacylglycerol and stored as fat
Protein is an important source of nitrogen in the diet. Protein within the body is constantly turning over (i.e. being degraded and resynthesized). Furthermore, there is a general demand for protein synthesis when an organism is growing. The Nitrogen Balance refers to the relationship between the supply and demand for nitrogen (i.e. protein) within an organism.
• A positive nitrogen balance means that the organism is taking in more protein that it needs for growth or turnover
• A negative nitrogen balance means that the organism is not getting enough protein for its normal turnover, or growth. This would represent a nutritional deficiency of protein
Carbohydrate
Carbohydrates are also an essential structural component of nucleic acids, nucleotides, glycoproteins and glycolipids. However, the principle role of carbohydrate in the diet is production of metabolic energy.
• Simple sugars are metabolized in the glycolytic pathway to release energy
• Complex carbohydrates are degraded into simple sugars, which then enter the glycolytic pathway
• Metabolism can make use of a wide variety of sugars for energy production. However, the brain relies solely on glucose for an energy source
• When dietary carbohydrate exceeds the supply needed for energy requirements, it is converted to glycogen and triacylglycerols for storage
• When dietary carbohydrate is in short supply, ketone bodies are formed from acetate units to provide fuel for the brain
Lipids
Fatty acids and triacylglycerols can be used as fuel by many tissues in the human body. Phospholipids are essential components of all biological membranes
• Excess dietary fat is stored as triacylglycerols in adipose tissue
• A deficiency of dietary fat is problematic because some fatty acids cannot be synthesized by the human body, and must be obtained through diet. These are termed
• essential fatty acids
• The human body cannot synthesize linoleic, linolenic or arachidonic fatty acids. These are key components of biological membranes, and arachidonic acid is a precursor of prostaglandins (an important class of hormones). These are therefore considered essential fatty acids
Fiber
"Dietary fiber" refers to molecules that cannot be broken down by enzymes in the human body.
• Cellulose (polysaccharide component of plant cell walls). Required for proper function of colon.
• Lignins (plant polymer of aromatic ring structures). Absorbs organic molecules in the digestive system (binds cholesterol).
Vitamins and minerals
Vitamins are essential nutrients that are required in the diet because they cannot be synthesized by human metabolic enzymes. Often, only trace levels are required, but a shortage can result in disease or death.
• A common categorization of human vitamins is whether they are
• water soluble or fat soluble compounds.
Coenzymes are low molecular weight molecules that provide unique chemical functionalities for certain enzyme/coenzyme complexes.
• Coenzymes may act as carriers of specific functional groups (e.g. methyl or acyl groups)
• They can provide chemically reactive groups that the common 20 amino acid side chains cannot provide
• Coenzymes are
• usually modified in the course of a reaction, and subsequently chemically regenerated back to their useful active form. Thus, this recycling of coenzymes means that only small concentrations are required.
• All the
• water soluble vitamins (with the exception of vitamin C) are coenzymes or precursors of coenzymes.
Summary of water soluble and fat soluble vitamins:
Common name
Chemical name
Related cofactor(s)
Water Soluble Vitamins
Vitamin B1
Thiamine
Thiamine pyrophosphate
Vitamin B2
Riboflavin
Flavin adenine dinucleotide (FAD)
Flavin mononucleotide (FMN)
Vitamin B6
Pyridoxal, pyridoxine, pyridoxamine
Pyridoxal phosphate
Vitamin B12
Cobalamin
5'-deoxyadenosylcobalamin
Methylcobalamin
Niacin
Nicotinic acid
Nicotinamide adenine dinucleotide (NAD+)
Nicotinamide adenine dinucleotide phosphate (NADP+)
Vitamin B3
Pantothenic acid
Coenzyme A
Biotin
Biotin-lysine conjugates (biocytin)
Lipoic acid
Lipoyl-lysine conjugates (lipoamide)
Folic acid
Tetrahydrofolate
Vitamin C
L-ascorbate
Fat Soluble Vitamins
Vitamin A
Retinol
Vitamin D2
Ergocalciferol
Vitamin D3
Cholecalciferol
Vitamin E
a-Tocopherol
Vitamin K
Vitamin B1: Thiamine and thiamine pyrophosphate
Thiamine is the precursor of thiamine pyrophosphate (TPP):
• TPP is a coenzyme
• It is a coenzyme for certain enzymes involved in carbohydrate metabolism
• It catalyzes the synthesis or cleavage of bonds to carbonyl carbons
Niacin (Nicotinic Acid) and nicotinamide coenzymes
Nicotinamide is an essential part of two important coenzymes: nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+).
• The reduced forms of these coenzymes are NADH and NADPH
• The coenzymes participate in
• redox reactions via the direct transfer of hydride (H-) ions either to or from the cofactor and a substrate. The hydride transfer carries two electrons along with it (a proton transfer in acid/base catalysis carries no electrons)
• The hydride transfer involves the C4 carbon of the nicotinamide ring. The quaternary amine of the nicotinamide ring acts as an electron sink to promote acceptance of a hydride ion, or to facilitate leaving of a hydride ion.
• Enzymes that are involved in such redox reactions are called
• dehydrogenases
• The nucleotide part of the molecule does not enter into any chemistry, but
• is important for recognition and binding to enzymes that will use FMN or FAD as a cofactor.
Vitamin B2: Riboflavin
Riboflavin is a constituent of riboflavin 5'-phosphate (flavin mononucleotide, or FMN) and flavin adenine dinucleotide (FAD). The nucleotide part of the molecule does not enter into any chemistry, but is important for recognition and binding to enzymes that will use FMN or FAD as a cofactor.
The isoalloxazine ring is the core structure of the different flavin molecules. It is yellow in color and the word "flavin" is derived from the latin word for yellow, flavus.
• Flavin coenzymes can exists in three different redox states, and each state has a different color (the reduced form is colorless)
• Flavin molecules can participate in both
• one- and two- electron transfer reactions
Vitamin B3: pantothenic acid and coenzyme A
Pantothenic acid is a component of coenzyme A (CoA). The two main functions of CoA are:
a-hydrogen of the acyl group for removal as a proton
1. Activation of acyl groups (R-COX) for transfer to nucleophilic acceptors
2. Activation of the
Both of these functions involve the reactive sulfhydryl group through the formation of thioester linkages with acyl groups
• The 4-phosphopantetheine part of CoA is also used in the same way in
• acyl carrier proteins (ACP's) involved in fatty acid biosynthesis
Vitamin B6: Pyridoxine and pyridoxal phosphate
The biologically active form of vitamin B6 is pyridoxal-5-phosphate (PLP), however, the nutritional requirements can be met by either pyridoxine, pyridoxal or pyridoxol.
PLP participates in a wide variety of reactions involving amino acids, including:
• Transamination
• a- and b-decarboxylation
• b- and g- elimination (not to be confused with painful elimination)
• Racemization
• Aldol reactions
These involve bonds to the amino acid Ca as well as side chain carbons. The wide variety of reactions is due to the ability of PLP to form stable Schiff base adducts with a-amino groups of amino acids:
• In PLP-dependent enzymes, the PLP is present in a Schiff base linkage with the
• e-amino group of an acitve site lysine
• Rearrangement to a Schiff base with the arriving amino acid substrate is a
• transaldiminization reaction
Vitamin B12: Cyanocobalamin
Vitamin B12 is not made by any animal or plant, it is produced by only a few species of bacteria. Once in the food chain, vitamin B12 is obtained by animals by eating other animals, but plants are sadly deficient. Therefore, herbivorous animals (and vegetarians) can suffer a deficiency. The structure contains a cobalt ion, coordinated within a corrin ring structure:
Vitamin B12 (cyanocobalamin) is converted in the body into two coenzymes:
1. 5'-deoxyadenosylcobalamin (the predominant form)
2. Methylcobalamin
Vitamin B12 coenzymes participate in three types of reactions:
1. Intramolecular rearrangements
2. Reductions of ribonucleotides to deoxyribonucleotides in certain bacteria
3. Methyl group transfers (these use methylcobalamin for this purpose)
Vitamin C (L-Ascorbate)
L-Ascorbate is a reducing sugar (has a reactive ene-diol structure) that is involved in the following biochemical processes:
• Hydroxylation of proline and lysine residues in collagen. Without these post-translational modifications the triple helix of collagen is unstable and connective tissue loses its integrity. This is the problem in the disease known as
• scurvy.
• Mobilization of iron, stimulation of immune system, anti-oxidant for scavenging of reactive free-radicals.
Almost all animals can synthesize vitamin C (its in the pathway of carbohydrate synthesis). Humans and great apes have suffered a mutation in the last enzyme in the pathway of synthesis for L-ascorbate (mutation occurred about 10-40 million years ago). Since that time, all great apes (of which humans are a member) must get L-ascorbate from their diet (fresh fruits and vegetable contain an abundance). Thus, for the great apes L-ascorbate is a "vitamin" (another way of looking at it is that all great apes suffer an in-born error in metabolism). Humans still have the gene for the enzyme to make vitamin C. However, it has suffered a couple of deletions that introduce a frame shift mutation, in addition to numerous point mutations.
Biotin
Biotin acts as a mobile carboxyl group carrier in a variety of enzymatic carboxylation reactions.
• Synthesized by intestinal bacteria (finaly, they do something for you)
• Biotin is bound covalently to the enzyme as a prosthetic group via an
• e-amino group of a lysine residue in the enzyme
• This biotin-lysine conjugated amino acid is termed a "biocytin" residue
• The lysine side-chain acts as a flexible "tether" for the biotin, and this flexibility allows the transfer of carboxylate groups within the enzyme
• It is the carrier for the most oxidized form of carbon
• - CO2 (using bicarbonate as the carboxylating agent). The carbon dioxide binds as a carboxy group to one of the ring nitrogens in the biotin
Lipoic Acid
Lipoic acid contains two sulfur atoms that can exist as a disulfide bonded pair, or as two free sulfhydrils. Conversion between the two forms involves a redox reaction (the two free sulfhydrils represent the reduced form). Lipoic acid is typically found covalently attached to a lysine side chain in enzymes that use it as a cofactor, as a lipoamide complex.
• Lipoic acid is an
• acyl group carrier (R-CO-X)
• It also functions to transfer electrons during oxidation and decarboxylation of
• a-keto acids
• Pyruvate dehydrogenase and
• a-ketoglutarate dehydrogenase use lipoic acid as a cofactor
• Its not clear whether a dietary deficiency of lipoic acid contributes to a disease state, so its not technically considered a vitamin
Folic Acid
Folic acid derivatives (i.e. "folates") are acceptors and donors of one-carbon units for all oxidation levels of carbon (except for the most oxidized form - CO2. See biotin above).
• The active coenzyme form is tetrahydrofolate (THF). Folate is reduced to THF by the action of tetrahydrofolate reductase.
• Three different oxidation states of carbon can bind to THF. These are oxidation states of -2 (methanol group), 0 (formaldehyde group) and +2 (formate group)
• These groups are attached to the THF molecule at either the N5 or N10 atom positions
• The biosynthetic pathways of
• methionine, homocysteine, purines, and thymine rely one one-carbon units being provided by THF.
Vitamin A: Retinol
Vitamin A occurs as an ester (Retinyl ester), aldehyde (Retinal) or acidic form (Retinoic acid).
• It is a fat soluble vitamin, and is synthesized from isoprene building blocks
• Obtained directly from an animal diet, or synthesized from
• b-carotene provided by plants
• It is essential to vision. Retinol transported to the eyes is oxidized by retinol dehydrogenase to produce trans-retinal. Trans-retinal is converted to 11-cis-retinal by retinal isomerase. The aldehyde group of retinal forms a Schiff base with a lysin of the protein opsin, to form rhodopsin (the light-sensitive pigment of vision).
• Vitamin A is essential for various biological processes - including fetal development and sperm development. But excessive vitamin A is toxic.
Vitamin D: Ergocalciferol (D2) and cholecalciferol (D3)
Cholecalciferol is produced in the skin of animals by the action of U.V. light on the precursor molecule 7-dehydrocholesterol.
• Light energy induces bond-breakage (between carbons 9 and 10) and formation of previtamin D3. Spontaneous isomerization produces D3
• Ergocalciferol is produced by the action of U.V. light on the plant sterol ergosterol.
• Since humans can produce D3 from 7-dehydrocholesterol, vitamin D3 is technically not really a vitamin
• Cholecalciferol is really a prohormone. Derivatives of this compound
• regulate calcium and phosphate metabolism.
• Inadequate intestinal absorption of calcium and phosphate can result in demineralization of bones, and the disease Rickets.
Vitamin E: Tocopherol
a-Tocopherol is a potent antioxidant, however, molecular details of its function are not clearly understood.
• Fatty acids in membranes are susceptible to oxidative damage
• Vitamin E is fat soluble and may protect membrane fatty acids from oxidation
• A deficiency of vitamin E results in red blood cells that are susceptible to oxidative damage
• Retinal damage in premature infants, due to supplemental oxygen, may be preventable by administering vitamin E
Vitamin K: Napthoquinone
Vitamin K is essential to the blood-clotting process. Vitamin K is required for the post-translational modification to produce g-carboxy glutamic acid from glutamic acid. Such modified residues can bind Ca2+, which is an essential part of the process in the clotting cascade.
g-carboxy glutamic acids in their structure
• Prothrombin ("factor II"), and factors VII, IX and X are serine proteases that participate in a protease activation cascade that is involved in blood coagulation, and have
Outside Links
http://www.mikeblaber.org/oldwine/BCH4053/bch4053.htm
Contributors and Attributions
• Dr Michael Blaber | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Metabolism/Nutrition.txt |
The secondary structure of DNA is actually very similar to the secondary structure of proteins. The protein single alpha helix structure held together by hydrogen bonds was discovered with the aid of X-ray diffraction studies. The X-ray diffraction patterns for DNA show somewhat similar patterns.
Introduction
In addition, chemical studies by E. Chargaff indicate several important clues about the structure of DNA. In the DNA of all organisms:
1. Chargaff's findings clearly indicate that some type of heterocyclic amine base pairing exists in the DNA structure. X-ray diffraction data shows that a repeating helical pattern occurs every 34 Angstrom units with 10 subunits per turn. Each subunit occupies 3.4 Angstrom units which is the same amount of space occupied by a single nucleotide unit. Using Chargaff's information and the X-ray data in conjunction with building actual molecular models, Watson and Crick developed the double helix as a model for DNA.
The double helix in DNA consists of two right-handed polynucleotide chains that are coiled about the same axis. The heterocyclic amine bases project inward toward the center so that the base of one strand interacts or pairs with a base of the other strand. According to the chemical and X-ray data and model building exercises, only specific heterocyclic amine bases may be paired.
Base Pairing Principle
The Base Pairing Principle is that adenine pairs with thymine (A - T) and guanine pairs with cytosine (G - C)
The base pairing is called complementary because there are specific geometry requirements in the formation of hydrogen bonds between the heterocylic amines. Heterocyclic amine base pairing is an application of the hydrogen bonding principle. In the structures for the complementary base pairs given in the graphic on the left, notice that the thymine - adenine pair interacts through two hydrogen bonds represented as (T=A) and that the cytosine-guanine pair interacts through three hydrogen bonds represented as (C=G).
Although other base pairing-hydrogen bonding combinations may be possible, they are not utilized because the bond distances do not correspond to those given by the base pairs already cited. The diameter of the helix is 20 Angstroms.
DNA Double Helix
The double-stranded helical model for DNA is shown in the graphic on the left. The easiest way to visualize DNA is as an immensely long rope ladder, twisted into a cork-screw shape. The sides of the ladder are alternating sequences of deoxyribose and phosphate (backbone) while the rungs of the ladder (bases) are made in two parts with each part firmly attached to the side of the ladder. The parts in the rung are heterocyclic amines held in position by hydrogen bonding. Although most DNA exists as open ended double helices, some bacterial DNA has been found as a cyclic helix. Occasionally, DNA has also been found as a single strand.
Problems
QUES. Describe the structure of the double helix of DNA in your own words including the terms: backbone, heterocyclic amines, complementary base pairings, hydrogen bonding, deoxyribose, phosphate.
Quiz: In RNA, which base hydrogen bonds with uracil? Carefully compare the structure of uracil to the others to find the one that is most similar.
Quiz: If DNA is heated, what happens to the double helix? Hint: The result is similar to the denaturing of a protein by the same method. What type of bonding holds the secondary structure of both proteins and DNA?
Outside Links
• The structure of the 'Dickerson Dodecamer' was originally reported in: Drew, H. R., Wing, R. M., Takano, T., Broka, C., Tanaka, S., Itakura, K. & Dickerson, R. E. (1981). Structure of a B-DNA dodecamer: conformation and dynamics. Proc. Natl. Acad. Sci. USA 78, 2179-2183. and the coordinates were acquired from the Brookhaven Protein Data Bank. The filename is 1BNA.
DNA: Mutations
This page takes a very brief look at what happens if the code in DNA becomes changed in some way, and the effect that would have on the proteins it codes for. Copying errors when DNA replicates or is transcribed into RNA can cause changes in the sequence of bases which makes up the genetic code. Radiation and some chemicals can also cause changes. The examples which follow show some of the easier-to-understand effects of this.
Contributors and Attributions
Jim Clark (Chemguide.co.uk) | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Nucleic_Acids/DNA/DNA%3A_Double_Helix.txt |
The hereditary material in a cell is coded in the sequence of the heterocyclic amines of DNA. There are normally 46 strands of DNA called chromosomes in human cells. Specific regions, called genes, on each chromosome contain the hereditary information which distinguishes individuals from each other. The genes also contain the coded information required for the synthesis of proteins and enzymes needed for the normal functions of the cells. Bacterial cells may have 1000 genes, while the human cell contains more than a million genes. A single E. coli (bacteria) chromosome of double helical DNA consists of 3.4 million base pairs.
Introduction
Prior to cell division, the DNA material in the original cell must be duplicated so that after cell division, each new cell contains the full amount of DNA material. The process of DNA duplication is usually called replication. The replication is termed semiconservative since each new cell contains one strand of original DNA and one newly synthesized strand of DNA. The original polynucleotide strand of DNA serves as a template to guide the synthesis of the new complementary polynucleotide of DNA. A template is a guide that may be used for example, by a carpenter to cut intricate designs in wood.
The DNA single strand template serves to guide the synthesis of a complementary strand of DNA. DNA polymerase III is an example of this process. More explanation in the next panel.
DNA Replication Process
Several enzymes and proteins are involved with the replication of DNA. At a specific point, the double helix of DNA is caused to unwind possibly in response to an initial synthesis of a short RNA strand using the enzyme helicase. Proteins are available to hold the unwound DNA strands in position. Each strand of DNA then serves as a template to guide the synthesis of its complementary strand of DNA. DNA polymerase III is used to join the appropriate nucleotide units together. The replication process is shown in graphic on the left.
Template #1 guides the formation of a new complementary #2 strand. The DNA template guides the formation of a DNA complementary strand - not an exact copy of itself. For example looking at template # 2, this process occurs because the heterocyclic amine, adenine (A), codes or guides the incorporation of only thymine (T) to synthesize new DNA #1. The replication of DNA is guided by the base pairing principle so that no other heterocyclic amine nucleotide can hydrogen bond and fit correctly with cytosine. The next heterocyclic amine, cytosine (C), guides the incorporation of guanine (G) while similar arguments apply to the other bases. Exactly the opposite reaction occurs using template #2 (far right margin) where cytosine (C) guides the incorporation of guanine (G) to form a new complementary #2 strand.
It is so important that the cells duplicate the DNA genetic material exactly, that the sequence of newly synthesized nucleotides is checked by two different polymerase enzymes. The second enzyme can check for and actually correct any mistake of mismatched base pairs in the sequence. The mismatched nucleotides are hydrolyzed and cut out and new correct ones are inserted.
DNA Replication
Although details of DNA replication is not thoroughly understood, because so many molecules are involved in the process. This example focuses on the bacteriophage T7 DNA replication complex because it consists of relatively few proteins. The mechanism of T7 DNA replication is a good model for other DNA replication. This molecule is based on the recent work of Doublie, et al. (1998). In the graphic below, the DNA polymerase enzyme is shown with a short section of DNA. The green color represents the DNA template, while the magenta color represents the newly synthesized DNA.
In the close up, guanine triphosphate nucleotide is shown on the active site, guided by the cytosine in the template matching through hydrogen bonds. Only a few of the enzyme protein side chain interactions with the nucleotide are shown. Magnesium ions are also active in stabilizing the triphosphate through ionic interactions. Eventually the two of the phosphates are hydrolyzed and the remaining phosphate is bonded in a phosphate ester bond to the deoxyribose on the end of the newly forming DNA chain.
Problems
1. In the green ribbon form of the enzyme, on the outside there are several series where the ribbons lie side by side. What protein structure is this?
2. On the inside of the "clamp", what protein structures are visible?
3. Using a single strand of DNA, write the sequence of heterocyclic amines to make its complementary strand.
Template: A C T A G G
Answers
1. These are beta pleated sheets in the tertiary structure.
2. There are numerous alpha helices that line the "hole" in the tertiary structure.
3. Complementary T G A T C C
Outside Links
• Link to: Great Animation of entire DNA Repllication - John Kyrk
• More details: E. coli DNA Polymerase III Beta Subunit The Sliding DNA Clamp © David Marcey, 1997
• Reference: Kong, X-P., Onrust, R., O'Donnell, M., and J. Kuriyan (1992). Three-Dimensional Structure of the Beta Subunit of E. coli DNA Polymerase III Holoenzyme: A Sliding DNA Clamp. Cell 69: 425-437.
• More Details: The Bacteriophage T7 DNA Replication Complex, Michael E. Ward and David Marcey. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Nucleic_Acids/DNA/DNA%3A_Replication.txt |
Deoxyribonucleic acid (DNA) is a macromolecule that consists of deoxyribonucleotide monomers linked to each other by phosphodiester bonds. The sequence of these nucleotides contains translates into a genetic blueprint by which a cell can synthesize proteins.
Mechanics
In cells, DNA exists as a double-stranded molecule that twists around its axis to form a helical structure. Each strand is a complement to the other; the nucleotides on one strand hydrogen-bond with complementary nucleotides on the opposite strand. The double helical "twist" occurs because of the angular geometry of each bonded nucleotide.
Uncoiled DNA can exist in either a linear form or as a closed-loop molecule (plasmid). Yet each complete turn of the double-helix spans either 10.7 base pairs (A-DNA) or 10.5 base pairs (B-DNA), so in order for a plasmid to close stably it must be a multiple of 10.7 or 10.5 base pairs in length.
A-DNA, B-DNA, Z-DNA
There are three major geometric configurations of DNA:
B-DNA is the "generic" double helical form of DNA that is typically presented in introductory biology textbooks and on television. It is the form that predominates in vivo (in live cells), and is unmethylated. Every complete turn of the helix spans ~10.5 base pairs. B-DNA is right-handed, and has a barely noticeable tilt from its vertical axis. B-DNA has a wide major groove at which proteins can bind.
Unlike B-DNA, A-DNA has a narrower major groove and a wider minor groove. Consequently, A-DNA binds proteins at the minor groove. A-DNA also has a significant tilt from its vertical axis and each helical rotation requires 10.7 base pairs. A-DNA typically forms either when DNA duplexes with RNA, or at low water concentrations.
Z-DNA earns its name from a zigzag-like appearance. It is narrower than either B-DNA or A-DNA, and is left-handed unlike the other two forms (both right-handed). Z-DNA also has a tilt from its vertical axis, but not as great as A-DNA. Z-DNA is sometimes formed in vivo, often due to alternating guanine and cytosine nucleotides or as a result of methylation.
Supercoiling
Without supporting proteins, DNA undergoes "supercoiling" and collapses onto itself. This is because its double-helical nature creates a torsional strain, similar to a twisted piece of rope.
CHROMATIN PACKAGING
In eukaryotic cells, linear DNA is packaged into a dense material called chromatin. This prevents supercoiling, keeps the DNA precisely organized, and prevents disastrous shearing during cell division. Specifically, DNA is wrapped around a histone pentamer (tetramer in some cases), forming a nucleosome. Below is a visual representation of the first degree of DNA packaging where multiple nucleosomes span the DNA molecule like beads on a string:
Histones interact with each other to form what is called the 30-nm fiber (referring to the thickness of the structure). Typically, less active genes are packed in a 30-nm fiber. When cell division occurs, the 30-nm is scaffolded to more structural proteins, until eventually the chromatin is packed into structures known as chromosomes.
Plasmids
Plasmids are typically found in bacteria, however some eukaryotes such as the yeast Saccharomyces cerevisiae also contain plasmids. Histones are not found in prokaryotes, and DNA is not packaged the way it is in eukaryotic cells. Therefore, plasmids are typically found in supercoiled form. Two common shapes are the toroid and the plectoneme:
SIGNIFICANCE
DNA shape affects how/whether proteins can bind to it, which has important consequences for gene transcription and regulation since condensed DNA cannot bind polymerases. DNA shape also affects its molecular mobility. This is an important consideration in gel electrophoresis, where linear DNA travels faster than plasmids of the same length in base pairs, and supercoiled plasmids travel faster than uncoiled plasmids.
Contributors
• Dmitry Ratner (UCD)
DNA Structure
DNA is the primary agent for all genetic material. This is common sense but it was not always that way.
O.T. Avery (1944)
The experiment of Griffith could not be taken further until methods were developed to separate and purify DNA and protein cellular components. Avery utilized methods to extract relatively pure DNA from pneumococcus to determine whether it was the "transforming agent" observed in Griffith's experiments.
The experiment:
• w.t. (smooth) type I -> extract the DNA component
• mutant (rough) type II + type I DNA + mouse = dead mouse
Isolation of bacteria from the dead mouse showed that they were type I w.t. (smooth) bacteria
A more sophisticated experiment:
Purified type I DNA was divided into two aliquots. One aliquot was treated with DNAse - an enzyme which non-specifically degrades DNA. The other aliquot was treated with Trypsin - a protease which (relatively) non-specfically degrades proteins.
• Type I DNA + DNAse + mutant (rough) type II + mouse = live mouse
• Type I DNA + Trypsin + mutant (rough) type II + mouse = dead mouse
The work of Avery provided strong evidence that the "transforming agent" was in fact DNA (and not protein). However, not everyone was convinced. Some people felt that a residual amount of protein might remain in the purified DNA, even after Trypsin treatment, and could be the "transforming agent".
A.D. Hershey and M. Chase (1952)
T2 is a virus which attacks the bacteria E. coli. The virus, or phage, looks like a tiny lunar landing module.
The viral particles adsorb to the surface of the E. coli cells. It was known that some material then leaves the phage and enters the cell. The "empty" phage particles on the surface cells can be physically removed by putting the cells into a blender and whipping them up. In any case, some 20 minutes after the phage adsorb to the surface of the bacteria the bacteria bursts open (lysis) and releases a multitude of progeny virus.
If the media in which the bacteria grew (and were infected) included 32P labeled ATP, progeny phage could be recovered with this isotope incorporated into its DNA (normal proteins contain only hydrogen, nitrogen, carbon, oxygen, and sulfur atoms). Likewise if the media contained 35S labeled methionine the resulting progeny phage could be recovered with this isotope present only in its protein components (normal DNA contains only hydrogen, nitrogen, carbon, oxygen and phosphorous atoms). | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Nucleic_Acids/DNA/DNA_Structure/DNA_History.txt |
This page looks at how the base sequences in DNA and RNA are used to code for particular amino acids when it comes to building protein chains.
Contributors and Attributions
Jim Clark (Chemguide.co.uk)
The Replication of DNA
This page takes a very simplified look at how DNA replicates (copies) itself. It gives only a brief over-view of the process, with no attempt to describe the mechanism.
Semi-conservative replication
We'll explain exactly what "semi-conservative" means when we have got some diagrams to look at. First imagine what happens if the two individual strands in the DNA double helix start to unzip. The diagram shows this happening in the middle of the DNA double helix - you mustn't assume that the top of the diagram is the end of the chain. It isn't. Further up the double helix, the two strands will still be joined together.
In fact, this is happening lots of times along the very long DNA molecule. Lengths of chain become separated to form what are known as "bubbles". If you feel the need to see this in more detail, read the rest of this page, and then have a quick look at the links above.
Some of the hydrogen bonds get broken and the two strands become partly separated. The red dotted lines on the diagram just point out the original base pairs. These are not bonds in any form. These base pairs are now much too far apart for any sort of bonding between them. Now suppose that you have a source of nucleotides - phosphate joined to deoxyribose joined to a base, including all the four sorts of bases needed for DNA.
The next diagram shows what would happen if a nucleotide containing guanine (G) and one containing cytosine (C) were attracted to the top two bases on the left-hand strand of the unzipped DNA - and then joined together.
How did they end up joined together? This is all under the control of a number of enzymes, one of which (DNA polymerase) is responsible for joining up nucleotides along the chain in this way. Now suppose the same sort of thing happened at the top of the right-hand strand. You would end up with . . .
Now compare the double strands that you are forming on the left- and right-hand sides. They are exactly the same . . . and if you were to continue this process, they would continue to be the same. And if you compare the patterns of bases in the new DNA being formed with what was in the original DNA before it started to unzip, everything is the same. This is inevitable because of the way the bases pair together.
What does semi-conservative replication mean?
Let's simplify the last diagram, and assume that the whole copying process is complete. The next diagram focusses on the short bit of the total DNA molecule that we have been looking at. A typical human DNA molecule is around 150 million base pairs long - you will have to imagine the rest of it! You have also got to remember that in reality the whole thing would have coiled into its double helix. Trying to draw that just makes everything look messy and complicated!
The original DNA is shown all in blue. The red strands in the daughter DNA are the ones which have been built on the original blue strands during the replication process.
You can see that each of the daughter molecules is made of half of the original DNA plus a new strand. That's all "semi-conservative replication" means. Half of the original DNA is conserved (kept) in each of the daughter molecules. The red and blue, of course, have no physical significance apart from as a way of making the diagrams clearer. All three of these DNA molecules will be identical in every way.
The Structure of DNA
This page, looking at the structure of DNA, is the first in a sequence of pages leading on to how DNA replicates (makes copies of) itself, and then to how information stored in DNA is used to make protein molecules.
Contributors and Attributions
Jim Clark (Chemguide.co.uk)
Transcription of DNA Into messenger RNA
This page takes a simple look at the structure of RNA and how the information in DNA is used to make messenger RNA
Contributors and Attributions
Jim Clark (Chemguide.co.uk) | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Nucleic_Acids/DNA/The_Genetic_Code.txt |
The nucleic acids are informational molecules because their primary structure contains a code or set of directions by which they can duplicate themselves and guide the synthesis of proteins. The synthesis of proteins - most of which are enzymes - ultimately governs the metabolic activities of the cell. In 1953, Watson, an American biologist, and Crick, an English biologist, proposed the double helix structure for DNA. This development set the stage for a new and continuing era of chemical and biological investigation. The two main events in the life of a cell - dividing to make exact copies of themselves, and manufacturing proteins - both rely on blueprints coded in our genes.
Nucleic Acids
The first isolation of what we now refer to as DNA was accomplished by Johann Friedrich Miescher circa 1870. He reported finding a weakly acidic substance of unknown function in the nuclei of human white blood cells, and named this material "nuclein". A few years later, Miescher separated nuclein into protein and nucleic acid components. In the 1920's nucleic acids were found to be major components of chromosomes, small gene-carrying bodies in the nuclei of complex cells. Elemental analysis of nucleic acids showed the presence of phosphorus, in addition to the usual C, H, N & O. Unlike proteins, nucleic acids contained no sulfur. Complete hydrolysis of chromosomal nucleic acids gave inorganic phosphate, 2-deoxyribose (a previously unknown sugar) and four different heterocyclic bases (shown in the following diagram). To reflect the unusual sugar component, chromosomal nucleic acids are called deoxyribonucleic acids, abbreviated DNA. Analogous nucleic acids in which the sugar component is ribose are termed ribonucleic acids, abbreviated RNA. The acidic character of the nucleic acids was attributed to the phosphoric acid moiety.
The two monocyclic bases shown here are classified as pyrimidines, and the two bicyclic bases are purines. Each has at least one N-H site at which an organic substituent may be attached. They are all polyfunctional bases, and may exist in tautomeric forms.
Base-catalyzed hydrolysis of DNA gave four nucleoside products, which proved to be N-glycosides of 2'-deoxyribose combined with the heterocyclic amines. Structures and names for these nucleosides will be displayed above by clicking on the heterocyclic base diagram. The base components are colored green, and the sugar is black. As noted in the 2'-deoxycytidine structure on the left, the numbering of the sugar carbons makes use of primed numbers to distinguish them from the heterocyclic base sites. The corresponding N-glycosides of the common sugar ribose are the building blocks of RNA, and are named adenosine, cytidine, guanosine and uridine (a thymidine analog missing the methyl group).
From this evidence, nucleic acids may be formulated as alternating copolymers of phosphoric acid (P) and nucleosides (N), as shown:
~ P – N – P – N'– P – N''– P – N'''– P – N ~
At first the four nucleosides, distinguished by prime marks in this crude formula, were assumed to be present in equal amounts, resulting in a uniform structure, such as that of starch. However, a compound of this kind, presumably common to all organisms, was considered too simple to hold the hereditary information known to reside in the chromosomes. This view was challenged in 1944, when Oswald Avery and colleagues demonstrated that bacterial DNA was likely the genetic agent that carried information from one organism to another in a process called "transformation". He concluded that "nucleic acids must be regarded as possessing biological specificity, the chemical basis of which is as yet undetermined." Despite this finding, many scientists continued to believe that chromosomal proteins, which differ across species, between individuals, and even within a given organism, were the locus of an organism's genetic information. It should be noted that single celled organisms like bacteria do not have a well-defined nucleus. Instead, their single chromosome is associated with specific proteins in a region called a "nucleoid". Nevertheless, the DNA from bacteria has the same composition and general structure as that from multicellular organisms, including human beings.
Views about the role of DNA in inheritance changed in the late 1940's and early 1950's. By conducting a careful analysis of DNA from many sources, Erwin Chargaff found its composition to be species specific. In addition, he found that the amount of adenine (A) always equaled the amount of thymine (T), and the amount of guanine (G) always equaled the amount of cytosine (C), regardless of the DNA source. As set forth in the following table, the ratio of (A+T) to (C+G) varied from 2.70 to 0.35. The last two organisms are bacteria.
Table: Nucleoside Base Distribution in DNA
Organism
Base Composition (mole %)
Base Ratios
Ratio (A+T)/(G+C)
A G T C A/T G/C
Human
30.9 19.9 29.4 19.8 1.05 1.00 1.52
Chicken
28.8 20.5 29.2 21.5 1.02 0.95 1.38
Yeast
31.3 18.7 32.9 17.1 0.95 1.09 1.79
Clostridium
perfringens
36.9 14.0 36.3 12.8 1.01 1.09 2.70
Sarcina
lutea
13.4 37.1 12.4 37.1 1.08 1.00 0.35
In a second critical study, Alfred Hershey and Martha Chase showed that when a bacterium is infected and genetically transformed by a virus, at least 80% of the viral DNA enters the bacterial cell and at least 80% of the viral protein remains outside. Together with the Chargaff findings this work established DNA as the repository of the unique genetic characteristics of an organism.
The Chemical Nature of DNA
The polymeric structure of DNA may be described in terms of monomeric units of increasing complexity. In the top shaded box of the following illustration, the three relatively simple components mentioned earlier are shown. Below that on the left , formulas for phosphoric acid and a nucleoside are drawn. Condensation polymerization of these leads to the DNA formulation outlined above. Finally, a 5'- monophosphate ester, called a nucleotide may be drawn as a single monomer unit, shown in the shaded box to the right. Since a monophosphate ester of this kind is a strong acid (pKa of 1.0), it will be fully ionized at the usual physiological pH (ca.7.4). Names for these DNA components are given in the table to the right of the diagram. Isomeric 3'-monophospate nucleotides are also known, and both isomers are found in cells. They may be obtained by selective hydrolysis of DNA through the action of nuclease enzymes. Anhydride-like di- and tri-phosphate nucleotides have been identified as important energy carriers in biochemical reactions, the most common being ATP (adenosine 5'-triphosphate).
Table: Names of DNA Base Derivatives
Base
Nucleoside
5'-Nucleotide
Adenine 2'-Deoxyadenosine 2'-Deoxyadenosine-5'-monophosphate
Cytosine 2'-Deoxycytidine 2'-Deoxycytidine-5'-monophosphate
Guanine 2'-Deoxyguanosine 2'-Deoxyguanosine-5'-monophosphate
Thymine 2'-Deoxythymidine 2'-Deoxythymidine-5'-monophosphate
A complete structural representation of a segment of the DNA polymer formed from 5'-nucleotides may be viewed by clicking on the above diagram. Several important characteristics of this formula should be noted.
• First, the remaining P-OH function is quite acidic and is completely ionized in biological systems.
• Second, the polymer chain is structurally directed. One end (5') is different from the other (3').
• Third, although this appears to be a relatively simple polymer, the possible permutations of the four nucleosides in the chain become very large as the chain lengthens.
• Fourth, the DNA polymer is much larger than originally believed. Molecular weights for the DNA from multicellular organisms are commonly 109 or greater.
Information is stored or encoded in the DNA polymer by the pattern in which the four nucleotides are arranged. To access this information the pattern must be "read" in a linear fashion, just as a bar code is read at a supermarket checkout. Because living organisms are extremely complex, a correspondingly large amount of information related to this complexity must be stored in the DNA. Consequently, the DNA itself must be very large, as noted above. Even the single DNA molecule from an E. coli bacterium is found to have roughly a million nucleotide units in a polymer strand, and would reach a millimeter in length if stretched out. The nuclei of multicellular organisms incorporate chromosomes, which are composed of DNA combined with nuclear proteins called histones. The fruit fly has 8 chromosomes, humans have 46 and dogs 78 (note that the amount of DNA in a cell's nucleus does not correlate with the number of chromosomes). The DNA from the smallest human chromosome is over ten times larger than E. coli DNA, and it has been estimated that the total DNA in a human cell would extend to 2 meters in length if unraveled. Since the nucleus is only about 5μm in diameter, the chromosomal DNA must be packed tightly to fit in that small volume.
In addition to its role as a stable informational library, chromosomal DNA must be structured or organized in such a way that the chemical machinery of the cell will have easy access to that information, in order to make important molecules such as polypeptides. Furthermore, accurate copies of the DNA code must be created as cells divide, with the replicated DNA molecules passed on to subsequent cell generations, as well as to progeny of the organism. The nature of this DNA organization, or secondary structure, will be discussed in a later section.
RNA, a Different Nucleic Acid
The high molecular weight nucleic acid, DNA, is found chiefly in the nuclei of complex cells, known as eucaryotic cells, or in the nucleoid regions of procaryotic cells, such as bacteria. It is often associated with proteins that help to pack it in a usable fashion. In contrast, a lower molecular weight, but much more abundant nucleic acid, RNA, is distributed throughout the cell, most commonly in small numerous organelles called ribosomes. Three kinds of RNA are identified, the largest subgroup (85 to 90%) being ribosomal RNA, rRNA, the major component of ribosomes, together with proteins. The size of rRNA molecules varies, but is generally less than a thousandth the size of DNA. The other forms of RNA are messenger RNA , mRNA, and transfer RNA , tRNA. Both have a more transient existence and are smaller than rRNA.
All these RNA's have similar constitutions, and differ from DNA in two important respects. As shown in the following diagram, the sugar component of RNA is ribose, and the pyrimidine base uracil replaces the thymine base of DNA. The RNA's play a vital role in the transfer of information (transcription) from the DNA library to the protein factories called ribosomes, and in the interpretation of that information (translation) for the synthesis of specific polypeptides. These functions will be described later.
A complete structural representation of a segment of the RNA polymer formed from 5'-nucleotides may be viewed by clicking on the above diagram
The Secondary Structure of DNA
In the early 1950's the primary structure of DNA was well established, but a firm understanding of its secondary structure was lacking. Indeed, the situation was similar to that occupied by the proteins a decade earlier, before the alpha helix and pleated sheet structures were proposed by Linus Pauling. Many researchers grappled with this problem, and it was generally conceded that the molar equivalences of base pairs (A & T and C & G) discovered by Chargaff would be an important factor. Rosalind Franklin, working at King's College, London, obtained X-ray diffraction evidence that suggested a long helical structure of uniform thickness. Francis Crick and James Watson, at Cambridge University, considered hydrogen bonded base pairing interactions, and arrived at a double stranded helical model that satisfied most of the known facts, and has been confirmed by subsequent findings.
Base Pairing
Careful examination of the purine and pyrimidine base components of the nucleotides reveals that three of them could exist as hydroxy pyrimidine or purine tautomers, having an aromatic heterocyclic ring. Despite the added stabilization of an aromatic ring, these compounds prefer to adopt amide-like structures. These options are shown in the following diagram, with the more stable tautomer drawn in blue.
A simple model for this tautomerism is provided by 2-hydroxypyridine. As shown on the left below, a compound having this structure might be expected to have phenol-like characteristics, such as an acidic hydroxyl group. However, the boiling point of the actual substance is 100º C greater than phenol and its acidity is 100 times less than expected (pKa = 11.7). These differences agree with the 2-pyridone tautomer, the stable form of the zwitterionic internal salt. Further evidence supporting this assignment will be displayed by clicking on the diagram. Note that this tautomerism reverses the hydrogen bonding behavior of the nitrogen and oxygen functions (the N-H group of the pyridone becomes a hydrogen bond donor and the carbonyl oxygen an acceptor).
The additional evidence for the pyridone tautomer, that appears above by clicking on the diagram, consists of infrared and carbon nmr absorptions associated with and characteristic of the amide group. The data for 2-pyridone is given on the left. Similar data for the N-methyl derivative, which cannot tautomerize to a pyridine derivative, is presented on the right.
Once they had identified the favored base tautomers in the nucleosides, Watson and Crick were able to propose a complementary pairing, via hydrogen bonding, of guanosine (G) with cytidine (C) and adenosine (A) with thymidine (T). This pairing, which is shown in the following diagram, explained Chargaff's findings beautifully, and led them to suggest a double helix structure for DNA.
Before viewing this double helix structure itself, it is instructive to examine the base pairing interactions in greater detail. The G#C association involves three hydrogen bonds (colored pink), and is therefore stronger than the two-hydrogen bond association of A#T. These base pairings might appear to be arbitrary, but other possibilities suffer destabilizing steric or electronic interactions. By clicking on the diagram two such alternative couplings will be shown. The C#T pairing on the left suffers from carbonyl dipole repulsion, as well as steric crowding of the oxygens. The G#A pairing on the right is also destabilized by steric crowding (circled hydrogens).
A simple mnemonic device for remembering which bases are paired comes from the line construction of the capital letters used to identify the bases. A and T are made up of intersecting straight lines. In contrast, C and G are largely composed of curved lines. The RNA base uracil corresponds to thymine, since U follows T in the alphabet.
The Double Helix
After many trials and modifications, Watson and Crick conceived an ingenious double helix model for the secondary structure of DNA. Two strands of DNA were aligned anti-parallel to each other, i.e. with opposite 3' and 5' ends , as shown in part a of the following diagram. Complementary primary nucleotide structures for each strand allowed intra-strand hydrogen bonding between each pair of bases. These complementary strands are colored red and green in the diagram. Coiling these coupled strands then leads to a double helix structure, shown as cross-linked ribbons in part b of the diagram. The double helix is further stabilized by hydrophobic attractions and pi-stacking of the bases. A space-filling molecular model of a short segment is displayed in part c on the right.
The helix shown here has ten base pairs per turn, and rises 3.4 Å in each turn. This right-handed helix is the favored conformation in aqueous systems, and has been termed the B-helix. As the DNA strands wind around each other, they leave gaps between each set of phosphate backbones. Two alternating grooves result, a wide and deep major groove (ca. 22Å wide), and a shallow and narrow minor groove (ca. 12Å wide). Other molecules, including polypeptides, may insert into these grooves, and in so doing perturb the chemistry of DNA. Other helical structures of DNA have also been observed, and are designated by letters (e.g. A and Z).
DNA Replication
In their 1953 announcement of a double helix structure for DNA, Watson and Crick stated, "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.". The essence of this suggestion is that, if separated, each strand of the molecule might act as a template on which a new complementary strand might be assembled, leading finally to two identical DNA molecules. Indeed, replication does take place in this fashion when cells divide, but the events leading up to the actual synthesis of complementary DNA strands are sufficiently complex that they will not be described in any detail.
As depicted in the following drawing, the DNA of a cell is tightly packed into chromosomes. First, the DNA is wrapped around small proteins called histones (colored pink below). These bead-like structures are then further organized and folded into chromatin aggregates that make up the chromosomes. An overall packing efficiency of 7,000 or more is thus achieved. Clearly a sequence of unfolding events must take place before the information encoded in the DNA can be used or replicated.
Once the double stranded DNA is exposed, a group of enzymes act to accomplish its replication. These are described briefly here:
• Topoisomerase: This enzyme initiates unwinding of the double helix by cutting one of the strands.
• Helicase: This enzyme assists the unwinding. Note that many hydrogen bonds must be broken if the strands are to be separated..
• SSB: A single-strand binding-protein stabilizes the separated strands, and prevents them from recombining, so that the polymerization chemistry can function on the individual strands.
• DNA Polymerase: This family of enzymes link together nucleotide triphosphate monomers as they hydrogen bond to complementary bases. These enzymes also check for errors (roughly ten per billion), and make corrections.
• Ligase: Small unattached DNA segments on a strand are united by this enzyme.
Polymerization of nucleotides takes place by the phosphorylation reaction described by the following equation.
Di- and triphosphate esters have anhydride-like structures and are consequently reactive phosphorylating reagents, just as carboxylic anhydrides are acylating reagents. Since the pyrophosphate anion is a better leaving group than phosphate, triphosphates are more powerful phosphorylating agents than are diphosphates. Formulas for the corresponding 5'-derivatives of adenosine will be displayed by Clicking Here, and similar derivatives exist for the other three common nucleosides. The DNA polymerization process that builds the complementary strands in replication, could in principle take place in two ways. Referring to the general equation above, R1 could represent the next nucleotide unit to be attached to the growing DNA strand, with R2 being this strand. Alternatively, these assignments could be reversed. In practice, the former proves to be the best arrangement. Since triphosphates are very reactive, the lifetime of such derivatives in an aqueous environment is relatively short. However, such derivatives of the individual nucleosides are repeatedly synthesized by the cell for a variety of purposes, providing a steady supply of these reagents. In contrast, the growing DNA segment must maintain its functionality over the entire replication process, and can not afford to be changed by a spontaneous hydrolysis event. As a result, these chemical properties are best accommodated by a polymerization process that proceeds at the 3'-end of the growing strand by 5'-phosphorylation involving a nucleotide triphosphate. This process is illustrated by the following animation, which may be activated by clicking on the diagram or reloading the page.
The polymerization mechanism described here is constant. It always extends the developing DNA segment toward the 3'-end (i.e. when a nucleotide triphosphate attaches to the free 3'-hydroxyl group of the strand, a new 3'-hydroxyl is generated). There is sometimes confusion on this point, because the original DNA strand that serves as a template is read from the 3'-end toward the 5'-end, and authors may not be completely clear as to which terminology is used.
Because of the directional demand of the polymerization, one of the DNA strands is easily replicated in a continuous fashion, whereas the other strand can only be replicated in short segmental pieces. This is illustrated in the following diagram. Separation of a portion of the double helix takes place at a site called the replication fork. As replication of the separate strands occurs, the replication fork moves away (to the left in the diagram), unwinding additional lengths of DNA. Since the fork in the diagram is moving toward the 5'-end of the red-colored strand, replication of this strand may take place in a continuous fashion (building the new green strand in a 5' to 3' direction). This continuously formed new strand is called the leading strand. In contrast, the replication fork moves toward the 3'-end of the original green strand, preventing continuous polymerization of a complementary new red strand. Short segments of complementary DNA, called Okazaki fragments, are produced, and these are linked together later by the enzyme ligase. This new DNA strand is called the lagging strand.
When you consider that a human cell has roughly 109 base pairs in its DNA, and may divide into identical daughter cells in 14 to 24 hours, the efficiency of DNA replication must be extraordinary. The procedure described above will replicate about 50 nucleotides per second, so there must be many thousand such replication sites in action during cell division. A given length of double stranded DNA may undergo strand unwinding at numerous sites in response to promoter actions. The unraveled "bubble" of single stranded DNA has two replication forks, so assembly of new complementary strands may proceed in two directions. The polymerizations associated with several such bubbles fuse together to achieve full replication of the entire DNA double helix. A cartoon illustrating these concerted replications will appear by clicking on the above diagram. Note that the events shown proceed from top to bottom in the diagram.
Repair of DNA Damage and Replication Errors
One of the benefits of the double stranded DNA structure is that it lends itself to repair, when structural damage or replication errors occur. Several kinds of chemical change may cause damage to DNA:
• Spontaneous hydrolysis of a nucleoside removes the heterocyclic base component.
• Spontaneous hydrolysis of cytosine changes it to a uracil.
• Various toxic metabolites may oxidize or methylate heterocyclic base components.
• Ultraviolet light may dimerize adjacent cytosine or thymine bases.
All these transformations disrupt base pairing at the site of the change, and this produces a structural deformation in the double helix.. Inspection-repair enzymes detect such deformations, and use the undamaged nucleotide at that site as a template for replacing the damaged unit. These repairs reduce errors in DNA structure from about one in ten million to one per trillion.
RNA and Protein Synthesis
The genetic information stored in DNA molecules is used as a blueprint for making proteins. Why proteins? Because these macromolecules have diverse primary, secondary and tertiary structures that equip them to carry out the numerous functions necessary to maintain a living organism. As noted in the protein chapter, these functions include:
• Structural integrity (hair, horn, eye lenses etc.).
• Molecular recognition and signaling (antibodies and hormones).
• Catalysis of reactions (enzymes)..
• Molecular transport (hemoglobin transports oxygen).
• Movement (pumps and motors).
The critical importance of proteins in life processes is demonstrated by numerous genetic diseases, in which small modifications in primary structure produce debilitating and often disastrous consequences. Such genetic diseases include Tay-Sachs, phenylketonuria (PKU), sickel cell anemia, achondroplasia, and Parkinson disease. The unavoidable conclusion is that proteins are of central importance in living cells, and that proteins must therefore be continuously prepared with high structural fidelity by appropriate cellular chemistry.
Early geneticists identified genes as hereditary units that determined the appearance and / or function of an organism (i.e. its phenotype). We now define genes as sequences of DNA that occupy specific locations on a chromosome. The original proposal that each gene controlled the formation of a single enzyme has since been modified as: one gene = one polypeptide. The intriguing question of how the information encoded in DNA is converted to the actual construction of a specific polypeptide has been the subject of numerous studies, which have created the modern field of Molecular Biology.
The Central Dogma and Transcription
Francis Crick proposed that information flows from DNA to RNA in a process called transcription, and is then used to synthesize polypeptides by a process called translation. Transcription takes place in a manner similar to DNA replication. A characteristic sequence of nucleotides marks the beginning of a gene on the DNA strand, and this region binds to a promoter protein that initiates RNA synthesis. The double stranded structure unwinds at the promoter site., and one of the strands serves as a template for RNA formation, as depicted in the following diagram. The RNA molecule thus formed is single stranded, and serves to carry information from DNA to the protein synthesis machinery called ribosomes. These RNA molecules are therefore called messenger-RNA (mRNA).
To summarize: a gene is a stretch of DNA that contains a pattern for the amino acid sequence of a protein. In order to actually make this protein, the relevant DNA segment is first copied into messenger-RNA. The cell then synthesizes the protein, using the mRNA as a template.
An important distinction must be made here. One of the DNA strands in the double helix holds the genetic information used for protein synthesis. This is called the sense strand, or information strand (colored red above). The complementary strand that binds to the sense strand is called the anti-sense strand (colored green), and it serves as a template for generating a mRNA molecule that delivers a copy of the sense strand information to a ribosome. The promoter protein binds to a specific nucleotide sequence that identifies the sense strand, relative to the anti-sense strand. RNA synthesis is then initiated in the 3' direction, as nucleotide triphosphates bind to complementary bases on the template strand, and are joined by phosphate diester linkages. An animation of this process for DNA replication was presented earlier. A characteristic "stop sequence" of nucleotides terminates the RNA synthesis. The messenger molecule (colored orange above) is released into the cytoplasm to find a ribosome, and the DNA then rewinds to its double helix structure.
In eucaryotic cells the initially transcribed m-RNA molecule is usually modified and shortened by an "editing" process that removes irrelevant material. The DNA of such organisms is often thousands of times larger and more complex than that composing the single chromosome of a procaryotic bacterial cell. This difference is due in part to repetitive nucleotide sequences (ca. 25% in the human genome). Furthermore, over 95% of human DNA is found in intervening sequences that separate genes and parts of genes. The informational DNA segments that make up genes are called exons, and the noncoding segments are called introns. Before the mRNA molecule leaves the nucleus, the nonsense bases that make up the introns are cut out, and the informationally useful exons are joined together in a step known as RNA splicing. In this fashion shorter mRNA molecules carrying the blueprint for a specific protein are sent on their way to the ribosome factories.
The Central Dogma of molecular biology, which at first was formulated as a simple linear progression of information from DNA to RNA to Protein, is summarized in the following illustration. The replication process on the left consists of passing information from a parent DNA molecule to daughter molecules. The middle transcription process copies this information to a mRNA molecule. Finally, this information is used by the chemical machinery of the ribosome to make polypeptides.
As more has been learned about these relationships, the central dogma has been refined to the representation displayed on the right. The dark blue arrows show the general, well demonstrated, information transfers noted above. It is now known that an RNA-dependent DNA polymerase enzyme, known as a reverse transcriptase, is able to transcribe a single-stranded RNA sequence into double-stranded DNA (magenta arrow). Such enzymes are found in all cells and are an essential component of retroviruses (e.g. HIV), which require RNA replication of their genomes (green arrow). Direct translation of DNA information into protein synthesis (orange arrow) has not yet been observed in a living organism. Finally, proteins appear to be an informational dead end, and do not provide a structural blueprint for either RNA or DNA.
In the following section the last fundamental relationship, that of structural information translation from mRNA to protein, will be described
Translation
Translation is a more complex process than transcription. This would, of course, be expected. After all, the coded messages produced by the German Enigma machine could be copied easily, but required a considerable decoding effort before they could be read with understanding. In a similar sense, DNA replication is simply a complementary base pairing exercise, but the translation of the four letter (bases) alphabet code of RNA to the twenty letter (amino acids) alphabet of protein literature is far from trivial. Clearly, there could not be a direct one-to-one correlation of bases to amino acids, so the nucleotide letters must form short words or codons that define specific amino acids. Many questions pertaining to this genetic code were posed in the late 1950's:
• How many RNA nucleotide bases designate a specific amino acid? If separate groups of nucleotides, called codons, serve this purpose, at least three are needed. There are 43 = 64 different nucleotide triplets, compared with 42 = 16 possible pairs.
• Are the codons linked separately or do they overlap? Sequentially joined triplet codons will result in a nucleotide chain three times longer than the protein it describes. If overlapping codons are used then fewer total nucleotides would be required.
• If triplet segments of mRNA designate specific amino acids in the protein, how are the codons identified? For the sequence ~CUAGGU~ are the codons CUA & GGU or ~C, UAG & GU~ or ~CU, AGG & U~?
• Are all the codon words the same size? In Morse code the most widely used letters are shorter than less common letters. Perhaps nature employs a similar scheme.
Physicists and mathematicians, as well as chemists and microbiologists all contributed to unravelling the genetic code. Although earlier proposals assumed efficient relationships that correlated the nucleotide codons uniquely with the twenty fundamental amino acids, it is now apparent that there is considerable redundancy in the code as it now operates. Furthermore, the code consists exclusively of non-overlapping triplet codons. Clever experiments provided some of the earliest breaks in deciphering the genetic code. Marshall Nirenberg found that RNA from many different organisms could initiate specific protein synthesis when combined with broken E.coli cells (the enzymes remain active). A synthetic polyuridine RNA induced synthesis of poly-phenylalanine, so the UUU codon designated phenylalanine. Likewise an alternating ~CACA~ RNA led to synthesis of a ~His-Thr-His-Thr~ polypeptide.
The following table presents the present day interpretation of the genetic code. Note that this is the RNA alphabet, and an equivalent DNA codon table would have all the U nucleotides replaced by T. Methionine and tryptophan are uniquely represented by a single codon. At the other extreme, leucine is represented by eight codons. The average redundancy for the twenty amino acids is about three. Also, there are three stop codons that terminate polypeptide synthesis.
RNA Codons for Protein Synthesis
Second Position
U C A G
F
i
r
s
t
P
o
s
i
t
i
o
n
U
UUU Phe [F]
UUC Phe [F]
UUA Leu [L]
UUG Leu [L]
UCU Ser [S]
UCC Ser [S]
UCA Ser [S]
UCG Ser [S]
UAU Tyr [Y]
UAC Tyr [Y]
UAA Stop
UAG Stop
UGU Cys [C]
UGC Cys [C]
UGA Stop
UGG Trp [W]
U
C
A
G
T
h
i
r
d
P
o
s
i
t
i
o
n
C
CUU Leu [L]
CUC Leu [L]
CUA Leu [L]
CUG Leu [L]
CCU Pro [P]
CCC Pro [P]
CCA Pro [P]
CCG Pro [P]
CAU His [H]
CAC His [H]
CAA Gln [Q]
CAG Gln [Q]
CGU Arg [R]
CGC Arg [R]
CGA Arg [R]
CGG Arg [R]
U
C
A
G
A
AUU Ile [I]
AUC Ile [I]
AUA Ile [I]
AUG Met [M]
ACU Thr [T]
ACC Thr [T]
ACA Thr [T]
ACG Thr [T]
AAU Asn [N]
AAC Asn [N]
AAA Lys [K]
AAG Lys [K]
AGU Ser [S]
AGC Ser [S]
AGA Arg [R]
AGG Arg [R]
U
C
A
G
G
GUU Val [V]
GUC Val [V]
GUA Val [V]
GUG Val [V]
GCU Ala [A]
GCC Ala [A]
GCA Ala [A]
GCG Ala [A]
GAU Asp [D]
GAC Asp [D]
GAA Glu [E]
GAG Glu [E]
GGU Gly [G]
GGC Gly [G]
GGA Gly [G]
GGG Gly [G]
U
C
A
G
The translation process is fundamentally straightforward. The mRNA strand bearing the transcribed code for synthesis of a protein interacts with relatively small RNA molecules (about 70-nucleotides) to which individual amino acids have been attached by an ester bond at the 3'-end. These transfer RNA's (tRNA) have distinctive three-dimensional structures consisting of loops of single-stranded RNA connected by double stranded segments. This cloverleaf secondary structure is further wrapped into an "L-shaped" assembly, having the amino acid at the end of one arm, and a characteristic anti-codon region at the other end. The anti-codon consists of a nucleotide triplet that is the complement of the amino acid's codon(s). Models of two such tRNA molecules are shown to the right. When read from the top to the bottom, the anti-codons depicted here should complement a codon in the previous table.
Cloverleaf cartoons of three other tRNA molecules will be shown on the right by clicking on the diagram.
A cell's protein synthesis takes place in organelles called ribosomes. Ribosomes are complex structures made up of two distinct and separable subunits (one about twice the size of the other). Each subunit is composed of one or two RNA molecules (60-70%) associated with 20 to 40 small proteins (30-40%). The ribosome accepts a mRNA molecule, binding initially to a characteristic nucleotide sequence at the 5'-end (colored light blue in the following diagram). This unique binding assures that polypeptide synthesis starts at the right codon. A tRNA molecule with the appropriate anti-codon then attaches at the starting point and this is followed by a series of adjacent tRNA attachments, peptide bond formation and shifts of the ribosome along the mRNA chain to expose new codons to the ribosomal chemistry.
The following diagram is designed as a slide show illustrating these steps. The outcome is synthesis of a polypeptide chain corresponding to the mRNA blueprint. A "stop codon" at a designated position on the mRNA terminates the synthesis by introduction of a "Release Factor".
To visit an informative Tour of the Ribosome site, created by Wayne Decatur, Univ. Mass. Amherst Click Here.
Post-translational Modification
Once a peptide or protein has been synthesized and released from the ribosome it often undergoes further chemical transformation. This post-translational modification may involve the attachment of other moieties such as acyl groups, alkyl groups, phosphates, sulfates, lipids and carbohydrates. Functional changes such as dehydration, amidation, hydrolysis and oxidation (e.g. disulfide bond formation) are also common. In this manner the limited array of twenty amino acids designated by the codons may be expanded in a variety of ways to enable proper functioning of the resulting protein. Since these post-translational reactions are generally catalyzed by enzymes, it may be said: "Virtually every molecule in a cell is made by the ribosome or by enzymes made by the ribosome."
Modifications, like phosphorylation and citrullination, are part of common mechanisms for controlling the behavior of a protein. As shown on the left below, citrullination is the post-translational modification of the amino acid arginine into the amino acid citrulline. Arginine is positively charged at a neutral pH, whereas citrulline is uncharged, so this change increases the hydrophobicity of a protein. Phosphorylation of serine, threonine or tyrosine residues renders them more hydrophilic, but such changes are usually transient, serving to regulate the biological activity of the protein. Other important functional changes include iodination of tyrosine residues in the peptide thyroglobulin by action of the enzyme thyroperoxidase. The monoiodotyrosine and diiodotyrosine formed in this manner are then linked to form the thyroid hormones T3 and T4, shown on the right below.
Amino acids may be enzymatically removed from the amino end of the protein. Because the "start" codon on mRNA codes for the amino acid methionine, this amino acid is usually removed from the resulting protein during post-translational modification. Peptide chains may also be cut in the middle to form shorter strands. Thus, insulin is initially synthesized as a 105 residue preprotein. The 24-amino acid signal peptide is removed, yielding a proinsulin peptide. This folds and forms disulfide bonds between cysteines 7 and 67 and between 19 and 80. Such dimeric cysteines, joined by a disulfide bond, are named cystine. A protease then cleaves the peptide at arg31 and arg60, with loss of the 32-60 sequence (chain C). Removal of arg31 yields mature insulin, with the A and B chains held together by disulfide bonds and a third cystine moiety in chain A. The following cartoon illustrates this chain of events.
Nisin is a polypeptide (34 amino acids) made by the bacterium Lactococcus lactis. Nisin kills gram positive bacteria by binding to their membranes and targeting lipid II, an essential precursor of cell wall synthesis. Such antimicrobial peptides are a growing family of compounds which have received the name lantibiotics due to the presence of lanthionine, a nonproteinogenic amino acid with the chemical formula HO2C-CH(NH2)-CH2-S-CH2-CH(NH2)-CO2H. Lanthionine is composed of two alanine residues that are crosslinked on their β-carbon atoms by a thioether linkage (i.e. it is the monosulfide analog of the disulfide cystine). Lantibiotics are unique in that they are ribosomally synthesized as prepeptides, followed by post-translational processing of a number of amino acids (e.g. serine, threonine and cysteine) into dehydro residues and thioether crossbridges. Nisin is the only bacteriocin that is accepted as a food preservative. Several nisin subtypes that differ in amino acid composition and biological activity are known. A typical structure is drawn below, and a Jmol model will be presented by clicking on the diagram.
The bacterial cell wall is a cross-linked glycan polymer that surrounds bacterial cells, dictates their cell shape, and prevents them from breaking due to environmental changes in osmotic pressure. This wall consists mainly of peptidoglycan or murein, a three-dimensional polymer of sugars and amino acids located on the exterior of the cytoplasmic membrane.
The monomer units are composed of two amino sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), shown on the right. Transglycosidase enzymes join these units by glycoside bonds, and they are further interlinked to each other via peptide cross-links between the pentapeptide moieties that are attached to the NAM residues. Peptidoglycan subunits are assembled on the cytoplasmic side of the bacterial membrane from a polyisoprenoid anchor. Lipid II, a membrane-anchored cell-wall precursor that is essential for bacterial cell-wall biosynthesis, is one of the key components in the synthesis of peptidoglycan. Peptidoglycan synthesis via polymerization of Lipid II is illustrated in the following diagram. Cross-linking of the peptide side chains is then effected by transpeptidase enzymes. A model of Lipid II complexed with nisin may be examined as part of the previous Jmol display.
In order for bacteria to divide by binary fission and increase their size following division, links in the peptidoglycan must be broken, new peptidoglycan monomers must be inserted, and the peptide cross links must be resealed. Transglycosidase enzymes catalyze the formation of glycosidic bonds between the NAM and NAG of the peptidoglycan monomers and the NAG and NAM of the existing peptidoglycan. Finally, transpeptidase enzymes reform the peptide cross-links between the rows and layers of peptidoglycan making the wall strong. Many antibiotic drugs, including penicillin, target the chemistry of cell wall formation. The effectiveness of choosing Lipid II for an antibacterial strategy is highlighted by the fact that it is the target for at least four different classes of antibiotic, including the clinically important glycopeptide antibiotic vancomycin. The growing problem of bacterial resistance to many current drugs, including vancomycin, has led to increasing interest in the therapeutic potential of other classes of compound that target Lipid II. Lantibiotics such as nisin are part of this interest.
For a speculative discussion of why nature selected the components and functional groups found in the nucleic acids Click Here.
Analysis of Structural Similarities and Differences between DNA and RNA
Background
We know that living organisms have the ability to reproduce and to pass many of their characteristics on to their offspring. From this we may infer that all organisms have genetic substances and an associated chemistry that enable inheritance to occur. It is instructive to consider the essential requirements such genetic materials must fullfill.
Information
Biologically useful information, especially instructions for protein synthesis, must be incorporated in the material.
Stability
The inherited information must be stable (unchanged) over the lifetime of the organism if accurate copies are to be conveyed to the offspring. Infrequent changes may take place (see mutability).
Reproduction
A method of faithfully replicating the information encoded in the material, and transmitting this copy to the offspring must exist.
Mutability
Despite the inherent stability noted above, the material must be capable of incorporating stable structural change, and passing this change on to succeeding generations.
Since this genetic substance has been identified as the nucleic acids DNA and RNA, it is instructive to examine the manner in which these polymers satisfy the above requirements.
Information Storage
The complexity of life suggests that even simple organisms will require very large inheritance libraries. Although the four nucleotides that make up of DNA might appear to be too simple for this task, the enormous size of the polymer and the permutations of the monomers within the chain meet the challenge easily. After all, the words and graphics in this document are all presented to the computer as combinations of only two characters, zeros and ones (the binary number system). DNA has four letters in its alphabet (A, C, G & T), so the number of words that can be formed increase exponentially with the number of letters per word. Thus, there are 42 or 16 two letter words, and 43 or 64 three letter words.
Assuring the stability of information encoded by the DNA alphabet presents a serious challenge. If the letters of this alphabet are to be strung together in a specific way on the polymer chain, chemical reactions for attaching (and removing) them must be available. Simple carboxylic ester or amide links might appear suitable for this purpose (note step-growth polymerization), but these are used in lipids and polypeptides, so a separate enzymatic machinery would be needed to keep the information processing operations apart from other molecular transformations.
The overall stability of such covalent links presents a more serious problem. Under physiological conditions (aqueous, pH near 7.4 & 27 to 37º C) esters are slowly hydrolyzed. Amides are more stable, but even a hydrolytic cleavage of one bond per hour would be devastating to a polymer having tens of thousands to millions such links. Furthermore, short difunctional linking groups, such as carbonates, oxylates and malonates show enhanced reactivity, and their parent acids are unstable or toxic.
Ester Hydrolysis at 35º C and pH 7
Ester
Rate of Hydrolysis
Relative Rate
Ethyl Acetate
CH3CO2C2H5
1.0*10-2 5*106
Trimethyl Phosphate
(CH3O)3PO
3.4*10-4 2*105
Dimethyl Phosphate
(CH3O)2PO2(-)
2.0*10-9 1.0
Phosphate is an ubiquitous inorganic nutrient. Mono, di and triesters of the corresponding acid (phosphoric acid) are all known. Because of their acidity (pKa ≈ 2), the mono and diesters are negatively charged at physiological pH, rendering them less susceptible to nucleophilic attack. The influence of negative charge on the rate of nucleophilic hydrolysis of some representative esters is shown in the table on the right. Clearly, a polymer in which monomer units are joined by negatively charged diphosphate ester links should be substantially more stable than one composed of carboxylate ester bonds. The negative charge found on all biological phosphate derivatives serves other purposes as well.
The diphosphate ester links that join the nucleotides units of DNA are formed by phosphorylation reactions involving nucleotide triphosphate reagents. These reagents are the phosphoric acid analogs of carboxylic acid anhydrides, a functional group that would not survive the aqueous environment of a cell. The high density of negative charge on the triphosphate function not only solubilizes the organic moiety to which it is attached, but also reduces the rate at which it is hydrolyzed.
Living cells must conserve and employ their chemical reagents within a volume defined and enclosed by a membrane barrier. These lipid bilayer membranes have hydrophobic interiors, which resist the passage of ions. Indeed, special trans-membrane structures called ion channels exist so that controlled ion transport across a membrane may take place. Small neutral organic molecules, such as adenosine, cytidine and guanosine, may pass through lipid membranes, albeit at a reduced rate, but their mono, di and triphosphate derivatives are more tightly sequestered in the cell.
Why is 2'-Deoxyribose the Sugar Moiety in DNA?
Common perhydroxylated sugars, such as glucose and ribose, are formed in nature as products of the reductive condensation of carbon dioxide we call photosynthesis. The formation of deoxysugars requires additional biological reduction steps, so it is reasonable to speculate why DNA makes use of the less common 2'-deoxyribose, when ribose itself serves well for RNA. At least two problems associated with the extra hydroxyl group in ribose may be noted. First, the additional bulk and hydrogen bonding character of the 2'-OH interfere with a uniform double helix structure, preventing the efficient packing of such a molecule in the chromosome. Second, RNA undergoes spontaneous hydrolytic cleavage about one hundred times faster than DNA. This is believed due to intramolecular attack of the 2'-hydroxyl function on the neighboring phosphate diester, yielding a 2',3'-cyclic phosphate. If stability over the lifetime of an organism is an essential characteristic of a gene, then nature's selection of 2'-deoxyribose for DNA makes sense. The following diagram illustrates the intramolecular cleavage reaction in a strand of RNA.
Structural stability is not a serious challenge for RNA. The transcripted information carried by mRNA must be secure for only a few hours, as it is transported to a ribosome. Once in the ribosome it is surrounded by structural and enzymatic segments that immediately incorporate its codons for protein synthesis. The tRNA molecules that carry amino acids to the ribosome are similarly short lived, and are in fact continuously recycled by the cellular chemistry.
The Thymine vs. Uracil Issue
Structural formulas for the three pyrimidine bases, cytosine, thymine and uracil are shown on the right. The carbon atoms that are part of these compounds may be categorized as follows. All of these compounds are apparently put together from a three-carbon malonate-like precursor (blue colored bonds) and a single high oxidation state carbon species (colored red). Such biosynthetic intermediates are well established. Thymine is unique in having an additional carbon, the green methyl group. Biosynthesis of this compound must involve additional steps, thus adding constructional complexity to the DNA molecules in which it replaces uracil.
The reason for the substitution of thymine for uracil in DNA may be associated with the repair mechanisms by which the cell corrects damage to its DNA. One source of error in the code is the slow hydrolysis of heterocyclic enamines, such as cytosine and guanine, to their corresponding lactams. This changes the structure of the base, and disrupts base pairing in a manner that can be identified and then repaired. However, the hydrolysis product from cytosine is uracil, and this mismatched species must somehow be distinguished from the uracil-like base that belongs in the DNA. The extra methyl group serves this role nicely.
For a more complete discussion of some of the issues touched on here see an article titled "Why Nature Chose Phosphates", authored by F. H .Westheimer, which appeared in the March 6th, 1987 issue of Science.
Nucleic Acids
The nucleic acids are informational molecules because their primary structure contains a code or set of directions by which they can duplicate themselves and guide the synthesis of proteins. The synthesis of proteins - most of which are enzymes - ultimately governs the metabolic activities of the cell. In 1953, Watson, an American biologist, and Crick, an English biologist, proposed the double helix structure for DNA. This development set the stage for a new and continuing era of chemical and biological investigation. The two main events in the life of a cell - dividing to make exact copies of themselves, and manufacturing proteins - both rely on blueprints coded in our genes.
Introduction
There are two types of nucleic acids which are polymers found in all living cells. Deoxyribonucleic Acid (DNA) is found mainly in the nucleus of the cell, while Ribonucleic Acid (RNA) is found mainly in the cytoplasm of the cell although it is usually synthesized in the nucleus. DNA contains the genetic codes to make RNA and the RNA in turn then contains the codes for the primary sequence of amino acids to make proteins.
Nucleic Acid Parts List
The best way to understand the structures of DNA and RNA is to identify and examine individual parts of the structures first. The complete hydrolysis of nucleic acids yields three major classes of compounds: pentose sugars, phosphates, and heterocyclic amines (or bases).
Phosphate
A major requirement of all living things is a suitable source of phosphorus. One of the major uses for phosphorus is as the phosphate ion which is incorporated into DNA and RNA.
Pentose Sugars
There are two types of pentose sugars found in nucleic acids. This difference is reflected in their names--deoxyribonucleic acid indicates the presence of deoxyribose; while ribonucleic acid indicates the presence of ribose. In the graphic below, the structures of both ribose and deoxyribose are shown. Note the red -OH on one and the red -H on the other are the only differences. The alpha and beta designations are interchangeable and are not a significant difference between the two.
Heterocyclic Amines
Heterocyclic amines are sometimes called nitrogen bases or simply bases. The heterocyclic amines are derived from two root structures: purines or pyrimidines. The purine root has both a six and a five member ring; the pyrimidine has a single six member ring. There are two major purines, adenine (A) and guanine (G), and three major pyrimidines, cytosine (C), uracil (U), and thymine (T). The structures are shown in the graphic on the left. As you can see, these structures are called "bases" because the amine groups as part of the ring or as a side chain have a basic property in water
.
A major difference between DNA and RNA is that DNA contains thymine, but not uracil, while RNA contains uracil but not thymine. The other three heterocyclic amines, adenine, guanine, and cytosine are found in both DNA and RNA. For convenience, you may remember, the list of heterocyclic amines in DNA by the words: The Amazing Gene Code (TAGC). | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Nucleic_Acids/Nucleic_Acids/Nucleic_Acids.txt |
Nucleotides are the basic monomer building block units in the nucleic acids. A nucleotide consists of a phosphate, pentose sugar, and a heterocyclic amine.
Adenosine 5'-monophosphate (AMP)
The phosphoric acid forms a phosphate-ester bond with the alcohol on carbon #5 in the pentose. A nitrogen in the heterocyclic amines displaces the -OH group on carbon #1 of the pentose. The reaction is shown in the graphic below. If the sugar is ribose, the general name is ribonucleotide and deoxyribonucleotide if the sugar is deoxyribose. The other four nucleotides are synthesized in a similar fashion.
• Simple Representation of Nucleotide Polymer
Just as the exact amino acid sequence is important in proteins, the sequence of heterocyclic amine bases determines the function of the DNA and RNA. This sequence of bases on DNA determines the genetic information carried in each cell. Currently, much research is under way to determine the heterocyclic amine sequences in a variety of RNA and DNA molecules. The Genome Project as already succeeded in determining the DNA sequences in humans and other organisms. Future research will be to determine the exact functions of each DNA segment as these contain the codes for protein synthesis.
Protein Synthesis 1
This page looks at how the information coded in messenger RNA is used to build protein chains.
Contributors and Attributions
Jim Clark (Chemguide.co.uk)
Protein-RNA Recognition
RNA-protein interactions are behind a number of vital processes in the cell. Without the ability of particular proteins to bind RNA, the RNA would no longer be able to carry out its important functions as a component of the ribosome1,2 and spliceosome.3 Other examples of important RNA-protein interactions include binding of tRNA to aminoacyl-tRNA synthetases, a process vital to translation of genetic information into proteins necessary for continued biological function4 and regulation of post-transcriptional control of gene expression via the binding of RNA to riobonucleoproteins, or RNPs.5
Although not as well characterized as the binding between DNA and proteins, RNA-protein binding has been a field that has seen a great deal of growth in recent years. Although it was originally expected that RNA-protein binding motifs might fall neatly into categories the way DNA motifs did, the wide range of secondary and tertiary RNA structures that can be recognized by proteins requires more variety in binding motifs of the proteins, and the rules used to categorize them become correspondingly more complex.6 At this time all major families of RNA-binding proteins have been structurally characterized and these characterizations have led to a much better understanding of RNA recognition.7
Contributors and Attributions
• Therese Gerbich- Truman State University, Kirksville, MO
• Ashley Hoaglin- Truman State University, Kirksville, MO
RNA - Transcription
The biosynthesis of RNA, called transcription, proceeds in much the same fashion as the replication of DNA and also follows the base pairing principle. Again, a section of DNA double helix is uncoiled and only one of the DNA strands serves as a template for RNA polymerase enzyme to guide the synthesis of RNA. After the synthesis is complete, the RNA separates from the DNA and the DNA recoils into its helix.
Introduction
The differences in the composition of RNA and DNA have already been noted. In addition, RNA is not usually found as a double helix but as a single strand. However, the single polynucleotide strand may fold back on itself to form portions which have a double helix structure like the tertiary structure of proteins.
The transcription of a single RNA strand is illustrated in the graphic on the left. One major difference is that the heterocyclic amine, adenine, on DNA codes for the incorporation of uracil in RNA rather than thymine as in DNA. Remember that thymine is not found in RNA and do not confuse the replacement of uracil in RNA for thymine in DNA in the transcription process. For example, thymine in DNA still codes for adenine on RNA not uracil, while the adenine on DNA codes for uracil in RNA. Note that the new RNA (red) is identical to non coding DNA with the exception of uracil where thymine was located in DNA.
There are three major types of RNA which will be fully explained in a later section. Although RNA is synthesized in the nucleus, it migrates out of the nucleus into the cytoplasm where it is used in the synthesis of proteins.
RNA Transcription Process
The RNA transcription process occurs in three stages: initiation, chain elongation, and termination.
The first stage occurs when the RNA Polymerase-Promoter Complex binds to the promoter gene in the DNA. This also allows for the finding of the start sequence for the RNA polymerase. The promoter enzyme will not work unless the sigma protein is present (shown in blue in graphic). Specific sequences on the non coding strand of DNA are recognized as the signal to start the unwinding process.
The recognition sequences are as follows:
Non-coding DNA -5' recognition sections in bold
GGCCGCTTGACAAAAGTGTTAAATTGTGCTATACT
Once the process has been initiated, then the RNA polymerase elongation enzyme takes over and is described in the next panel.
RNA Polymerase - Elongation
The elongation begins when the RNA polymerase "reads" the template DNA. Only one strand of the DNA is read for the base sequence. The RNA which is synthesized is the complementary strand of the DNA. The RNA (top strand) and DNA (bottom strand) sequences in the model are:
5' -GACCAGGCA-3'
3'-TCTGGTCCGTAAA-5'
In the graphic, the magenta color is the template DNA, while the green is the RNA strand. In the next reaction step, uracil triphosphate (UTP) is the next to be added to the RNA by bind and pairing with the adenine (A) nucleotide on the template DNA strand.
A phosphodiester bond is formed; the RNA chain is than elongated to 10 nucleotides; and diphosphate left over would dissociate. Note: The coordinates used in this display have only the alpha carbons of the proteins RNA Polymerase.
Outside Links
• Link with more details - RNA Polymerase - Promoter Complex
• Credit: Murakami, K. S., Masuda, S., Campbell, E. A., Muzzin, O., Darst, S. A. (2002) "Structural Basis of Transcription Initiation: An RNA Polymerase Holoenzyme-DNA Complex "Science 2961285.
• More details: Yeast RNA Polymerase II Elongation Complex
• Credit: Gnatt, A. L., Cramer, P., Fu, J., Bushnell, D. A., Kornberg, R. D. (2001) "Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution" Science 292:1876.
Contributors
• Charles Ophardt, Professor Emeritus, Elmhurst College; Virtual Chembook | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Nucleic_Acids/Nucleotides.txt |
Three general types of RNA exist: messenger, ribosomal, and transfer.
Messenger RNA
Messenger RNA (mRNA) is synthesized from a gene segment of DNA which ultimately contains the information on the primary sequence of amino acids in a protein to be synthesized. The genetic code as translated is for m-RNA not DNA. The messenger RNA carries the code into the cytoplasm where protein synthesis occurs.
Genetic Code
Each gene (or distinct segment) on DNA contains instructions for making one specific protein with order of amino acids coded by the precise sequence of heterocyclic amines on the nucleotides. Since proteins have a variety of functions including those of enzymes mistakes in the primary sequence of amino acids in proteins may have lethal effects.
How can a polymeric nucleotide with only four different heterocyclic amines specify the sequence of 20 or more different amino acids? If each nucleotide coded for a single amino acid, then obviously only 4 of the 20 amino acids could be accommodated. If the nucleotides were used in groups of two, there are 16 different combinations possible which is still inadequate.
It has been determined that the genetic code is actually based upon triplets of nucleotides which provide 64 different codes using the 4 nucleotides. During the 1960's, a tremendous effort was devoted to proving that the code was read as triplets, and also to solving the genetic code. The genetic code was originally translated for the bacteria E. Coli, but its universality has since been established. The genetic code is "read" from a type of RNA called messenger RNA (mRNA). Each nucleotide triplet, called a codon, can be "read" and translated into an amino acid to be incorporated into a protein being synthesized. The genetic code is shown in Figure 7.
Several distinctive features of the genetic code are clearly evident. First, all of the 64 codons or triplets have a known function, with 61 coding for amino acids and the other 3 serving as a stop or termination signal for protein synthesis. Secondly, the code is degenerate, meaning that there are usually several codons for each amino acid. Only methionine and tryptophan have a single codon. More specifics on the importance of the degeneracy of the genetic code will be discussed in a later section.
Ribosomal RNA
In the cytoplasm, ribsomal RNA (rRNA) and protein combine to form a nucleoprotein called a ribosome. The ribosome serves as the site and carries the enzymes necessary for protein synthesis. In the graphic on the left, the ribosome is shown as made from two sub units, 50S and 30 S. There are about equal parts rRNA and protein. The far left graphic shows the complete ribosome with three tRNA attached.
The ribosome attaches itself to m-RNA and provides the stabilizing structure to hold all substances in position as the protein is synthesized. Several ribosomes may be attached to a single RNA at any time. In upper right corner is the 30S sub unit with mRNA and tRNA attached.
Note: The coordinates used in this display have only the alpha carbons of the proteins (CA) and the DNA backbone atoms.
Transfer RNA
Transfer RNA (tRNA) contains about 75 nucleotides, three of which are called anticodons, and one amino acid. The tRNA reads the code and carries the amino acid to be incorporated into the developing protein.
There are at least 20 different tRNA's - one for each amino acid. The basic structure of a tRNA is shown in the left graphic. Part of the tRNA doubles back upon itself to form several double helical sections. On one end, the amino acid, phenylalanine, is attached. On the opposite end, a specific base triplet, called the anticodon, is used to actually "read" the codons on the mRNA.
The tRNA "reads" the mRNA codon by using its own anticodon. The actual "reading" is done by matching the base pairs through hydrogen bonding following the base pairing principle. Each codon is "read" by various tRNA's until the appropriate match of the anticodon with the codon occurs. In this example, the tRNA anticodon (AAG) reads the codon (UUC) on the mRNA. The UUC codon codes for phenylalanine which is attached to the tRNA. Remember that the codons read from the mRNA make up the genetic code as read by humans.
Problems
Quiz: Read and translate the codons on mRNA into the appropriate amino acids.
G U A C G A A A A
Quiz: Read and translate the codon on mRNA into the appropriate amino acid.
AGA. What is the anticodon on the appropriate tRNA? | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Nucleic_Acids/RNA/Types_of_RNA.txt |
A very broad definition of a drug would include "all chemicals other than food that affect living processes." If the affect helps the body, the drug is a medicine. However, if a drug causes a harmful effect on the body, the drug is a poison. The same chemical can be a medicine and a poison depending on conditions of use and the person using it. Another definition would be "medicinal agents used for diagnosis, prevention, treatment of symptoms, and cure of diseases." Contraceptives would be outside of this definition unless pregnancy were considered a disease.
• Anti-Cancer Drugs I
• Anti-Cancer Drugs II
• Antidepressants
Antidepressant drugs are used to restore mentally depressed patients to an improved mental status. Depression results from a deficiency of norepinephrine at receptors in the brain. Mechanisms that increase their effective concentration at the receptor sites should alleviate depression.
• Barbiturates and Benzodiazepines
Barbiturates are central nervous system depressants and are similar, in many ways, to the depressant effects of alcohol.
• Drugs Acting Upon the Central Nervous System
Chemical influences are capable of producing a myriad of effects on the activity and function of the central nervous system. Since our knowledge of different regions of brain function and the neurotransmitters in the brain is limited, the explanations for the mechanisms of drug action may be vague. The known neurotransmitters are: acetylcholine which is involved with memory and learning; norepinephrine which is involved with mania-depression and emotions.
• Drug Activity
Page notifications Off Barbiturates and Benzodiazepines Drug Receptor Interactions picture_as_pdf Batch Donate Table of contents A very broad definition of a drug would include "all chemicals other than food that affect living processes." If the affect helps the body, the drug is a medicine. However, if a drug causes a harmful effect on the body, the drug is a poison. The same chemical can be a medicine and a poison depending on conditions of use and the person using it.
• Drug Receptor Interactions
Drugs interact with receptor sites localized in macromolecules which have protein-like properties and specific three dimensional shapes. A minimum three point attachment of a drug to a receptor site is required. In most cases a rather specific chemical structure is required for the receptor site and a complementary drug structure. Slight changes in the molecular structure of the drug may drastically change specificity.
• Enzyme Inhibition
Although activation of enzymes may be exploited therapeutically, most effects are produced by enzyme inhibition.
• Hallucinogenic Drugs
Hallucinogenic agents, also called psychomimetic agents, are capable of producing hallucinations, sensory illusions and bizarre thoughts. The primary effect of these compounds is to consistently alter thought and sensory perceptions.
• Local Anesthetics
Local anesthetics are agents that reversibly block the generation and conduction of nerve impulses along a nerve fiber. They depress impulses from sensory nerves of the skin, surfaces of mucosa, and muscles to the central nervous system. These agents are widely used in surgery, dentistry, and ophthalmology to block transmission of impulses in peripheral nerve endings.
• Misc Antibiotics
Antibiotics are specific chemical substances derived from or produced by living organisms that are capable of inhibiting the life processes of other organisms.
• Narcotic Analgesic Drugs
Narcotic agents are potent analgesics which are effective for the relief of severe pain. Analgesics are selective central nervous system depressants used to relieve pain. The term analgesic means "without pain". Even in therapeutic doses, narcotic analgesics can cause respiratory depression, nausea, and drowsiness.
• Penicillin
The penicillins were the first antibiotics discovered as natural products from the mold Penicillium.
• Sulfa Drugs
Sulfonamides are synthetic antimicrobial agents with a wide spectrum encompassing most gram-positive and many gram-negative organisms. These drugs were the first efficient treatment to be employed systematically for the prevention and cure of bacterial infections.
Thumbnail: Ritalin SR 20 mg, a brand-name sustained-release formulation of methylphenidate. (CC SA-BY 3.0; Sponge).
Pharmaceuticals
The available anticancer drugs have distinct mechanisms of action which may vary in their effects on different types of normal and cancer cells. A single "cure" for cancer has proved elusive since there is not a single type of cancer but as many as 100 different types of cancer. In addition, there are very few demonstrable biochemical differences between cancerous cells and normal cells. For this reason the effectiveness of many anticancer drugs is limited by their toxicity to normal rapidly growing cells in the intestinal and bone marrow areas. A final problem is that cancerous cells which are initially suppressed by a specific drug may develop a resistance to that drug. For this reason cancer chemotherapy may consist of using several drugs in combination for varying lengths of time.
Introduction
Chemotherapy drugs, are sometimes feared because of a patient's concern about toxic effects. Their role is to slow and hopefully halt the growth and spread of a cancer. There are three goals associated with the use of the most commonly-used anticancer agents.
1. Damage the DNA of the affected cancer cells.
2. Inhibit the synthesis of new DNA strands to stop the cell from replicating, because the replication of the cell is what allows the tumor to grow.
3. Stop mitosis or the actual splitting of the original cell into two new cells. Stopping mitosis stops cell division (replication) of the cancer and may ultimately halt the progression of the cancer.
Unfortunately, the majority of drugs currently on the market are not specific, which leads to the many common side effects associated with cancer chemotherapy. Because the common approach of all chemotherapy is to decrease the growth rate (cell division) of the cancer cells, the side effects are seen in bodily systems that naturally have a rapid turnover of cells iincluding skin, hair, gastrointestinal, and bone marrow. These healthy, normal cells, also end up damaged by the chemotherapy program.
Categories of Chemotherapy Drugs
In general, chemotherapy agents can be divided into three main categories based on their mechanism of action.
• Stop the synthesis of pre DNA molecule building blocks
These agents work in a number of different ways. DNA building blocks are folic acid, heterocyclic bases, and nucleotides, which are made naturally within cells. All of these agents work to block some step in the formation of nucleotides or deoxyribonucleotides (necessary for making DNA). When these steps are blocked, the nucleotides, which arethe building blocks of DNA and RNA, can not be synthesized. Thus the cells can not replicate because they can nnot make DNA without the nucleotides. Examples of drugs in this class include 1) methotrexate (Abitrexate®),2) fluorouracil (Adrucil®), 3) hydroxyurea (Hydrea®), and 4) mercaptopurine (Purinethol®).
• Directly damage the DNA in the nucleus of the cell
These agents chemically damage DNA and RNA. They disrupt replication of the DNA and either totally halt replication or cause the manufacture of nonsense DNA or RNA (i.e. the new DNA or RNA does not code for anything useful). Examples of drugs in this class include cisplatin (Platinol®) and 7) antibiotics - daunorubicin (Cerubidine®), doxorubicin (Adriamycin®), and etoposide (VePesid®).
• Effect the synthesis or breakdown of the mitotic spindles
Mitotic spindles serve as molecular railroads with "North and South Poles" in the cell when a cell starts to divide itself into two new cells. These spindles are very important because they help to split the newly copied DNA such that a copy goes to each of the two new cells during cell division. These drugs disrupt the formation of these spindles and therefore interrupt cell division. Examples of drugs in this class of 8) miotic disrupters include: Vinblastine (Velban®), Vincristine (Oncovin®) and Pacitaxel (Taxol®).
Enzyme L-asparaginase
In the 1950's a biochemical difference in metabolism related to the amino acid asparagine was found. Normal cells apparently can synthesize asparagine while leukemia cells cannot. If leukemia cells are deprived of asparagine, they will eventually die. In an almost unrecognized and parallel discovery, it was found that blood serum from guinea pigs and other South American rodents had antileukemia properties. The enzyme L-asparaginase was eventually identified as the anticancer agent. L-asparaginase was isolated and tested successfully on human leukemias. Eventually the enzyme asparaginase was also found and isolated from the bacteria, E. coli.
If the enzyme L-asparaginase is given to humans, various types of leukemias can be controlled. Tumor cells, more specifically lymphatic tumor cells, require huge amounts of asparagines to keep up with their rapid, malignant growth. This means they use both asparagine from the diet as well as what they can make themselves (which is limited) to satisfy their large asparagines demand. L-asparaginase is an enzyme that destroys asparagine external to the cell. Normal cells are able to make all the asparagine they need internally whereas tumor cells become depleted rapidly and die.The enzyme converts asparagine in the blood into aspartic acid by a deamination reaction. The leukemia cells are thus deprived of their supply of asparagine and will die.
Methotrexate
Methotrexate inhibits folic acid reductase which is responsible for the conversion of folic acid to tetrahydrofolic acid. At two stages in the biosynthesis of purines (adenine and guanine) and at one stage in the synthesis of pyrimidines (thymine, cytosine, and uracil), one-carbon transfer reactions occur which require specific coenzymes synthesized in the cell from tetrahydrofolic acid.
Tetrahydrofolic acid itself is synthesized in the cell from folic acid with the help of an enzyme, folic acid reductase. Methotrexate looks a lot like folic acid to the enzyme, so it binds to it thinking that it is folic acid. In fact, methotrexate looks so good to the enzyme that it binds to it quite strongly and inhibits the enzyme. Thus, DNA synthesis cannot proceed because the coenzymes needed for one-carbon transfer reactions are not produced from tetrahydrofolic acid because there is no tetrahydrofolic acid. Again, without DNA, no cell division.
5-Fluorouracil
5-Fluorouracil (5-FU; Adrucil®, Fluorouracil, Efudex®, Fluoroplex®) is an effective pyrimidine antimetabolite. Fluorouracil is synthesized into the nucleotide, 5-fluoro-2-deoxyuridine. This product acts as an antimetabolite by inhibiting the synthesis of 2-deoxythymidine because the carbon - fluorine bond is extremely stable and prevents the addition of a methyl group in the 5-position. The failure to synthesize the thymidine nucleotide results in little or no production of DNA. Two other similar drugs include: gemcitabine (Gemzar®) and arabinosylcytosine (araC). They all work through similar mechanisms.
Contributors
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Pharmaceuticals/Anti-Cancer_Drugs_I.txt |
Hydroxyurea
Hydroxyurea blocks an enzyme which converts the cytosine nucleotide into the deoxy derivative. In addition, DNA synthesis is further inhibited because hydroxyurea blocks the incorporation of the thymidine nucleotide into the DNA strand.
Mercaptopurine
Mercaptopurine, a chemical analog of the purine adenine, inhibits the biosynthesis of adenine nucleotides by acting as an antimetabolite. In the body, 6-MP is converted to the corresponding ribonucleotide. 6-MP ribonucleotide is a potent inhibitor of the conversion of a compound called inosinic acid to adenine Without adenine, DNA cannot be synthesized. 6-MP also works by being incorporated into nucleic acids as thioguanosine, rendering the resulting nucleic acids (DNA, RNA) unable to direct proper protein synthesis.
Thioguanine
Thioguanine is an antimetabolite in the synthesis of guanine nucleotides.
Alkylating Agents
Alkylating agents involve reactions with guanine in DNA. These drugs add methyl or other alkyl groups onto molecules where they do not belong. This in turn inhibits their correct utilization by base pairing and causes a miscoding of DNA.
1. In the first mechanism an alkylating agent attaches alkyl groups to DNA bases. This alteration results in the DNA being fragmented by repair enzymes in their attempts to replace the alkylated bases.
2. A second mechanism by which alkylating agents cause DNA damage is the formation of cross-bridges, bonds between atoms in the DNA. In this process, two bases are linked together by an alkylating agent that has two DNA binding sites. Cross-linking prevents DNA from being separated for synthesis or transcription.
3. The third mechanism of action of alkylating agents causes the mispairing of the nucleotides leading to mutations.
There are six groups of alkylating agents: nitrogen mustards; ethylenimes; alkylsulfonates; triazenes; piperazines; and nitrosureas. Cyclosporamide is a classical example of the role of the host metabolism in the activation of an alkylating agent and is one or the most widely used agents of this class. It was hoped that the cancer cells might posses enzymes capable of accomplishing the cleavage, thus resulting in the selective production of an activated nitrogen mustard in the malignant cells. Compare the top and bottom structures in the graphic on the left.
Antibiotics
A number of antibiotics such as anthracyclines, dactinomycin, bleomycin, adriamycin, mithramycin, bind to DNA and inactivate it. Thus the synthesis of RNA is prevented. General properties of these drugs include: interaction with DNA in a variety of different ways including intercalation (squeezing between the base pairs), DNA strand breakage and inhibition with the enzyme topoisomerase II. Most of these compounds have been isolated from natural sources and antibiotics. However, they lack the specificity of the antimicrobial antibiotics and thus produce significant toxicity.
The anthracyclines are among the most important antitumor drugs available. Doxorubicin is widely used for the treatment of several solid tumors while daunorubicin and idarubicin are used exclusively for the treatment of leukemia. These agents have a number of important effects including: intercalating (squeezing between the base pairs) with DNA affecting many functions of the DNA including DNA and RNA synthesis. Breakage of the DNA strand can also occur by inhibition of the enzyme topoisomerase II.
Dactinomycin (Actinomycin D)
At low concentrations dactinomycin inhibits DNA directed RNA synthesis and at higher concentrations DNA synthesis is also inhibited. All types of RNA are affected, but ribosomal RNA is more sensitive. Dactinomycin binds to double stranded DNA , permitting RNA chain initiation but blocking chain elongation. Binding to the DNA depends on the presence of guanine.
Mitotic Disrupters
Plant alkaloids like vincristine prevent cell division, or mitosis. There are several phases of mitosis, one of which is the metaphase. During metaphase, the cell pulls duplicated DNA chromosomes to either side of the parent cell in structures called "spindles". These spindles ensure that each new cell gets a full set of DNA. Spindles are microtubular fibers formed with the help of the protein "tubulin". Vincristine binds to tubulin, thus preventing the formation of spindles and cell division.
Taxol
Paclitaxel (taxol) was first isolated from the from the bark of the Pacific Yew (Taxus brevifolia). Docetaxel is a more potent analog that is produced semisynthetically. In contrast to other microtubule antagonists, taxol disrupts the equilibrium between free tubulin and mircrotubules by shifting it in the direction of assembly, rather than disassembly. As a result, taxol treatment causes both the stabilization of microtubules and the formation of abnormal bundles of microtubules. The net effect is still the disruption of mitosis.
Mechanism of Intercalating Agents
Intercalating agents wedge between bases along the DNA. The intercalated drug molecules affect the structure of the DNA, preventing polymerase and other DNA binding proteins from functioning properly. The result is prevention of DNA synthesis, inhibition of transcription and induction of mutations. Examples include: Carboplatin and Cisplatin.
These related drugs covalently bind to DNA with preferential binding to the N-7 position of guanine and adenine. They are able to bind to two different sites on DNA producing cross-links, either intrastrand (within the same DNA molecule which results in inhibition of DNA synthesis and transcription.
Contributors
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Pharmaceuticals/Anti-Cancer_Drugs_II.txt |
Antidepressant drugs are used to restore mentally depressed patients to an improved mental status. Depression results from a deficiency of norepinephrine at receptors in the brain. Mechanisms that increase their effective concentration at the receptor sites should alleviate depression. Antidepressant drugs act by one or more of the following stimulation type mechanisms:
1. Increase release of norepinephrine: Amphetamines and electroconvulsive therapy act by this mechanism. Amphetamines mimic norepinephrine.
2. Prevent inactivation of norepinephrine:Monoamine oxidase (MAO) inhibitors are thought to act as antidepressant agents in part by preventing the breakdown and inactivation of norepinephrine.
3. Prevent the re uptake of norepinephrine:The action of norepinephrine at the receptor site is terminated by the re uptake of norepinephrine by the neuron from which it was originally released.
Tricyclic Antidepressants
The tricyclic antidepressants are the most effective drugs presently available for the treatment of depression. These act by increasing the release of norepinephrine. Amphetamine and cocaine can also act in this manner. Imipramine, amitriptylin, and other closely related drugs are among the drugs currently most widely used for the treatment of major depression.
• imipramine (Tofranil)
• desipramine (Norpramin)
The activity of the tricyclic drugs depends on the central ring of seven or eight atoms which confers an angled or twisted conformation. The side chain must have at least 2 carbons although 3 appear to be better. The amine group may be either tertiary or secondary. All tricyclic antidepressants block the re-uptake of norepinephrine at nerve terminals. However, the potency and selectivity for the inhibition of the uptake of norepinephrine, serotonin, and dopamine vary greatly among the agents. The tertiary amine tricyclics seem to inhibit the serotonin uptake pump, whereas the secondary amine ones seem better in switching off the NE pump. For instance, imipramine is a potent and selective blocker of serotonin transport, while desipramine inhibits the uptake of norepinephrine.
Serotonin
Serotonin (5-hydroxytryptamine or 5-HT) is a monoamine neurotransmitter found in cardiovascular tissue, in endothelial cells, in blood cells, and in the central nervous system. The role of serotonin in neurological function is diverse, and there is little doubt that serotonin is an important CNS neurotransmitter. Although some of the serotonin is metabolized by monoamine oxidase, most of the serotonin released into the post-synaptic space is removed by the neuron through a re-uptake mechanism inhibited by the tricyclic antidepressants and the newer, more selective antidepressant re uptake inhibitors such as fluoxetine and sertraline.
Selective Serotonin Reuptake Inhibitors
In recent years, selective serotonin reuptake inhibitors have been introduced for the treatment of depression. Prozac is the most famous drug in this class. Clomiprimine, fluoxetine (Prozac), sertraline and paroxetine selectively block the re uptake of serotonin, thereby increasing the levels of serotonin in the central nervous system. Note the similarities and differences between the tricyclic antidepressants and the selective serotonin re uptake inhibitors. Clomipramine has been useful in the treatment of obsessive-compulsive disorders.
Monoamine Oxidase Inhibitors
Monoamine oxidase (MAO) causes the oxidative deamination of norephinephrine, serotonin, and other amines. This oxidation is the method of reducing the concentration of the neurotransmitter after it has sent the signal at the receptor site. A drug which inhibits this enzyme has the effect of increasing the concentration of the norepinephrine which in turn causes a stimulation effect. Most MAO inhibitors are hydrazine derivatives. Hydrazine is highly reactive and may form a strong covalent bond with MAO with consequent inhibition for up to 5 days.
These drugs are less effective and produce more side effects than the tricyclic antidepressants. For example, they lower blood pressure and were at one time used to treat hypertension. Their use in psychiatry has also become very limited as the tricyclic antidepressants have come to dominate the treatment of depression and allied conditions. Thus, MAOIs are used most often when tricyclic antidepressants give unsatisfactory results.
Phenelzine is the hydrazine analog of phenylethylamine, a substrate of MAO. This and several other MAOIs, such as isocarboxazide, are structurally related to amphetamine and were synthesized in an attempt to enhance central stimulant properties.
• phenelzine (Nardil)
• isocarboxazid (Marplan)
Contributors
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Pharmaceuticals/Antidepressants.txt |
Hypnotic and sedative drugs are non-selective, general depressants of the central nervous system. If the dose is relatively low, a sedative action results in a reduction in restlessness and emotional tension. A larger dose of the same drug produces a hypnotic sleep inducing effect. As the dosage is increased further, the result is anesthesia or death if the dosage is sufficiently high.
Introduction
The barbiturates once enjoyed a long period of extensive use as sedative-hypnotic drugs; however, except for a few specialized uses, they have been largely replaced by the much safer benzodiazepines. Barbiturates are central nervous system depressants and are similar, in many ways, to the depressant effects of alcohol. To date, there are about 2,500 derivatives of barbituric acid of which only 15 are used medically. The first barbiturate was synthesized from barbituric acid in 1864. The original use of barbiturates was to replace drugs such as opiates, bromides, and alcohol to induce sleep.
The hyponotic and sedative effects produced by barbiturates are usually ascribed to their interference of nerve transmission to the cortex. Various theories for the action of barbiturates include: changes in ion movements across the cell membrane; interactions with cholinergic and non cholinergic receptor sites; impairment of biochemical reactions which provide energy; and depression of selected areas of the brain. The structures of the barbiturates can be related to the duration of effective action. Although over 2000 derivatives of barbituric acid have been synthesized only about a dozen are currently used. All of the barbiturates are related to the structure of barbituric acid shown below.
The duration of effect depends mainly on the alkyl groups attached to carbon # 5 which confer lipid solubility to the drug. The duration of effective action decreases as the total number of carbons at C # 5 increases. To be more specific, a long effect is achieved by a short chain and/or phenyl group. A short duration effect occurs when there are the most carbons and branches in the alkyl chains
Benzodiazepines
The term benzodiazepine refers to the portion of the structure composed of a benzene ring (A) fused to a seven-membered diazepine ring (B). However, since all of the important benzodiazepines contain a aryl substituent ring C) and a 1, 4-diazepine ring, the term has come to mean the aryl-1,4-benzodiazepines. There are several useful benzodiazepines available: chlordiazepoxide (Librium) and diazapam (Valium).
The actions of benzodiazepines are a result of increased activation of receptors by gamma-aminobutyric acid (GABA). Benzodiazepine receptors are located on the alpha subunit of the GABA receptor located almost exclusively on postsynaptic nerve endings in the CNS (especially cerebral cortex). Benzodiazepines enhance the GABA transmitter in the opening of postsynaptic chloride channels which leads to hyperpolarization of cell membranes. That is, they "bend" the receptor slightly so that GABA molecules attach to and activate their receptors more effectively and more often.
Contributors
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook
Drug Activity
A very broad definition of a drug would include "all chemicals other than food that affect living processes." If the affect helps the body, the drug is a medicine. However, if a drug causes a harmful effect on the body, the drug is a poison. The same chemical can be a medicine and a poison depending on conditions of use and the person using it. Another definition would be "medicinal agents used for diagnosis, prevention, treatment of symptoms, and cure of diseases." Contraceptives would be outside of this definition unless pregnancy were considered a disease.
Disease Classification
A disease is a condition of impaired health resulting from a disturbance in the structure or function of the body. Diseases may be classified into the following major categories:
1. Chemotherapy, broadly defined, means the treatment of any disease by chemicals including infectious and non-infectious diseases. The original definition applied only to drugs which were used in the treatment of infectious diseases. The proper term for the treatment of non-infectious diseases is pharmacodynamics.
Drug Classification
1. Drugs have three or more names including a: chemical name, brand or trade name, and generic or common name. The chemical name is assigned according to rules of nomenclature of chemical compounds. The brand name is always capitalized and is selected by the manufacturer. The generic name refers to a common established name irrespective of its manufacturer.
In most cases, a drug bearing a generic name is equivalent to the same drug with a brand name. However, this equivalency is not always true. Although drugs are chemically equivalent, different manufacturing processes may cause differences in pharmacological action. Several differences may be crystal size or form, isomers, crystal hydration, purity-(type and number of impurities), vehicles, binders, coatings, dissolution rate, and storage stability.
Mode of Drug Action
One major problem of pharmacology is that no drug produces a single effect. The primary effect is the desired therapeutic effect. Secondary effects are all other effects beside the desired effect which may be either beneficial or harmful. Drugs are chosen to exploit differences between normal metabolic processes and any abnormalities which may be present. Since the differences may not be very great, drugs may be nonspecific in action and alter normal functions as well as the undesirable ones. This leads to undesirable side effects.
The biological effects observed after a drug has been administered are the result of an interaction between that chemical and some part of the organism. Mechanisms of drug action can be viewed from different perspectives, namely, the site of action and the general nature of the drug-cell interaction.
Killing Foreign Organisms
Chemotherapeutic agents act by killing or weakening foreign organisms such as bacteria, worms, viruses. The main principle of action is selective toxicity, i.e. the drug must be more toxic to the parasite than to the host.
Stimulation and Depression
Drugs act by stimulating or depressing normal physiological functions. Stimulation increases the rate of activity while depression reduces the rate of activity.
Sites of Drug Action
Enzyme Inhibition
Drugs act within the cell by modifying normal biochemical reactions. Enzyme inhibition may be reversible or non reversible; competitive or non-competitive. Antimetabolites may be used which mimic natural metabolites. Gene functions may be suppressed.
Drug-Receptor Interaction
Drugs act on the cell membrane by physical and/or chemical interactions. This is usually through specific drug receptor sites known to be located on the membrane. A receptor is the specific chemical constituents of the cell with which a drug interacts to produce its pharmacological effects. Some receptor sites have been identified with specific parts of proteins and nucleic acids. In most cases, the chemical nature of the receptor site remains obscure.
Non-specific Interactions
Drugs act exclusively by physical means outside of cells. These sites include external surfaces of skin and gastrointestinal tract. Drugs also act outside of cell membranes by chemical interactions. Neutralization ofstomach acid by antacids is a good example.
Contributors
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Pharmaceuticals/Barbiturates_and_Benzodiazepines.txt |
The vast majority of drugs show a remarkably high correlation of structure and specificity to produce pharmacological effects. Experimental evidence indicates that drugs interact with receptor sites localized in macromolecules which have protein-like properties and specific three dimensional shapes. A minimum three point attachment of a drug to a receptor site is required. In most cases a rather specific chemical structure is required for the receptor site and a complementary drug structure. Slight changes in the molecular structure of the drug may drastically change specificity.
Introduction
Several chemical forces may result in a temporary binding of the drug to the receptor. Essentially any bond could be involved with the drug-receptor interaction. Covalent bonds would be very tight and practically irreversible. Since by definition the drug-receptor interaction is reversible, covalent bond formation is rather rare except in a rather toxic situation. Since many drugs contain acid or amine functional groups which are ionized at physiological pH, ionic bonds are formed by the attraction of opposite charges in the receptor site.
Polar-polar interactions as in hydrogen bonding are a further extension of the attraction of opposite charges. The drug-receptor reaction is essentially an exchange of the hydrogen bond between a drug molecule, surrounding water, and the receptor site.
Finally hydrophobic bonds are formed between non-polar hydrocarbon groups on the drug and those in the receptor site. These bonds are not very specific but the interactions do occur to exclude water molecules. Repulsive forces which decrease the stability of the drug-receptor interaction include repulsion of like charges and steric hindrance. Steric hindrance refers to certain 3-dimensional features where repulsion occurs between electron clouds, inflexible chemical bonds, or bulky alkyl groups.
Drug Interaction with Receptor Site
• A neurotransmitter has a specific shape to fit into a receptor site and cause a pharmacological response such as a nerve impulse being sent. The neurotransmitter is similar to a substrate in an enzyme interaction. After attachment to a receptor site, a drug may either initiate a response or prevent a response from occurring. A drug must be a close "mimic" of the neurotransmitter.
• An agonist is a drug which produces a stimulation type response. The agonist is a very close mimic and "fits" with the receptor site and is thus able to initiate a response.
• An antagonist drug interacts with the receptor site and blocks or depresses the normal response for that receptor because it only partially fits the receptor site and can not produce an effect. However, it does block the site preventing any other agonist or the normal neurotransmitter from interacting with the receptor site.
Drugs Acting Upon the Central Nervous System
The central nervous system directs the functions of all tissues of the body. The peripheral nervous system receives thousands of sensory inputs and transmits them to the brain via the spinal cord. The brain processes this incoming information and discards 99% as unimportant. After sensory information has been evaluated, selected areas of the central nervous system initiate nerve impulses to organs or tissue to make an appropriate response.
Chemical influences are capable of producing a myriad of effects on the activity and function of the central nervous system. Since our knowledge of different regions of brain function and the neurotransmitters in the brain is limited, the explanations for the mechanisms of drug action may be vague. The known neurotransmitters are: acetylcholine which is involved with memory and learning; norepinephrine which is involved with mania-depression and emotions; and serotonin which is involved with biological rhythms, sleep, emotion, and pain.
Central Nervous System Stimulants
Stimulants are drugs that exert their action through excitation of the central nervous system. Psychic stimulants include caffeine, cocaine, and various amphetamines. These drugs are used to enhance mental alertness and reduce drowsiness and fatigue. However, increasing the dosage of caffeine above 200 mg (about 2 cups of coffee) does not increase mental performance but may increase nervousness, irritability, tremors, and headache. Heavy coffee drinkers become psychically dependent upon caffeine. If caffeine is withheld, a person may experience mild withdrawal symptoms characterized by irritability, nervousness, and headache.
Caffeine and the chemically related xanthines, theophylline and theobromine, decrease in the order given in their stimulatory action. They may be included in some over-the-counter drugs. The action of caffeine is to block adenosine receptors as an antagonist. As caffeine has a similar structure to the adenosine group. This means that caffeine will fit adenosine receptors as well as adenosine itself. It inhibits the release of neurotransmitters from presynaptic sites but works in concert with norepinephrine or angiotensin to augment their actions. Antagonism of adenosine receptors by caffeine would appear to promote neurotransmitter release, thus explaining the stimulatory effects of caffeine.
Amphetamines
The stimulation caused by amphetamines is caused by excessive release of norepinephrine from storage sites in the peripheral nervous system. It is not known whether the same action occurs in the central nervous system. Two other theories for their action are that they are degraded slower than norepinephrine or that they could act on serotonin receptor sites. Therapeutic doses of amphetamine elevate mood, reduce feelings of fatigue and hunger, facilitate powers of concentration, and increase the desire and capacity to carry out work. They induce exhilarating feelings of power, strength, energy, self-assertion, focus and enhanced motivation. The need to sleep or eat is diminished.
Levoamphetamine (Benzedrine), dextroamphetamine (Dexedrine), and methamphetamine (Methedrine) are collectively referred to as amphetamines. Benzedrine is a mixture of both the dextro and levoamphetamine isomers. The dextro isomer is several times more potent than the levo isomer.
The misuse and abuse of amphetamines is a significant problem which may include the house wife taking diet pills, athletes desiring an improved performance, the truck driver driving non-stop coast to-coast, or a student cramming all night for an exam. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Pharmaceuticals/Drug_Receptor_Interactions.txt |
Although activation of enzymes may be exploited therapeutically, most effects are produced by enzyme inhibition.
Direct Enzyme Inhibition
Inhibition caused by drugs may be either reversible or irreversible. A reversible situation occurs when an equilibrium can be established between the enzyme and the inhibitory drug. A competitive inhibition occurs when the drug, as "mimic" of the normal substrate competes with the normal substrate for the active site on the enzyme. Concentration effects are important for competitive inhibition.
In noncompetitive inhibition, the drug combines with an enzyme, at a different site other than the active site. The normal substrate can not displace the drug from this site and can not interact with the active either since the shape of the enzyme has been altered. Among the many types of drugs that act as enzyme inhibitors the following may be included: antibiotics, acetylchlolinesterase agents, certain antidepressants such as monoamine oxidase inhibitors and some diuretics.
Suppression of Gene Function
Many drugs act as suppressors of gene function including antibiotics, fungicides, antimalarials and antivirals. Gene function may be suppressed in several steps of protein synthesis or inhibition of nucleic acid biosynthesis. Many substances which inhibit nucleic acid biosynthesis are very toxic since the drug is not very selective in its action between the parasite and host.
Antimetabolites
The strategy of chemotherapy consists of exploiting the biochemical differences between the host and parasite cells. Metabolites are any substances used or produced by biochemical reactions. A drug which possesses a remarkably close chemical similarity (mimic) to the normal metabolite is called an antimetabolite. The antimetabolite enters a normal synthetic reaction by "fooling" an enzyme and producing a counterfeit metabolite. The counterfeit metabolite inhibits another enzyme or is an unusable fraudulent end product which cannot be utilized by the cell for growth or reproduction. Such antimetabolites have been used as antibacterial or anticancer agents.
Hallucinogenic Drugs
Hallucinogenic agents, also called psychomimetic agents, are capable of producing hallucinations, sensory illusions and bizarre thoughts. The primary effect of these compounds is to consistently alter thought and sensory perceptions. Some of these drugs are used in medicine to produce model psychoses as aids in psychotherapy. Another purpose is to investigate the relationship of mind, brain, and biochemistry with the purpose of elucidating mental diseases such as schizophrenia.
Introduction
A large body of evidence links the action of hallucinogenic agents to effects at serotonin receptor sites in the central nervous system. Whether the receptor site is stimulated or blocked is not exactly known. The serotonin receptor site may consist of three polar or ionic areas to complement the structure of serotonin as shown in the graphic on the left.
Mescalin and Psilocybin
The drugs shown in the graphic can be isolated from natural sources: lysergic acid amide from morning glory seeds, psilocybin from the "magic mushroom", Psilocybe mexicana. The hallucinogenic molecules fit into the same receptors as the neuro-transmitter, and over-stimulate them, leading to false signals being created.
Mescaline is isolated from a peyote cactus. The natives of Central America first made use of these drugs in religious ceremonies, believing the vivid, colorful hallucinations had religious significance. The Aztecs even had professional mystics and prophets who achieved their inspiration by eating the mescaline-containing peyote cactus (Lophophora williamsii). Indeed, the cactus was so important to the Aztecs that they named it teo-nancacyl, or "God's Flesh". This plant was said to have been distributed to the guests at the coronation of Montezuma to make the ceremony seem even more spectacular.
Lysergic acid diethylamide (LSD)
LSD is one of the most powerful hallucinogenic drugs known. LSD stimulates centers of the sympathetic nervous system in the midbrain, which leads to pupillary dilation, increase in body temperature, and rise in the blood-sugar level. LSD also has a serotonin-blocking effect. The hallucinogenic effects of lysergic acid diethylamide (LSD) are also the result of the complex interactions of the drug with both the serotoninergic and dopaminergic systems.
During the first hour after ingestion, the user may experience visual changes with extreme changes in mood. The user may also suffer impaired depth and time perception, with distorted perception of the size and shape of objects, movements, color, sound, touch and the user's own body image.
Serotonin
Serotonin (5-hydroxytryptamine or 5-HT) is a monoamine neurotransmitter found in cardiovascular tissue, in endothelial cells, in blood cells, and in the central nervous system. The role of serotonin in neurological function is diverse, and there is little doubt that serotonin is an important CNS neurotransmitter. Although some of the serotonin is metabolized by monoamine oxidase, most of the serotonin released into the post-synaptic space is removed by the neuron through a re uptake mechanism inhibited by the tricyclic antidepressants and the newer, more selective antidepressant re uptake inhibitors such as fluoxetine and sertraline.
Outside Links
• en.Wikipedia.org/wiki/Lysergi...d_diethylamide
• en.Wikipedia.org/wiki/Serotonin | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Pharmaceuticals/Enzyme_Inhibition.txt |
Unlike other drugs which act in the region of the synapse, local anesthetics are agents that reversibly block the generation and conduction of nerve impulses along a nerve fiber. They depress impulses from sensory nerves of the skin, surfaces of mucosa, and muscles to the central nervous system. These agents are widely used in surgery, dentistry, and ophthalmology to block transmission of impulses in peripheral nerve endings.
Introduction
Most local anesthetics can be represented by the following general formula. In both the official chemical name and the proprietary name, a local anesthetic drug can be recognized by the "-caine" ending. The ester linkage can also be an amide linkage. The most recent research indicates that the local anesthetic binds to a phospholipid in the nerve membrane and inhibits the ability of the phospholipid to bind Ca+2 ions.
Practically all of the free-base forms of the drugs are liquids. For this reason most of these drugs are used as salts (chloride, sulfate, etc.) which are water soluble, odorless, and crystalline solids. As esters these drugs are easily hydrolyzed with consequent loss of activity. The amide form of the drug is more stable and resistant to hydrolysis.
Benzocaine and Lidocaine
Two local anesthtics are shown below.
Misc Antibiotics
Antibiotics are specific chemical substances derived from or produced by living organisms that are capable of inhibiting the life processes of other organisms. The first antibiotics were isolated from microorganisms but some are now obtained from higher plants and animals. Over 3,000 antibiotics have been identified but only a few dozen are used in medicine. Antibiotics are the most widely prescribed class of drugs comprising 12% of the prescriptions in the United States.
Macrolides
Macrolides are products of actinomycetes (soil bacteria) or semi-synthetic derivatives of them. Erythromycin is an orally effective antibiotic discovered in 1952 in the metabolic products of a strain of Streptocyces erythreus, originally obtained from a soil sample.
Erythromycin and other macrolide antibiotics inhibit protein synthesis by binding to the 23S rRNA molecule (in the 50S subunit) of the bacterial ribosome blocking the exit of the growing peptide chain. of sensitive microorganisms. (Humans do not have 50 S ribosomal subunits, but have ribosomes composed of 40 S and 60 S subunits). Certain resistant microorganisms with mutational changes in components of this subunit of the ribosome fail to bind the drug. The association between erythromycin and the ribosome is reversible and takes place only when the 50 S subunit is free from tRNA molecules bearing nascent peptide chains. Gram-positive bacteria accumulate about 100 times more erythromycin than do gram-negative microorganisms. The non ionized from of the drug is considerably more permeable to cells, and this probably explains the increased antimicrobial activity that is observed in alkaline pH.
Tetracyclines
Tetracyclines have the broadest spectrum of antimicrobial activity. These may include: Aureomycin, Terramycin, and Panmycin. Four fused 6-membered rings, as shown in the figure below, form the basic structure from which the various tetracyclines are made. The various derivatives are different at one or more of four sites on the rigid, planar ring structure. The classical tetracyclines were derived from Streptomyces spp., but the newer derivatives are semisynthetic as is generally true for newer members of other drug groups.
Tetracyclines inhibit bacterial protein synthesis by blocking the attachment of the transfer RNA-amino acid to the ribosome. More precisely they are inhibitors of the codon-anticodon interaction. Tetracyclines can also inhibit protein synthesis in the host, but are less likely to reach the concentration required because eukaryotic cells do not have a tetracycline uptake mechanism.
Streptomycin
Streptomycin is effective against gram-negative bacteria, although it is also used in the treatment of tuberculosis. Streptomycin binds to the 30S ribosome and changes its shape so that it and inhibits protein synthesis by causing a misreading of messenger RNA information.
Chloramphenicol
Chloromycetin is also a broad spectrum antibiotic that possesses activity similar to the tetracylines. At present, it is the only antibiotic prepared synthetically. It is reserved for treatment of serious infections because it is potentially highly toxic to bone marrow cells. It inhibits protein synthesis by attaching to the ribosome and interferes with the formation of peptide bonds between amino acids. It behaves as an antimetabolite for the essential amino acid phenylalanine at ribosomal binding sites.
Table 1: Some clinically important antibiotics
Antibiotic Producer organism Activity Site or mode of action
Penicillin Penicillium chrysogenum Gram-positive bacteria Wall synthesis
Cephalosporin Cephalosporium acremonium Broad spectrum Wall synthesis
Griseofulvin Penicillium griseofulvum Dermatophytic fungi Microtubules
Bacitracin Bacillus subtilis Gram-positive bacteria Wall synthesis
Polymyxin B Bacillus polymyxa Gram-negative bacteria Cell membrane
Amphotericin B Streptomyces nodosus Fungi Cell membrane
Erythromycin Streptomyces erythreus Gram-positive bacteria Protein synthesis
Neomycin Streptomyces fradiae Broad spectrum Protein synthesis
Streptomycin Streptomyces griseus Gram-negative bacteria Protein synthesis
Tetracycline Streptomyces rimosus Broad spectrum Protein synthesis
Vancomycin Streptomyces orientalis Gram-positive bacteria Protein synthesis
Gentamicin Micromonospora purpurea Broad spectrum Protein synthesis
Rifamycin Streptomyces mediterranei Tuberculosis Protein synthesis
Contributors
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Pharmaceuticals/Local_Anesthetics.txt |
Narcotic agents are potent analgesics which are effective for the relief of severe pain. Analgesics are selective central nervous system depressants used to relieve pain. The term analgesic means "without pain". Even in therapeutic doses, narcotic analgesics can cause respiratory depression, nausea, and drowsiness. Long term administration produces tolerance, psychic, and physical dependence called addiction.
Introduction
Narcotic agents may be classified into four categories:
1. Morphine and codeine - natural alkaloids of opium.
2. Synthetic derivatives of morphine such as heroin.
3. Synthetic agents which resemble the morphine structure.
4. Narcotic antagonists which are used as antidotes for overdoses of narcotic analgesics.
The main pharmacological action of analgesics is on the cerebrum and medulla of the central nervous system. Another effect is on the smooth muscle and glandular secretions of the respiratory and gastro-intestinal tract. The precise mechanism of action is unknown although the narcotics appear to interact with specific receptor sites to interfere with pain impulses.
Receptor Site
A schematic for an analgesic receptor site may look as shown in the graphic below with morphine. Three areas are needed: a flat areas to accommodate a flat nonpolar aromatic ring, a cavity to accept another series of rings perpendicular, and an anionic site for polar interaction of the amine group.
Enkephalins
Recently investigators have discovered two compounds in the brain called enkephalins which resemble morphine in structure. Each one is a peptide composed of 5 amino acids and differ only in the last amino acid. The peptide sequences are: tyr-gly-gly-phe-leu and tyr-gly-gly-phe-met. Molecular models show that the structures of the enkephalins has some similarities with morphine. The main feature in common appears to be the aromatic ring with the -OH group attached (tyr). Methadone and other similar analgesics have 2 aromatic rings which would be similar to the enkephalins (tyr and phe).
Analgesics may relieve pain by preventing the release of acetylcholine. Enkephalin molecules are released from a nerve cell and bind to analgesic receptor sites on the nerve cell sending the impulse. The binding of enkephalin or morphine-like drugs changes the shape of the nerve sending the impulse in such a fashion as to prevent the cell from releasing acetylcholine. As a result, the pain impulse cannot be transmitted and the brain does not preceive pain.
Morphine and Codeine
Morphine exerts a narcotic action manifested by analgesia, drowsiness, changes in mood, and mental clouding. The major medical action of morphine sought in the CNS is analgesia. Opiates suppress the "cough center" which is also located in the brain stem, the medulla. Such an action is thought to underlie the use of opiate narcotics as cough suppressants. Codeine appears to be particularly effective in this action and is widely used for this purpose.
Narcotic analgesics cause an addictive physical dependence. If the drug is discontinued, withdrawal symptoms are experienced. Although the reasons for addiction and withdrawal symptoms are not completely known, recent experiments have provided some information. A nucleotide known as cyclicadenosine monophosphate (cAMP) is synthesized with the aid of the enzyme adenylate cyclase. Enkephalin and morphine-like drugs inhibit this enzyme and thus decrease the amount of cAMP in the cells. In order to compensate for the decreased cAMP, the cells synthesize more enzyme in an attempt to produce more cAMP. Since more enzyme has been produced, more morphine is required as an inhibitor to keep the cAMP at a low level. This cycle repeats itself causing an increase in the tolerance level and increasing the amounts of morphine required. If morphine is suddenly withheld, withdrawal symptoms are probably caused by a high concentration of cAMP since the synthesizing enzyme, adenylate cyclase, is no longer being inhibited.
Morphine and codeine are contained in opium from the poppy (Papaver Somniterum) plant found in Turkey, Mexico, Southeast Asia, China, and India. This plant is 3-4 feet tall with 5-8 egg shaped capsules on top. Ten days after the poppy blooms in June, incisions are made in the capsules permitting a milky fluid to ooze out. The following day the gummy mass (now brown) is carefully scraped off and pressed into cakes of raw opium to dry.
Opium contains over 20 compounds but only morphine (10%) and codeine (0.5%) are of any importance. Morphine is extracted from the opium and isolated in a relatively pure form. Since codeine is in such low concentration, it is synthesized from morphine by an ether-type methylation of an alcohol group. Codeine has only a fraction of the potency compared to morphine. It is used with aspirin and as a cough suppressant.
Heroin
Heroin is synthesized from morphine by a relatively simple esterification reaction of two alcohol (phenol) groups with acetic anhydride (equivalent to acetic acid). Heroin is much more potent than morphine but without the respiratory depression effect. A possible reason may be that heroin passes the blood-brain barrier much more rapidly than morphine. Once in the brain, the heroin is hydrolyzed to morphine which is responsible for its activity.
Synthetic narcotic analgesics include meperidine and methadone. Meperidine is the most common subsitute for morphine. It exerts several pharmacological effects: analgesic, local anesthetic, and mild antihistamine. This multiple activity may be explained by its structural resemblance to morphine, atropine, and histamine.
Methadone is more active and more toxic than morphine. It can be used for the relief of may types of pain. In addition it is used as a narcotic substitute in addiction treatment because it prevents morphine abstinence syndrome. Methadone was synthesized by German chemists during Wold War II when the United States and our allies cut off their opium supply. And it is difficult to fight a war without analgesics so the Germans went to work and synthesized a number of medications in use today, including demerol and darvon which is structurally simular to methadone. And before we go further lets clear up another myth. Methadone, or dolophine was not named after Adolf Hitler. The "dol" in dolophine comes from the latin root "dolor." The female name Dolores is derived from it and the term dol is used in pain research to measure pain e.g., one dol is 1 unit of pain.
Even methadone, which looks strikingly different from other opioid agonists, has steric forces which produce a configuration that closely resembles that of other opiates. See the graphic on the left and the top graphic on this page. In other words, steric forces bend the molecule of methadone into the correct configuration to fit into the opiate receptor. When you take methadone it first must be metabolized in the liver to a product that your body can use. Excess methadone is also stored in the liver and blood stream and this is how methadone works its 'time release trick' and last for 24 hours or more. Once in the blood stream metabolized methadone is slowly passed to the brain when it is needed to fill opiate receptors. Methadone is the only effective treatment for heroin addiction. It works to smooth the ups and down of heroin craving and allows the person to function normally.
Narcotic Antagonists
Narcotic Antagonists prevent or abolish excessive respiratory depression caused by the administration of morphine or related compounds. They act by competing for the same analgesic receptor sites. They are structurally related to morphine with the exception of the group attached to nitrogen.
Nalorphine precipitates withdrawal symptoms and produces behavioral disturbances in addition to the antogonism action. Naloxane is a pure antagonist with no morphine like effects. It blocks the euphoric effect of heroin when given before heroin. Naltrexone became clinically available in 1985 as a new narcotic antagonist. Its actions resemble those of naloxone, but naltrexone is well is well absorbed orally and is long acting, necessitating only a dose of 50 to 100 mg. Therefore, it is useful in narcotic treatment programs where it is desired to maintain an individual on chronic therapy with a narcotic antagonist. In individuals taking naltrexone, subsequent injection of an opiate will produce little or no effect. Naltrexone appears to be particularly effective for the treatment of narcotic dependence in addicts who have more to gain by being drug-free rather than drug dependant
Contributors and Attributions
• Charles Ophardt, Professor Emeritus, Elmhurst College; Virtual Chembook
• Poppies image from: leda.lycaeum.org | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Pharmaceuticals/Narcotic_Analgesic_Drugs.txt |
The penicillins were the first antibiotics discovered as natural products from the mold Penicillium.
Introduction
In 1928, Sir Alexander Fleming, professor of bacteriology at St. Mary's Hospital in London, was culturing Staphylococcus aureus. He noticed zones of inhibition where mold spores were growing. He named the mold Penicillium rubrum. It was determined that a secretion of the mold was effective against Gram-positive bacteria.
Figure 1: Beta Lactam Structure
Penicillins as well as cephalosporins are called beta-lactam antibiotics and are characterized by three fundamental structural requirements: the fused beta-lactam structure (shown in the blue and red rings, a free carboxyl acid group (shown in red bottom right), and one or more substituted amino acid side chains (shown in black). The lactam structure can also be viewed as the covalent bonding of pieces of two amino acids - cysteine (blue) and valine (red).
Penicillin-G where R = an ethyl pheny group, is the most potent of all penicillin derivatives. It has several shortcomings and is effective only against gram-positive bacteria. It may be broken down in the stomach by gastric acids and is poorly and irregularly absorbed into the blood stream. In addition many disease producing staphylococci are able to produce an enzyme capable of inactivating penicillin-G. Various semisynthetic derivatives have been produced which overcome these shortcomings.
Powerful electron-attracting groups attached to the amino acid side chain such as in phenethicillin prevent acid attack. A bulky group attached to the amino acid side chain provides steric hindrance which interferes with the enzyme attachment which would deactivate the pencillins i.e. methicillin. Refer to Table 2 for the structures. Finally if the polar character is increased as in ampicillin or carbenicillin, there is a greater activity against Gram-negative bacteria.
Penicillin Mode of Action
All penicillin derivatives produce their bacteriocidal effects by inhibition of bacterial cell wall synthesis. Specifically, the cross linking of peptides on the mucosaccharide chains is prevented. If cell walls are improperly made cell walls allow water to flow into the cell causing it to burst. Resemblances between a segment of penicillin structure and the backbone of a peptide chain have been used to explain the mechanism of action of beta-lactam antibiotics. The structures of a beta-lactam antibiotic and a peptide are shown on the left for comparison. Follow the trace of the red oxygens and blue nitrogen atoms.
Gram-positive bacteria possess a thick cell wall composed of a cellulose-like structural sugar polymer covalently bound to short peptide units in layers.The polysaccharide portion of the peptidoglycan structure is made of repeating units of N-acetylglucosamine linked b-1,4 to N-acetylmuramic acid (NAG-NAM). The peptide varies, but begins with L-Ala and ends with D-Ala. In the middle is a dibasic amino acid, diaminopimelate (DAP). DAP (orange) provides a linkage to the D-Ala (gray) residue on an adjacent peptide.
The bacterial cell wall synthesis is completed when a cross link between two peptide chains attached to polysaccharide backbones is formed. The cross linking is catalyzed by the enzyme transpeptidase. First the terminal alanine from each peptide is hydrolyzed and secondly one alanine is joined to lysine through an amide bond.
Peptidoglycan image courtesy of the University of Texas-Houston Medical School
Penicillin binds at the active site of the transpeptidase enzyme that cross-links the peptidoglycan strands. It does this by mimicking the D-alanyl-D-alanine residues that would normally bind to this site. Penicillin irreversibly inhibits the enzyme transpeptidase by reacting with a serine residue in the transpeptidase. This reaction is irreversible and so the growth of the bacterial cell wall is inhibited. Since mammal cells do not have the same type of cell walls, penicillin specifically inhibits only bacterial cell wall synthesis.
Bacterial Resistance
As early as the 1940s, bacteria began to combat the effectiveness of penicillin. Penicillinases (or beta-lactamases) are enzymes produced by structurally susceptable bacteria which renders penicillin useless by hydrolysing the peptide bond in the beta-lactam ring of the nucleus. Penicillinase is a response of bacterial adaptation to its adverse environment, namely the presence of a substance which inhibits its growth. Many other antibiotics are also rendered ineffective because of this same type of resistance.
Severe Allergic Shock
It is estimated that between 300-500 people die each year from penicillin-induced anaphylaxis, a severe allergic shock reaction to penicillin. In afflicted individuals, the beta-lactam ring binds to serum proteins, initiating an IgE-mediated inflammatory response. Penicillin and ala-ala peptide - Chime in new window
Cephalosporins
Cephalosporins are the second major group of beta-lactam antibiotics. They differ from penicillins by having the beta-lactam ring as a 6 member ring. The other difference, which is more significant from a medicinal chemistry stand point, is the existence of a functional group (R) at position 3 of the fused ring system. This now allows for molecular variations to effect changes in properties by diversifying the groups at position 3.
The first member of the newer series of beta-lactams was isolated in 1956 from extracts of Cephalosporium acremonium, a sewer fungus. Like penicillin, cephalosporins are valuable because of their low toxicity and their broad spectrum of action against various diseases. In this way, cephalosporin is very similar to penicillin. Cephalosporins are one of the most widely used antibiotics, and economically speaking, has about 29% of the antibiotic market. The cephalosporins are possibly the single most important group of antibiotics today and are equal in importance to penicillin.
The structure and mode of action of the cephalosporins are similar to that of penicillin. They affect bacterial growth by inhibiting cell wall synthesis, in Gram-positive and negative bacteria. Some brand names include: cefachlor, cefadroxil, cefoxitin, ceftriaxone. Cephalexin
Sulfa Drugs
Sulfonamides are synthetic antimicrobial agents with a wide spectrum encompassing most gram-positive and many gram-negative organisms. These drugs were the first efficient treatment to be employed systematically for the prevention and cure of bacterial infections.
Contributors and Attributions
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Pharmaceuticals/Penicillin.txt |
Photoreceptors are photosensing proteins that respond to the surrounding light environment aroun. Upon light absorption, photorceptors can go through large scale conformational changes, which happen via several intermediates over multiple timescales. Many studies have been done, and the sutdy of photoreceptors can to characterize the transient structural deformation of a protein environment that propagates and initiates the signal transduction pathways.
• Chemistry of Vision
Vision is such an everyday occurrence that we seldom stop to think and wonder how we are able to see the objects that surround us. Yet the vision process is a fascinating example of how light can produce molecular changes. The retina contain the molecules that undergo a chemical change upon absorbing light, but it is the brain that actually makes sense of the visual information to create an image.
• Photoreceptor Excitation
Upon excitation from a laser or other light energy source, photoreceptor molecules transition from a lower energy state to a higher energy state. During this process, electrons of photoreceptor absorbs the energy, and turn into excited state therefore change photoreceptors form. Now, let's see the basic concept of excitation of electrons.
• Photoreceptor Proteins
Photoreceptor proteins are light-sensitive proteins involved in the sensing and response to light in a variety of organisms.[1] Photoreceptor proteins can be find in both animals and plants. Human eye retina is a good example of photoreceptor protein. Many bacteria, such as halohodospira halophila, an extremophile bacterium contain Photoactive Yellow Protein.
• Vision and Light
Eyes receive light energy then transfer and passing the energy into neural impulses to brain. This page will show the role of light plays in vision.
Photoreceptors
Vision is such an everyday occurrence that we seldom stop to think and wonder how we are able to see the objects that surround us. Yet the vision process is a fascinating example of how light can produce molecular changes. The retina contain the molecules that undergo a chemical change upon absorbing light, but it is the brain that actually makes sense of the visual information to create an image.
Introduction
Light is one of the most important resources for civilization, it provides energy as it pass along by the sun. Light influence our everyday live. Living organisms sense light from the environment by photoreceptors. Light, as waves carry energy, contains energy by different wavelength. In vision, light is the stimulus input. Light energy goes into eyes stimulate photoreceptor in eyes. However, as an energy wave, energy is passed on through light at different wavelength.
Light, as waves carry energy, contains energy by different wavelength. From long wavelength to short wavelength, energy increase. 400 nm to 700 nm is visible spectrum.
Light energy can convert chemical to other forms. Vitamin A, also known as retinol, anti-dry eye vitamins, is a required nutrition for human health. The predecessor of vitamin A is present in the variety of plant carotene. Vitamin A is critical for vision because it is needed by the retina of eye. Retinol can be convert to retinal, and retinal is a chemical necessary for rhodopsin. As light enters the eye, the 11-cis-retinal is isomerized to the all-"trans" form.
Mechanism of Vision
The molecule cis-retinal can absorb light at a specific wavelength. When visible light hits the cis-retinal, the cis-retinal undergoes an isomerization, or change in molecular arrangement, to all-trans-retinal. The new form of trans-retinal does not fit as well into the protein, and so a series of geometry changes in the protein begins. The resulting complex is referred to a bathrhodopsin (there are other intermediates in this process, but we'll ignore them for now).
The reaction above shows Lysine side-chain from the opsin react with 11-cis-retinal when stimulated. By removing the oxygen atom form the retinal and two hydrogen atom form the free amino group of the lysine, the linkage show on the picture above is formed, and it is called Schiff base.
Signal Transduction Pathway
As the protein changes its geometry, it initiates a cascade of biochemical reactions that results in changes in charge so that a large potential difference builds up across the plasma membrane. This potential difference is passed along to an adjoining nerve cell as an electrical impulse. The nerve cell carries this impulse to the brain, where the visual information is interpreted.
The light image is mapped on the surface of the retina by activating a series of light-sensitive cells known as rods and cones or photoreceptors. The rods and cones convert the light into electrical impulses which are transmitted to the brain via nerve fibers. The brain then determines, which nerve fibers carried the electrical impulse activate by light at certain photoreceptors, and then creates an image.
The retina is lined with many millions of photoreceptor cells that consist of two types: 7 million cones provide color information and sharpness of images, and 120 million rods are extremely sensitive detectors of white light to provide night vision. The tops of the rods and cones contain a region filled with membrane-bound discs, which contain the molecule cis-retinal bound to a protein called opsin. The resulting complex is called rhodopsin or "visual purple".
In human eyes, rod and cones react to light stimulation, and a series of chemical reactions happen in cells. These cells receive light, and pass on signals to other receiver cells. This chain of process is class signal transduction pathway. Signal transduction pathway is a mechanism that describe the ways cells react and response to stimulation.
Contributors
• {{template.ContribOphardt()}}
Chemistry of Vision
Conversion of Vitamin A into Cis-Retinal
Vitamin A, trans-retinol, is carried to the rods in the eyes from storage in the liver. First it is converted to cis-retinol by a process of isomerization, which means that the trans isomer is converted to a cis isomer. The molecule must break the pi bond, rotate on the single bond, and reform the pi bond. The cis-retinol, an alcohol, is then oxidized to cis-retinal, an aldehyde.
Isomerization of Retinal
Photochemical events in vision involve the protein opsin and the cis/trans isomers of retinal. The cis-retinal fits into a receptor site of opsin. Upon absorption of a photon of light in the visible range, cis-retinal can isomerize to all-trans-retinal. In the cis-retinal, the hydrogens (light gray in the molecular model on the left) are on the same side of the double bond (yellow in the molecular model).
In the trans-retinal, the hydrogens are on opposite sides of the double bond. In fact, all of the double bonds are in the trans-configuration in this isomer: the hydrogens, or hydrogen and -CH3, are always on opposite sides of the double bonds (hence, the name "all-trans-retinal").
Note how the shape of the molecule changes as a result of this isomerization. The molecule changes from an overall bent structure to one that is more or less linear. All of this is the result of trigonal planar bonding (120 o bond angles) about the double bonds.
This photochemical reaction is best understood in terms of molecular orbitals, orbital energy, and electron excitation. In cis-retinal, absorption of a photon promotes a p electron in the pi bond to a higher-energy orbital. This excitation "breaks" the pi component of the double bond and is temporarily converted into a single bond. This means the molecule can now rotate around this single bond, which it does by swiveling through 180o.
The double bond then reforms and locks the molecule back into position in a trans configuration of the all-trans-retinal. This isomerization occurs in a few picoseconds (10-12 s) or less. Energy from light is crucial for this isomerization process: absorption of a photon leads to breaking the double bond and consequent isomerization about half the time (in the dark is almost never happens. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Photoreceptors/Chemistry_of_Vision/Cis-Trans_Isomerization_of_Retinal.txt |
Photochemical events in vision involve the protein opsin and the cis/trans isomers of retinal. Opsin does not absorb visible light, but when it is bonded with 11-cis-retinal to form rhodopsin, which has a very broad absorption band in the visible region of the spectrum. The peak of the absorption is around 500 nm, which matches the output of the sun closely.
Upon absorption of a photon of light in the visible range, cis-retinal can isomerize to all-trans-retinal. The shape of the molecule changes as a result of this isomerization. The molecule changes from an overall bent structure to one that is more or less linear. All of this is the result of trigonal planar bonding (120 o bond angles) about the double bonds.
Protein Geometry Changes Following Retinal Isomerization
As we shall see below, the isomerization of retinal has an important effect on special proteins in the rod cell: the isomerization event actually causes the proteins to change their shape. This shape change ultimately leads to the generation of a nerve impulse. Hence, the next step in understanding the vision process for monochrome vision is to describe these proteins, and how they change their shape after retinal isomerizes.
Opsin consists of 348 amino acids, covalently linked together to form a single chain. This chain has seven hydrophobic, or water-repelling, alpha-helical regions that pass through the lipid membrane of the pigment-containing discs. This region consists primarily of nonpolar amino acids, which do not attract the polar water molecule. The cis-retinal is situated among these alpha helixes in the hydrophobic region. It is covalently linked to Lysine 296, one of the amino acids in the opsin peptide chain. The linkage is as a Schiff base reaction.
When the cis-retinal absorbs a photon it isomerizes to the all-trans configuration without (at first) any accompanying change in the structure of the protein. Rhodopsin containing the all-trans isomer of retinal is known as bathorhodopsin. However, the trans isomer does not fit well into the protein, due to its rigid, elongated shape. While it is contained in the protein, the all-trans-retinal adopts a twisted conformation, which is energetically unfavorable. The molecule undergoes a series of shape changes to try and better fit the binding site. Therefore, a series of changes in the protein occurs to expel the trans-retinal from the protein.
These rapid movements of the retinal are transferred to the protein, and from there into the lipid membrane and nerve cells to which it is attached. This generates nerve impulses which travel along the optic nerve to the brain, and we perceive them as visual signals - sight. The free all-trans-retinal is then converted back into the cis form by a series of enzyme-catalyzed reactions, whereupon is reattaches to another opsin ready for the next photon to begin the process again.
Activated rhodopsin causes electrical impulses in the following way:
1. The cell membrane (outer layer) of a rod cell has an electric charge. When light activates rhodopsin, it causes a reduction in cyclic GMP, which causes this electric charge to increase. This produces an electric current along the cell. When more light is detected, more rhodopsin is activated and more electric current is produced.
2. This electric impulse eventually reaches a ganglion cell, and then the optic nerve.
3. The nerves reach the optic chasm, where the nerve fibers from the inside half of each retina cross to the other side of the brain, but the nerve fibers from the outside half of the retina stay on the same side of the brain.
4. These fibers eventually reach the back of the brain (occipital lobe). This is where vision s interpreted and is called the primary visual cortex. Some of the visual fibers go to other parts of the brain to help to control eye movements, response of the pupils and iris, and behavior.
Outside Links
• Rhodopsin changes - NEUROBIOLOGY, Molecules, Cells and Systems, Gary G. Matthews | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Photoreceptors/Chemistry_of_Vision/Photochemical_Changes_in_Opsin.txt |
Upon excitation from a laser or other light energy source, photoreceptor molecules transition from a lower energy state to a higher energy state. During this process, electrons of photoreceptor absorbs the energy, and turn into excited state therefore change photoreceptors form. Now, let's see the basic concept of excitation of electrons.
In order to convert between stereoisomers, photons are required to excite an electron to a higher energy state. The lifetime of an excited electron ranges from a few femtoseconds to several hours. When the electron relaxes to a lower energy state, a photon is emitted equal in energy to the difference between the two states.An example of an energy diagram is shown below.
In the retina, electronic excitation converts trans-retinal to cis-retinal by breaking the π Bond of an alkene and rotating the molecule about its σ bond to change the relationship of neighboring groups from cis to trans.
Molecular Orbitals and Light-induced bond rotation
Ethene is a simple molecule that contains only one double bond, and serves as a convenient model to explain electronic excitations and subsequent changes in molecular geometry.
[3]
Yellow lobes indicate σ bonding regions and purple lobes represent π bonding regions. In the molecule's lowest-energy conformation, each carbon's unhybridized p orbitals are coplanar, allowing ethene to form a stabilized π bond; the molecule is subsequently "locked" in htis conformation. When one of the electrons in the π bonding orbital is excited to a higher energy orbital, the bond is destabilized, allowing rotation about the carbon-carbon axis. The two figures below show this rotation from the π-stabilized conformation to the rotated, destabilized rotation.[4]
Molecular orbital diagrams for ethene are given in the figure below: the ground state configuration is on the left, and an excited state configuration on the right. In the ground state, the highest occupied molecular orbital (HOMO) is the carbon-carbon π bonding orbital; the lowest unoccupied molecular orbital (LUMO) is the carbon-carbon π antibonding orbital. Upon exposure to light, an electron is excited from the fully-occupied HOMO to the LUMO as shown.
[5]
Photoreceptor Photocycle
Upon excitation photoreceptors under go several changes in conformation, forming photocycles. Photoreceptors can be stimulated by unique wavelengths of light. For example, photoactive yellow protein (PYP) is a UV-blue light photoreceptor: ultraviolet and blue light can initiate photocycle formation in PYP.
Photoreceptor Proteins
Photoreceptor proteins are light-sensitive proteins involved in the sensing and response to light in a variety of organisms.[1] Photoreceptor proteins can be find in both animals and plants. Human eye retina is a good example of photoreceptor protein. Many bacteria, such as halohodospira halophila, an extremophile bacterium contain Photoactive Yellow Protein.
Photoreceptor proteins are optimally suited to study the role of dynamical alterations in protein structure in relation to their function. First, such proteins can be triggered with (laser) flash illumination, and therefore excellent time-resolution is achievable in studies of the dynamical alterations in their structure. Second, because they are signal-transduction proteins, one may anticipate large conformational transitions to be involved in their signaling state formation (and its subsequent decay), which is indeed borne out by the experiments. Third, the (changing) color of these proteins often is an excellent indicator as to which time scale is relevant to resolve structural transitions. Significant and unsurpassed insight along these lines has been obtained for a number of different photoreceptor proteins. Hence, they can, indeed, be considered as “star actors” in the pursuit to understand, in general terms, the atomic details of the dynamics of functional conformational transitions [i.e., (partial) un/folding] in these proteins required for their functioning.[4]
Structure
Photoreceptor protein contains two parts, the protein part, and non-protein, chromophore part. The non-protein part can response to light throught photoisomerization, or photoexcitation.
[2]
The figure above show protein part in secondary structure, and chromophorepart in line structure.
Photoreceptor in Plants
Plants are important living organisms on earth. As autotrophs, part of plants absorb sunlight through photosynthesis to convert water and Carbon dioxide to oxygen and other chemicals. The part of plants that responsible for absorb and use sunlight is photoreceptor. There are many types of photoreceptors in plants, such as Chlorophyll.
PHYTOCHROMES
BLUE-LIGHT RECEPTORS
• In plant seeds, the photoreceptor phytochrome is responsible for the process termed photomorphogenesis. This occurs when a seed initially situated in an environment of complete darkness is exposed to light. A brief exposure to electromagnetic radiation, particularly that whose wavelength is within the red and far-red lights, results in the activation of the photorecepter phytochrome within the seed. This in turn sends a signal through the signal transduction pathway into the nucleus, and triggers hundreds of genes responsible for growth and development.[3] | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Photoreceptors/Photoreceptor_Excitation.txt |
Vision is such an everyday occurrence that we seldom stop to think and wonder how we are able to see the objects that surround us. Yet the vision process is a fascinating example of how light can produce molecular changes. The retina contain the molecules that undergo a chemical change upon absorbing light, but it is the brain that actually makes sense of the visual information to create an image.
Introduction
Light is one of the most important resources for civilization, it provides energy as it pass along by the sun. Light influence our everyday live. Living organisms sense light from the environment by photoreceptors. Light, as waves carry energy, contains energy by different wavelength. In vision, light is the stimulus input. Light energy goes into eyes stimulate photoreceptor in eyes. However, as an energy wave, energy is passed on through light at different wavelength.
Light, as waves carry energy, contains energy by different wavelength. From long wavelength to short wavelength, energy increase. 400 nm to 700 nm is visible spectrum.
Light energy can convert chemical to other forms. Vitamin A, also known as retinol, anti-dry eye vitamins, is a required nutrition for human health. The predecessor of vitamin A is present in the variety of plant carotene. Vitamin A is critical for vision because it is needed by the retina of eye. Retinol can be convert to retinal, and retinal is a chemical necessary for rhodopsin. As light enters the eye, the 11-cis-retinal is isomerized to the all-"trans" form.
Mechanism of Vision
The molecule cis-retinal can absorb light at a specific wavelength. When visible light hits the cis-retinal, the cis-retinal undergoes an isomerization, or change in molecular arrangement, to all-trans-retinal. The new form of trans-retinal does not fit as well into the protein, and so a series of geometry changes in the protein begins. The resulting complex is referred to a bathrhodopsin (there are other intermediates in this process, but we'll ignore them for now).
The reaction above shows Lysine side-chain from the opsin react with 11-cis-retinal when stimulated. By removing the oxygen atom form the retinal and two hydrogen atom form the free amino group of the lysine, the linkage show on the picture above is formed, and it is called Schiff base.
Signal Transduction Pathway
As the protein changes its geometry, it initiates a cascade of biochemical reactions that results in changes in charge so that a large potential difference builds up across the plasma membrane. This potential difference is passed along to an adjoining nerve cell as an electrical impulse. The nerve cell carries this impulse to the brain, where the visual information is interpreted.
The light image is mapped on the surface of the retina by activating a series of light-sensitive cells known as rods and cones or photoreceptors. The rods and cones convert the light into electrical impulses which are transmitted to the brain via nerve fibers. The brain then determines, which nerve fibers carried the electrical impulse activate by light at certain photoreceptors, and then creates an image.
The retina is lined with many millions of photoreceptor cells that consist of two types: 7 million cones provide color information and sharpness of images, and 120 million rods are extremely sensitive detectors of white light to provide night vision. The tops of the rods and cones contain a region filled with membrane-bound discs, which contain the molecule cis-retinal bound to a protein called opsin. The resulting complex is called rhodopsin or "visual purple".
Figure 3: A fundus photograph of the back of the retina. The white area is the beginning of the optical nerve (optic disc). The image in this photo is the right eye of eric anthamatten. (CC-BY-SA-4.0; TheGoose aPrisoner).
In human eyes, rod and cones react to light stimulation, and a series of chemical reactions happen in cells. These cells receive light, and pass on signals to other receiver cells. This chain of process is class signal transduction pathway. Signal transduction pathway is a mechanism that describe the ways cells react and response to stimulation.
Contributors
• {{template.ContribOphardt()}} | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Photoreceptors/Vision_and_Light.txt |
Photosynthesis is a process that occurs in plants, algae, and some bacteria. These photosynthetic organisms (called autotrophs) use the sun's energy to convert carbon dioxide (CO2) into organic compounds, such as carbohydrates. An example of carbohydrates would be simple sugars such as glucose, mannose, or galactose.
Introduction
Photosynthesis by these organisms is essential for life on Earth. They take in CO2 ,which are waste products from animals and humans, and create oxygen so that we can breathe. However, there are certain bacteria that perform anoxygenic photosynthesis, meaning they consume CO2 but do not release O2.
Essentially, photosynthesis is the opposite of cellular respiration, which is carried out through glycolysis, the Krebs cycle, and the electron transport chain (ETC). All processes of photosynthesis are carried out in the chloroplasts of plants.
Figure 1. The photosynthesis equation. Can be found at commons.wikimedia.org/wiki/Fi...C3%ADntese.jpg
The process
The process of photosynthesis occurs through:
1. Light reactions, which contain Photosystem I and Photosystem II. During the light reactions, energy from light is used to make ATP and NADPH, which are used to provide energy for the making of glucose, for exmple. During the dark reactions, carbon is synthesized into glucose, sucrose, and starch.
2. Dark reactions occurs through the Calvin-Benson-Bassham cycle, which results in CO2 fixation.
Where Photosynthesis occurs: The Chloroplast
Figure 2. The parts of a chloroplast. Used with permission from Wikipedia Commons.
The chloroplasts are organelles in plants that harvest light energy to produce ATP and fix carbon in eukaryotic photosynthetic cells. Chloroplasts that exist in green plants are usually globular or discoid. They have a dual membrane system. Important features of the chloroplast include:
1. Chlorophyll: Chlorophyll is a pigment that absorbs light in the blue and red wavelengths, which appears green to our eyes.The primary types are chlorophyll a and chlorophyll b.
2. Thylakoid membranes: This is where the chlorophyll, which are carriers for electron transport and are the required components for synthesis of ATP and NADPH, are stored.
3. Thylakoids: Essentially, they are flattened sacs made up of an extensive network of membranes.
4. Stroma: This is the fluid matrix of the chloroplast. It is analogous to the cytoplasm of a cell. It contains most of the necessary enzymes to carry out the dark reactions.
5. Grana: These are stacks of thylakoids. They look like poker chips stacked up. (Sing. Granum).
6. Lumen: This is the inside of a thylakoid.
7. Lamella: This is the linkage between one thylakoid in a granum to another. (Pl. Lamellae.)
8. Photosystems: These are light-capturing units of the thylakoid membrane.
Photosynthetic prokaryotes such as cyanobacteria do not have chloroplasts but they do have photosynthetic apparati bound to their plasma membrane. In the chloroplasts, electrons flow from H2O, through several electron acceptors, to NADP+, which serves as the final electron acceptor. This entire process requires energy, which is provided by the sun.
Photons and Light
Quantities of light are defined in terms of photons, which are particles of light. They are represented by the symbol hv. When photons strike the molecules and are absorbed, the electrons of those molecules get excited to a higher energy level. When the light source is removed, the electrons may return to their original states, give off energy as heat or light, or be transferred to other molecules. Visible light is the region of importance in photosynthesis. The range is from 350 to 800 nm (nanometers). The energy of light increases with shorter wavelengths and decreases with longer wavelengths.
Figure 3. The electromagnetic spectrum. Used with permission. Can be found at commons.wikimedia.org/wiki/File:Electromagnetic-Spectrum.png
Primary Pigments
In the thylakoid membranes of photosynthetic cells, there are light-absorbing molecules called pigments. Green pigments are in a class called chlorophyll. Chlorophylls a and b are found in most green plants. Bacteriochlorophyll is found in photosynthetic bacteria.
Figure 4. Structure of chlorophyll a, chlorophyll b, and bacteriochlorophyll, respectively. Used with permission from Wikipedia Commons.
Secondary Pigments
Photosynthetic cells also contain secondary, or accessory, pigments, which absorb light where chlorophyll is not as useful. There are two types of secondary pigments: carotenoids and phycobilins. All carotenoids have 40 carbons and absorb in the range of 400 to 500 nm. That is why their colors are red, orange, and yellow. Two categories that represent carotenoids are xanthophylls and beta-carotenes. Phycobilins absorb in the range of 550 to 630 nm.
Figure 5. Zeaxanthin, a xanthophyll which is one of the carotenoids.
Problems
1. True or False: Sucrose is a direct product of photosynthesis.
2. Chlorophyll can be found in the:
a) Grana
b) Lumen
c) Stroma
d) Thylakoid membrane
e) Outer membrane
3. Photosynthesis can be observed in the region of:
a) Ultraviolet light
b) Infrared
c) Visible light
d) Gamma rays
e) Radio waves
4. True or False: Chlorophyll a, chlorophyll b and bacteriochlorophyll are found in green plants.
5. Fill in the blank: The color of carotenoids is usually _____, _____, or _____.
Answers
1. False
2. d
3. c
4. False
5. red, orange, yellow
Contributors and Attributions
• Tiffany Lui, University of California, Davis
Photosynthesis overview
The light reactions, also known as photolysis reactions, convert energy from the sun into chemical energy in the form of NADPH and ATP. These reactions must take place in the light and in chloroplasts of plants.
Contributors and Attributions
• Tiffany Lui, University of California, Davis | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Photosynthesis/Photosynthesis_overview/The_Light_Reactions.txt |
Photosystem is the form of pigments on the thylakoid membrane1. It collects energy over the wavelengths and concentrates it to one molecule which uses the energy to pass one of its electrons on to a series of enzymes1. Photosystem II occurs with two series of enzymes followed by Photosystem I in order to create energy for a plant1. In Photosystem II which also called water- plastoquinone oxidoreductase, the generated hydrogen ions help to create a proton gradient that is used by ATP synthase to generate ATP, and the transferred energized electrons are used to reduce 2NADP+ to 2NADPH.
Photosystem II is the first membrane protein complex in oxygenic photosynthetic organisms in nature. It produces atmospheric oxygen to catalyze the photo-oxidation of water by using light energy. It oxidizes two molecules of water into one molecule of molecular oxygen. The four electrons removed from the water molecules are transferred by an electron transport chain which is formed hydrogen ions and molecular oxygen to plastoquinone2. By obtaining these electrons from water, photosystem II provides the electrons for all of photosynthesis to occur4.
Photosystem II is composed of 20 subunits such as D1, D2, CP43, CP47, and PsbO3. Subunit D1 (beta-carotene, quinine and manganese center) reacts in the center of protein and binds Chlorophyll P680 and pheophytin, and Subunit D2 reacts in the center Protein. D1 and D2 form the core of this membrane protein3. D1 (colored in red) is homologous to the L subunit of the bacterial photosystem where as D2 (colored in blue) is homologous to the M subunit of the bacterial photosystem3. Chlorophylls is bounded by D1 and D2 and colored in green in the Figure A shown below3. CP43 binds with manganese center and CP47 appears in Photosystem I3. Last, PsbO (colored in purple) occurs in Manganese center to stabilize Protein.
These subunits contains 99 cofactors and coenzymes; “35 chlorophyll a, 12 beta – carotene, two pheophytin, three plastoquinone, two heme, bicarbonate, 25 lipid and seven n-dodecyl – beta – D – maltoside detergent molecules, the six components of the Mn4Ca cluster, and one Fe2+ and two putative Ca2+ ion per monomer”1. Chlorophyll absorbs light4, Beta – carotene absorbs photoexcitation energy4, and heme contains iron4. Pheophytin is transferred an electron from P680 which is formed of 2 chlorophylls that absorb light at the wavelength of 680nm4. It is a primary electron acceptor and contains chlorophyll with the Magnesium replaced by two protons5. Then the electron is transferred to Plastoquinone (PQ) at QA site then QB site4. Plastoquinone can be one or two electron acceptor or donor from Photosystem II to the cytochrome bf complex in mobile intra-thylakoid membrane5. The arrival of a second electron at QB site with the uptake of two protons produces PQH24. When Plastoquinone is fully reduced to PQH2, it is called Plastoquinol. Therefore, the overall reaction for Photosystem II is shown below;
2PQ + 2H2O -> O2 + 2PQH2 (3)
When the electron is transferred from P680 to Phephytin, a positive charge is formed on P680+ which is a strong oxidant that extracts electrons from water at manganese center5. Manganese center is the oxygen evolving center (OEC) and the site of water oxidation. It includes 4 manganese ions, a calcium ion, a chloride ion, and a tyrosine radical5. It is the core of this redox center because it has four stable oxidation states such as Mn2+, Mn3+, Mn4+, and Mn5+.5 Each time the P680 is excited and an electron is kicked out, the positively charged pair extracts an electron from the manganese center5.
The manganese center is oxidized one electron at a time so it requires four steps to complete the oxidation. A tyrosine residue is not shown participates in the proton electron transfers, therefore; the structures are designated S0 through S4 to indicate the number of electrons removed6. We know there are five different oxidation states because of S0 through S4. When S4 is attained, an oxyzen molecule is released and two new molecules of water bind.
The site of plastoquinone reduction is on the stroma side of the membrane6. The manganese complex is on the thylakoid lumen side of the membrane6. For every four electrons harvested from water, two molecules of PQH2 are formed extracting four protons from the stroma6. The four protons formed during the oxidation of water are released into the thylakoid lumen6. This distribution of protons across the thylakoid membrane generates a pH gradient with a low pH in the lumen and a high pH in the stroma6.
The oxygen evolving complex of photosystem II contains Mn4, a redox-active tyrosine, and Ca2+/Cl- ions, but its molecular structure has not yet determined8. However, by looking at Figure B above, the point group for Photosystem II can be determined as C2 with a metal, Mn7. The Figure B describes an oblique surface-rendered view of the 3D structure of the C. reinhardtii supercompex6. The supercomplex is dimeric, therefore; it is found to be C2 point group symmetric containing two sets of subunits6.
The primary emphasis of the Raman study in Photosystem II is on the low frequency range from 220 to 620 (cm-1)8. The low frequency region is examined for both S1 and S2. The Raman spectra of Photosystem II in the S1 state represents a few unique low-frequency bands that do not represent in S2 state8. This indicates that it is coordinated by two H2O or OH-. The Raman Mn-depleted Photosystem II and Photosystem II in the S2 are almost the same8. This indicates that the S1 state of the Manganese has a near infrared electronic transition from the resonance enhanced Raman scattering can be induced8.
Photosystem II which is a part of Photosynthesis is one of the protein complexes. It has been the focus on many studies as a major biological energy source for life on the earth. This process requires water to obtain the electrons in order to provide the electrons for all of photosynthesis. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Photosynthesis/Photosystem_II/Photosystem_II_2.txt |
Photosystem II is crucial to life as we know it. This process is the only natural process capable of forming O2 from water and sunlight (Siegbahn, 2009).This capability is used to convert light energy to chemical energy in plants. This process also provides the energy to drive the conversion of carbon dioxide into the oxygen critical to animal life. Animals also derive benefit from chemical energy stored in the form of sugars within the cells of the plants through their consumption. This may also occur indirectly via the consumption of other animals who have previously consumed these plants.
The overall process is divided into light reactions which need light to occur, and dark reactions, also known as the Calvin Cycle, which do not directly need light. (Campbell, 2005) Electrons and protons are passed from the light reactions to NADPH which allows for their transport to the Calvin Cycle. (Campbell, 2005).
At the heart of this indispensable system is the protein p680 which drives the light reaction, and thereby the entirety of the process (Campbell, 2005). At the heart of p680 is the Oxygen Evolving Complex (OEC), shown below, which is a cluster of inorganic atoms. The precise form of the OEC continues to elude researchers, and many models have been proposed. The Dangler Model (Siegbahn, 2009) has been selected for this assignment because it gives the best opportunity to delve into the symmetrical aspects of the OEC. This model of the OEC consists of four manganese atoms, four oxygen atoms, and one calcium atom. A chlorine atom becomes involved in the reactions but is not directly a part of this cluster. (Vrettos, 2002)
Figure 1: Oxygen Evolving Complex. (simplified† from distorted cubic layout)
But what of the chemistry drives this vital process? We find that inorganic chemistry, most specifically that of manganese, plays the key role in this process. In many of its oxidation states, those above (II), manganese is known to have great oxidizing power (Housecraft, 2008). The Manganese atoms in the OEC have been found to be the (III) and (IV) oxidation states (Kulik, 2007). Using the oxidative power of these manganese atoms the OEC is able to drive the process through a series of reactions divided into steps S0-S4. Overall, the Manganese drives the general reaction: 2H2O + Light → O2 + 4H+ + 4e- (Micklitz, 1998)
Seeing the oxygen evolving complex arranged in the manner of the Dangler Model provides an excellent opportunity to assess the symmetry of this structure, at least locally. Knowledge of the symmetry is of importance when predicting or interpreting the results of IR and Raman Spectroscopy. Using the simplified depiction of the OEC in figure 1 it can be seen that the cluster is of a cubic shape with one magnesium stretching away from a corner (Siegbahn, 2009) the symmetry is as follows:
The complex without the blue Mn would be C3v
With the blue Mn the complex is C1
The entire protein also has C1 symmetry
Through knowing the structure and employing the character tables, the IR and Raman spectroscopic results can be predicted for this complex. In the event that the simplified version of the distorted cubic structure were are to be the precise layout of the OEC, we could can expect to see the following.
• These results can also be run through a quick visual check to see that point group symmetries that have a center of inversion have irreducible forms in which each individual term does not yield both a Raman and IR band. These predicted bands meet that criteria. Another useful application of the fundamentals of inorganic chemistry to understanding the form of the oxygen evolving center is to look at the bonding energy diagram of the manganese to an adjacent oxygen.
Through an understanding of the basics of inorganic chemistry and looking at ongoing research we can see the pivotal role inorganic chemistry plays in the chemistry of life. Looking at a periodic table with each atom marked as to whether it is essential to all investigated species yields 5 elements in the which reside in the d-block, metals. The same table shows an additional 5 metals being essential to at least one biological species (Schore, 2007)
Through this example, and likely many, many more, it becomes obvious that there is no distinct chasm that inorganic and organic chemistry can not bridge. Life is a powerful and diverse process rooted in chemistry and as such takes advantage of opportunities not provided by the typical carbon, hydrogen, oxygen, nitrogen arsenal of organic chemistry.
†: The Dangler Model does not predict all atoms for the OEC to be equidistant, not for all the angles to perfectly fit the 90º required of a perfect cube. It can be described as distorted cubic. By representing it as a simple cube, there is access to a greater variety of symmetries to evaluate. The image is a simplified depiction of that found in the Siegbahn paper in the references section.
Contributors and Attributions
• Kenneth King | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Photosynthesis/Photosystem_II/Photosystem_II_3.txt |
Common amino acids
There are 20 common amino acids. They are composed of C, H, O, N and S atoms. They are structurally and chemically different, and also differ in size and volume. Some are branched structures, some are linear, some have ring structures. One of the 20 common amino acids is actually an imino acid. A typical grouping of their chemical nature is as follows:
• Nonpolar (hydrocarbons and one sulfur-containing amino acid). Dispersion forces and hydrophobic effects predominate in their interactions. They cannot H-bond with water and these side chains have a characteristic hydrophobic effect in water.
• Polar uncharged. Contain functional groups that can H-bond with water and other amino acids. Include C, H, O, N and S atoms.
• Acidic. Contain a carboxylic acid functional group with a negative charge at neutral pH. Can H-bond with water, can form ionic interactions, and can also serve as nucleophiles or participate in acid-base chemistry.
• Basic. Nitrogen containing bases (e.g. guanidino, imidazole or amino groups) with a net positive charge at neutral pH. Can serve as proton donors in chemical reactions, and form ionic interactions.
The amino acids have a name, as well as a three letter or single letter mnemonic code:
Type
Name
R-group Structure
Nonpolar
Leucine
Leu, L
Isoleucine
Ile, I
Valine
Val, V
Alanine
Ala, A
Methionine
Met, M
Phenylalanine
Phe, F
Tryptophan
Trp, W
Proline
Pro, P
Glycine
Gly, G
(note: sometimes included in polar group)
Polar, uncharged
Serine
Ser, S
Asparagine
Asn, N
Glutamine
Gln, Q
Threonine
Thr, T
Cysteine
Cys, C
Tyrosine
Tyr, Y
Acidic
Aspartic acid
Asp, D
Glutamic acid
Glu, E
Basic
Lysine
Lys, K
Arginine
Arg, R
Histidine
His, H
Uncommon amino acids
In addition to the 20 common amino acids, there are several uncommon ones found:
• Hydroxylysine and hydroxyproline. These are found in the protein collagen. Collagen is a fibrous protein made up of three polypeptides that form a stable assembly, but only if the proline and lysine residues are hydroxylated. (requires vitamin C for reduction of these amino acids to hydroxy form)
• Thyroxine, an iodinated derivative of tyrosine, found in thyroglobulin (produced by thyroid gland; requires iodine in diet)
• g-carboxyglutamic acid (i.e. glutamic acid with two carboxyl groups) found in certain blood clotting enzymes (requires vitamin K for production)
• N-methyl arginine and n-acetyl lysine. Found in some DNA binding proteins known as histones
Amino acid derivatives not found in proteins
Some amino acids are made that are not intended for incorporation into proteins, rather they have important functionalities on their own
• Serotonin (derivative of tryptophan) and g-amino butyric acid (a derivative of glutamic acid) are both neurotransmitters
• Histamine (derivative of histidine) involved in allergic response
• Adrenaline (derivative of tyrosine) a hormone
• Various antibiotics are amino acid derivatives (penicillin)
Contributors and Attributions
Thumbnail: 3D model of L-tryptophan. (Public Domain; Benjah-bmm27).
1. Backgrounds of Amino Acids
• 1. Backgrounds of Amino Acids
This page explains what amino acids are, concentrating on the 2-amino acids that are biologically important. It looks in some detail at their simple physical properties such as solubility and melting points.
• Charged Nature of Amino Acids
Proteins are probably the most important class of biochemical molecules, although of course lipids and carbohydrates are also essential for life. Proteins are the basis for the major structural components of animal and human tissue. Proteins are natural polymer molecules consisting of amino acid units. The number of amino acids in proteins may range from two to several thousand.
• Stereochemistry of Amino Acids
With the exception of glycine, all the 19 other common amino acids have a uniquely different functional group on the central tetrahedral alpha carbon.
• Structure of Amino Acids
Amino acid monomers are chemically linked to form linear polymers known as proteins.
Thumbnail: 3D model of L-tryptophan. (Public Domain; Benjah-bmm27).
Properties of Amino Acids
This page explains what amino acids are, concentrating on the 2-amino acids that are biologically important. It looks in some detail at their simple physical properties such as solubility and melting points.
Structures and names
Amino acids are exactly what they say they are! They are compounds containing an amino group, -NH2, and a carboxylic acid group, -COOH. The biologically important amino acids have the amino group attached to the carbon atom next door to the -COOH group. They are known as 2-amino acids. They are also known (slightly confusingly) as alpha-amino acids. These are the ones we will concentrated on.
The two simplest of these amino acids are 2-aminoethanoic acid and 2-aminopropanoic acid. Because of the biological importance of molecules like these, they are normally known by their traditional biochemical names. 2-aminoethanoic acid, for example, is usually called glycine, and 2-aminopropanoic acid is usually known as alanine.
The general formula for a 2-amino acid is:
. . . where "R" can be quite a complicated group containing other active groups like -OH, -SH, other amine or carboxylic acid groups, and so on. It is definitely NOT necessarily a simple hydrocarbon group!
Physical properties
Melting points
The amino acids are crystalline solids with surprisingly high melting points. It is difficult to pin the melting points down exactly because the amino acids tend to decompose before they melt. Decomposition and melting tend to be in the 200 - 300°C range. For the size of the molecules, this is very high. Something unusual must be happening. If you look again at the general structure of an amino acid, you will see that it has both a basic amine group and an acidic carboxylic acid group.
There is an internal transfer of a hydrogen ion from the -COOH group to the -NH2 group to leave an ion with both a negative charge and a positive charge. This is called a zwitterion.
A zwitterion is a compound with no overall electrical charge, but which contains separate parts which are positively and negatively charged. This is the form that amino acids exist in even in the solid state. Instead of the weaker hydrogen bonds and other intermolecular forces that you might have expected, you actually have much stronger ionic attractions between one ion and its neighbors. These ionic attractions take more energy to break and so the amino acids have high melting points for the size of the molecules.
Solubility
Amino acids are generally soluble in water and insoluble in non-polar organic solvents such as hydrocarbons. This again reflects the presence of the zwitterions. In water, the ionic attractions between the ions in the solid amino acid are replaced by strong attractions between polar water molecules and the zwitterions. This is much the same as any other ionic substance dissolving in water. The extent of the solubility in water varies depending on the size and nature of the "R" group. The lack of solubility in non-polar organic solvents such as hydrocarbons is because of the lack of attraction between the solvent molecules and the zwitterions. Without strong attractions between solvent and amino acid, there won't be enough energy released to pull the ionic lattice apart.
Optical activity
If you look yet again at the general formula for an amino acid, you will see that (apart from glycine, 2-aminoethanoic acid) the carbon at the centre of the structure has four different groups attached. In glycine, the "R" group is another hydrogen atom.
This is equally true if you draw the structure of the zwitterion instead of this simpler structure.
Because of these four different groups attached to the same carbon atom, amino acids (apart from glycine) are chiral. The lack of a plane of symmetry means that there will be two stereoisomers of an amino acid (apart from glycine) - one the non-superimposable mirror image of the other. For a general 2-amino acid, the isomers are:
All the naturally occurring amino acids have the right-hand structure in this diagram. This is known as the "L-" configuration. The other one is known as the "D-" configuration. If you read around the other groups in a clockwise direction, you get the word CORN.
You can't tell by looking at a structure whether that isomer will rotate the plane of polarisation of plane polarised light clockwise or anticlockwise. All the naturally occurring amino acids have the same L- configuration, but they include examples which rotate the plane clockwise (+) and those which do the opposite (-).
For example:
• (+)alanine
• (-)cysteine
• (-)tyrosine
• (+)valine
It is quite common for natural systems to only work with one of the optical isomers (enantiomers) of an optically active substance like the amino acids. It isn't too difficult to see why that might be. Because the molecules have different spatial arrangements of their various groups, only one of them is likely to fit properly into the active sites on the enzymes they work with. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Proteins/Amino_Acids/Nomenclature_of_Amino_acids.txt |
Although we are studying only about 20 amino acids, there are about six more found in the body. Many others are also known from a variety of sources. Amino acids are the building blocks used to make proteins and peptides. The different amino acids have interesting properties because they have a variety of structural parts which result in different polarities and solubilities.
Each amino acid has at least one amine and one acid functional group as the name implies. See graphic on the left. The different properties result from variations in the structures of different R groups. The R group is often referred to as the amino acid "side chain". Amino acids have special common names, however, a three letter abbreviation for the name is used most of the time. Consult the amino acid table on the next page for structure, names, and abbreviations.
Zwitterion
Amino acid physical properties indicate a "salt-like" behavior. Amino acids are crystalline solids with relatively high melting points, and most are quite soluble in water and insoluble in non-polar solvents. In solution, the amino acid molecule appears to have a charge which changes with pH. An intramolecular neutralization reaction leads to a salt-like ion called a zwitterion. The accepted practice is to show the amino acids in the zwitterion form:
1. The carboxyl group can lose a hydrogen ion to become negatively charged.
2. The amine group can accept a hydrogen ion to become positively charged.
Contributors
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook
Stereochemistry of Amino Acids
With the exception of glycine, all the 19 other common amino acids have a uniquely different functional group on the central tetrahedral alpha carbon (i.e. $C_{\alpha}$). The $C_{\alpha}$ is termed "chiral" to indicate there are four different constituents and that the Ca is asymmetric. Since the $C_{\alpha}$ is asymmetric there exists two possible, non-superimposable, mirror images of the amino acids:
Exercise $1$
How are these two uniquely different structures in the figure ago distinguished?
Answer
Based off the stereochemistyr at the $C_{\alpha}$.
The D, L system
Glyceraldehyde contains a chiral carbon, and therefore, there are two enantiomers of this molecule. One is labeled the "L" form, and the other the "D" form. This is the frame of reference used to describe amino acid enantiomers as being either the "L" or "D" form
Even though the two enantiomers would seem to be essentially equivalent to each other, all common amino acids are found in the "L" enantiomer in living systems. When looking down the H-C, a bond towards the $C_{\alpha}$ there is a mnemonic to identify the L-enantiomer of amino acids (note: in this view the three functional groups are pointing away from you, and not towards you; the H atom is omitted for clarity - but it would be in front of the C)
Starting with the carbonyl functional group, and going clockwise around the $C_{\alpha}$ of the L-enantiomer, the three functional groups spell out the word CORN. If you follow the above instructions, it will spell out CONR (a silly, meaningless word) for the D-enantiomer
Optical Activity
Enantiomeric molecules have an optical property known as optical activity - the ability to rotate the plane of plane polarized light. Clockwise rotation is known as "dextrorotatory" behavior and counterclockwise rotation is known as "levorotatory" behavior.
A source of potential confusion...
All common amino acids are the L-enantiomer (i.e. their $C_{\alpha}$ chiral center is the L-enantiomer), based on the structural comparison with L-glyceraldehyde. However, not all L-amino acids are Levorotatory, some are actually Dextrorotatory with regard to their optical activity. To (attempt) to avoid confusion, the optical activities are given as (+) for dextrorotatory, and (-) for levorotatory
• L(+)-alanine (this is the L-enantiomer and it is dextrorotatory)
• L(-)-serine (this is the L-enantiomer and it is levorotatory)
Multiple chiral centers
• Molecules with N chiral centers can exist in 2N isomeric structures
• Isomers that differ in configuration at only one chiral center are called diastereomers
The R,S system of naming chiral centers
A relative ranking of the "priority" of various functional groups is given as:
$\ce{SH > OH > NH2 > COOH > CHO > CH2OH > CH3 > H}$
• A chiral center has four different functional groups. Identify the functional group with the lowest priority
• View the chiral center down the bond from the chiral center to the lowest priority atom
• don't confuse this with the CORN mnemonic method of identifying the L-amino acid chirality by viewing from the H to the Ca )
• Assign priorities to the three other functional groups connected to the chiral center, using the above ranking
• If the priorities of these other groups goes in a clockwise rotation, the chirality is "R". If the priorities of these other groups goes counterclockwise, the chirality is "S". (Note that this assignment has nothing to do with optical activity, and is not using L-glyceraldehyde as a reference molecule)
Spectroscopic properties of amino acids
This refers to the ability of amino acids to absorb or emit electromagnetic energy at different wavelengths (i.e. energies)
• No amino acids absorb light in the visible spectrum (i.e. they are "colorless").
• If proteins have color (e.g. hemoglobin is red) it is because they contain a bound, non-protein atom, ion or molecule; iron in this case)
• All amino acids absorb in the infrared region (longer wavelengths, weaker energy than visible light)
• Some amino acids absorb in the ultraviolet spectrum (shorter wavelengths, higher energy than visible light)
• Absorption occurs as electrons rise to higher energy states
• Electrons in aromatic ring structures absorb in the u.v. spectrum. Such structures comprise the side chains of
• tryptophan, tyrosine and phenylalanine.
Separation and analysis of amino acid mixtures
The 20 common amino acids differ from one another in several important ways. Here are just two:
• Mass. The smallest amino acid (glycine) has a mass of 57 Da (i.e. g/mol), and the largest (tryptophan) has a mass of 186 Da
• Isoelectric point (pH at which the amino acid has a neutral charge). This is a function of all ionizable groups on the amino acid, including the amino and carboxyl functional groups in addition to any ionizable group on the side chain.
Amino Acid
Mass (Da)
Isoelectric Point
Amino Acid
Mass (Da)
Isoelectric Point
Aspartic Acid
114.11
2.98
Isoleucine
113.16
6.038
Glutamic Acid
129.12
3.08
Glycine
57.05
6.064
Cysteine
103.15
5.02
Alanine
71.09
6.107
Tyrosine
163.18
5.63
Proline
97.12
6.3
Serine
87.08
5.68
Histidine
137.14
7.64
Methionine
131.19
5.74
Lysine
128.17
9.47
Tryptophan
186.12
5.88
Arginine
156.19
10.76
Phenylalanine
147.18
5.91
Threonine
101.11
-
Valine
99.14
6.002
Asparagine
115.09
-
Leucine
113.16
6.036
Glutamine
128.14
-
We can use these differences in physical properties to fractionate complex mixtures of amino acids into individual amino acids
• In looking at the isoelectric point of the different amino acids it seems that they will have different partial charges at a given pH.
• For example, at pH 6.0 some will be negatively charged, and some positively charged.
• For those that are negatively charged, some will be slightly negative, and others strongly negative. Similarly, for those that are positively charged, some will be slightly positive, and others strongly positive
• The charge differences of the amino acids means that they will have different affinities for other cationic or anionic charges | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Proteins/Amino_Acids/Properties_of_Amino_Acids/Charged_Nature_of_Amino_Acids.txt |
• Acid-base Chemistry of Amino Acids
Amino acids by themselves have amino (pKa ~9.0-10.5) and carboxyl groups (pKa ~2.0-2.4) that can be titrated. At neutral pH the amino group is protonated, and the carboxyl group is deprotonated. The side chains of acid and basic amino acids, and some polar amino acids can also be titrated.
• Acid-Base Reactions of Amino Acids
This page looks at what happens to amino acids as you change the pH by adding either acids or alkalis to their solutions. For simplicity, the page only looks at amino acids which contain a single -NH2 group and a single -COOH group.
• Amino Acid Reactions
Amino acids react with each other in a typical acid-base neutralization reaction to form a salt.
Thumbnail: 3D model of L-tryptophan. (Public Domain; Benjah-bmm27).
Reactions of Amino Acids
This page looks at what happens to amino acids as you change the pH by adding either acids or alkalis to their solutions. For simplicity, the page only looks at amino acids which contain a single -NH2 group and a single -COOH group.
Amino acids as Zwitterions
An amino acid has both a basic amine group and an acidic carboxylic acid group.
There is an internal transfer of a hydrogen ion from the -COOH group to the -NH2 group to leave an ion with both a negative charge and a positive charge. This is called a zwitterion.
This is the form that amino acids exist in even in the solid state. If you dissolve the amino acid in water, a simple solution also contains this ion. A zwitterion is a compound with no overall electrical charge, but which contains separate parts which are positively and negatively charged.
Adding an alkali to an amino acid solution
If you increase the pH of a solution of an amino acid by adding hydroxide ions, the hydrogen ion is removed from the -NH3+ group.
You could show that the amino acid now existed as a negative ion using electrophoresis. In its simplest form, electrophoresis can just consist of a piece of moistened filter paper on a microscope slide with a crocodile clip at each end attached to a battery. A drop of amino acid solution is placed in the center of the paper.
Although the amino acid solution is colourless, its position after a time can be found by spraying it with a solution of ninhydrin. If the paper is allowed to dry and then heated gently, the amino acid shows up as a coloured spot. The amino acid would be found to travel towards the anode (the positive electrode).
Adding an acid to an amino acid solution
If you decrease the pH by adding an acid to a solution of an amino acid, the -COO- part of the zwitterion picks up a hydrogen ion.
This time, during electrophoresis, the amino acid would move towards the cathode (the negative electrode).
Shifting the pH from one extreme to the other
Suppose you start with the ion we've just produced under acidic conditions and slowly add alkali to it. That ion contains two acidic hydrogens - the one in the -COOH group and the one in the -NH3+ group. The more acidic of these is the one in the -COOH group, and so that is removed first - and you get back to the zwitterion.
So when you have added just the right amount of alkali, the amino acid no longer has a net positive or negative charge. That means that it wouldn't move towards either the cathode or anode during electrophoresis. The pH at which this lack of movement during electrophoresis happens is known as the isoelectric point of the amino acid. This pH varies from amino acid to amino acid. If you go on adding hydroxide ions, you will get the reaction we've already seen, in which a hydrogen ion is removed from the -NH3+ group.
You can, of course, reverse the whole process by adding an acid to the ion we've just finished up with. That ion contains two basic groups - the -NH2 group and the -COO- group. The -NH2 group is the stronger base, and so picks up hydrogen ions first. That leads you back to the zwitterion again.
. . . and, of course, you can keep going by then adding a hydrogen ion to the -COO- group.
Why isn't the Isoelectric Point of an Amino Acid at pH 7?
When an amino acid dissolves in water, the situation is a little bit more complicated than we tend to pretend at this level. The zwitterion interacts with water molecules - acting as both an acid and a base. As an acid:
The -NH3+ group is a weak acid and donates a hydrogen ion to a water molecule. Because it is only a weak acid, the position of equilibrium will lie to the left.
As a base:
The -COO- group is a weak base and takes a hydrogen ion from a water molecule. Again, the equilibrium lies to the left.
When you dissolve an amino acid in water, both of these reactions are happening. However, the positions of the two equilibria aren't identical - they vary depending on the influence of the "R" group. In practice, for the simple amino acids we have been talking about, the position of the first equilibrium lies a bit further to the right than the second one. That means that there will be rather more of the negative ion from the amino acid in the solution than the positive one.
In those circumstances, if you carried out electrophoresis on the unmodified solution, there would be a slight drift of amino acid towards the positive electrode (the anode). To stop that, you need to cut down the amount of the negative ion so that the concentrations of the two ions are identical. You can do that by adding a very small amount of acid to the solution, moving the position of the first equilibrium further to the left. Typically, the pH has to be lowered to about 6 to achieve this. For glycine, for example, the isoelectric point is pH 6.07; for alanine, 6.11; and for serine, 5.68.
Acid-base Chemistry of Amino Acids
Amino acids by themselves have amino (pKa ~9.0-10.5) and carboxyl groups (pKa ~2.0-2.4) that can be titrated. At neutral pH the amino group is protonated, and the carboxyl group is deprotonated. The side chains of acid and basic amino acids, and some polar amino acids can also be titrated:
Amino acid Functional Group Side chain pKa
Cysteine -SH 8.3
Serine -OH 13
Threonine -OH 13
Tyrosine -OH 10.1
Aspartic acid -COOH 3.9
Glutamic acid -COOH 4.3
Histidine Imidazole ring 6.0
Arginine Guanidino 12.5
Lysine -NH2 10.5
Physiological pH is near neutral. It would appear that only histidine is of physiological relevance. However, pKa values can be shifted significantly by neighboring charged groups in complex molecular structures.
Reactions of amino acids
• Free amino acids (excluding proline) share similar chemical reactivities due to the common amino and carboxyl groups.
• Different amino acid side chains have different chemical reactivities. Therefore,
• reactivities of different proteins reflects the composition of the unique sequence of amino acids in their structure.
Some common carboxyl-group reactivities:
A common side chain reaction involving cysteine:
NOTE: This can covalently link two polypeptide chains in a "disulfide bond" crosslink
Amino Acid Reactions
Amino acids react with each other in a typical acid-base neutralization reaction to form a salt.
The reaction is simply the transfer of the -H (positive ion) from the acid to the amine and the attraction of the positive and negative charges. The acid group becomes negative, and the amine nitrogen becomes positive because of the positive hydrogen ion.
For example in the graphic on the left - top, glycine (gly) and alanine (ala) may just interact in the zwitterion form by an attraction of the positive (amine) of the alanine and negative (carboxyl acid) charges to form the salt.
Salt formation of Side Chains
A more important interaction for protein tertiary structure is the interaction of the acid and base "side chains". If the amino acid has an extra acid or amine on the "side chain", these are used in the salt formation. For example in the left-bottom graphic, Aspartic acid (asp) has a side chain that forms a salt with the amine on the lysine (lys) side chain. The hydrogen ion (red) moves to the amine nitrogen resulting in the salt with the attraction of the positive and negative charges.
Disulfide Bridges and Oxidation-Reduction
The amino acid cysteine undergoes oxidation and reduction reactions involving the -SH (sulfhydryl group). The oxidation of two sulfhydryl groups results in the formation of a disulfide bond by the removal of two hydrogens. The oxidation of two cysteine amino acids is shown in the graphic on the left. An unspecified oxidizing agent (O) provides an oxygen which reacts with the hydrogen (red) on the -SH group to form water. The sulfurs (yellow) join to make the disulfide bridge. This is an important bond to recognize in protein tertiary structure.
The reduction of a disulfide bond is the opposite reaction which again leads to two separate cysteine molecules. Remember that reduction is the addition of hydrogen. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Proteins/Amino_Acids/Reactions_of_Amino_Acids/Acid-Base_Reactions_of_Amino_Acids.txt |
• Angiotnesin Peptide
• Enkephalines
• Membrane Transport
Membrane transport is essential for cellular life. As cells proceed through their life cycle, a vast amount of exchange is necessary to maintain function. Transport may involve the incorporation of biological molecules and the discharge of waste products that are necessary for normal function. 1 Membrane transport refers to the movement of particles (solute) across or through a membranous barrier. 2 These membranous barriers, in the case of the cell for example, consist of a phospholipid bilayer. The phospholipids orient themselves in such a way so that the hydrophilic (polar) heads are nearest the extracellular and intracellular mediums, and the hydrophobic (non-polar) tails align between the two hydrophilic head groups. Membrane transport is dependent upon the permeability of the membrane, transmembrane solute concentration, and the size and charge of the solute. 2 Solute particles can traverse the membrane via three mechanisms: passive, facilitated, and active transport. 1 Some of these transport mechanisms require the input of energy and use of a transmembrane protein, whereas other mechanisms do not incorporate secondary molecules. 3
• Permanent Hair Wave
The formation of disulfide bonds has a direct application in producing curls in hair by the permanent wave process. Hair keratin consists of many protein alpha-helices. Three alpha-helices are interwoven into a left-handed coil called a protofibril. Eleven protofibrils are bonded and coiled together to make a microfibril. Hundreds of these microfibrils are cemented into an irregular bundle called a macrofibril. These in turn are mixed with dead and living cells to make a complete strand of hair.
• Sickle Cell Anemia
The incorrect amino acid sequence in a protein may lead to fatal consequences. For example, the inherited disease, sickle cell anemia, results from a single incorrect amino acid at the 6th position of the beta - protein chain out of 146. Hemoglobin consists of four protein chains - two beta and two alpha.
Case Studies: Proteins
Hypertension (high blood pressure) is a major human disease and despite much research, the problem is still not completely understood. A major part of the problem involves the action of an octapeptide, angiotensin II. This peptide hormone stimulates the constriction of blood vessels which leads to an increase in blood pressure. Angiotensin II is produced by the removal of two amino acids units on angiotensin I by an enzyme in the blood. Angiotensin I does not constrict blood vessels.
QUES. What is the difference between angiotensin I and angiotensin II? This difference is responsible for difference in physiological action.
Angiotensin I: asp - arg - val - tyr - ile - his - pro - phe - his - leu
Angiotensin II: asp - arg - val - tyr - ile - his - pro - phe.
Contributors and Attributions
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook
Enkephalines
Enkephalins (penta-peptides) have recently been discovered as regulators of nerve impulses involving pain in the brain. Apparently these peptides act as natural analgesics (pain-killers) and their action mimics that of morphine and other opiates. At present it is thought that the morphine-like effects are due to aromatic side chains on phenylanine and tyrosine which mimic a similar sturcture on morphine. Apparently, it does not make much difference whether the enkephalin contains methionine or leucine at the acid end of the peptide. The primary structures are:
methionine-enkephaline: tyr - gly - gly - phe - met
leucine-enkephalin: tyr - gly - gly - phe - leu
QUES. Write the peptide structure for methionine-enkephalin. Enkephalin - Met - Chime in new window
Quiz:Which amino acid is the N-terminal one?
Which amino acid is the C-terminal one? | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Proteins/Case_Studies%3A_Proteins/Angiotnesin_Peptide.txt |
Overview
Membrane transport is essential for cellular life. As cells proceed through their life cycle, a vast amount of exchange is necessary to maintain function. Transport may involve the incorporation of biological molecules and the discharge of waste products that are necessary for normal function.1
Membrane transport refers to the movement of particles (solute) across or through a membranous barrier.2 These membranous barriers, in the case of the cell for example, consist of a phospholipid bilayer. The phospholipids orient themselves in such a way so that the hydrophilic (polar) heads are nearest the extracellular and intracellular mediums, and the hydrophobic (non-polar) tails align between the two hydrophilic head groups.
Membrane transport is dependent upon the permeability of the membrane, transmembrane solute concentration, and the size and charge of the solute.2 Solute particles can traverse the membrane via three mechanisms: passive, facilitated, and active transport.1 Some of these transport mechanisms require the input of energy and use of a transmembrane protein, whereas other mechanisms do not incorporate secondary molecules.3
Passive transport
Passive transport is the simplest method of transport and is dependent upon the concentration gradient, and the size and charge of the solute.2 In passive transport, small uncharged solute particles diffuse across the membrane until both sides of the membrane have reached an equilibrium that is similar in concentration. The direction of solute travel is indicative of the concentration of that particular particle on each side of the membrane.
(1)(2)(3)
Figure 1. Passive diffusion of O2 and CO2 across a membrane over time 1-3.
Based on the thermodynamics of the system, particles will move from an area of high concentration to an area of low concentration in order to increase the entropy of the cell. Additionally, this particle movement will occur spontaneously as the free energy (Gibbs free energy; ∆G) of the system is negative.4
Where:
• Further, the amount of energy consumed or released by the system is as follows2:
• If ∆G<0, then particle movement is considered to be spontaneous; whereas, ∆G>0 particle movement requires the input of energy to move in the desired direction.
The properties of the membrane must also be considered when determining the rate of flow of the substrate.5 Darcy’s Law can be used to determine flow rate.
Where:
• The permeability coefficient is dependent upon the porous nature of the membrane and fluid.6
Where:
• Common permeability coefficients are displayed below7:
Membrane
Substrate
300
Escherichia coli
Glycerol
25
Escherichia coli
Glucose
1
Escherichia coli
Lactose
The diffusion of small charged particles, on the other hand, across a membrane is dependent upon the charge and transmembrane concentration of the solute.2 Again, however, the direction of solute travel is indicative of the thermodynamics of the system. Particles will travel from an area of high to low concentration, as well as travel so that the electrical potential across the membrane is diminished. As a result of this movement, the entropy of the system has increased.
Passive transport is independent of membrane proteins and the catabolism of biological molecules for energy.2 This energy deficient process commonly occurs in the blood brain barrier as specific molecules, such as sodium thiopental (Figure 2. Sodium Thiopental Structure at right), can diffuse across the membrane.8 Sodium thiopental is a barbiturate frequently used in the methods of lethal injection.9 Sodium thiopental is a negatively charged particle and proceeds across the blood brain barrier to neuronal synaptic clefts. Sodium thiopental is an agonist of γ-aminobutyric acid (GABA), which acts as a neurotransmitter inhibitor. Sodium thiopental acts an anesthetic to cause unconsciousness.
Additionally, passive diffusion occurs across the placenta as all solute particles are exchanged between mother and fetus.10 Placental physiology juxtaposes maternal and fetal capillaries in order to exchange solute particles, such as oxygen and carbon dioxide gas.11 These uncharged molecules proceed across maternal and fetal capillary membranes in the direction from high to low concentration. This spontaneous process occurs in accord with the aforementioned properties. Despite the cellular system, passive diffusion across the membranes of all biological cells is in accord with the characteristics outlined above.
Facilitated Diffusion
Facilitated diffusion, not to be confused with simple diffusion, is a form of passive transport mediated by transport proteins imbedded within the cellular membrane.12 Facilitated diffusion allows the passage of lipophobic molecules through the cell membrane’s lipid bilayer.2 Just as in passive transport, molecules, particles, and ions travel freely across the cellular membrane from high concentration to low concentration in an attempt to achieve equilibrium and thereby increase the entropy of the system. Also like passive transport, the Gibbs Free Energy of the system is negative, allowing the particle movement to be spontaneous.4 Facilitated diffusion, however, uses channel proteins to facilitate solute movement.
(1)
(2)
(3)
(4)
Figure 3. Facilitated Diffusion via channel protein across a membrane proceeding images 1-4.
Channel Proteins
Channel proteins are pores immersed in the lipid bilayer membrane and are the hallmark of facilitated diffusion.13 All channel proteins have two things in common:
1. It is of particular importance to note that channel proteins are not indiscriminate; each channel protein contains a selectivity filter.14 The selectivity filter is a collection of amino acid residues concentrated in the interior of the channel protein. As particles, often ions, pass into the channel protein, an electrostatic interaction occurs between the amino acid residues and the ion.15 The interaction would, for example, involve negatively charged amino acid residues in the case of ions like calcium (Na+) or potassium (K+), and positively charged amino acid residues in the case of chlorine (Cl-).2 The electrostatic interaction between amino acid residues and ions allows the channel protein to identify the ion in question by measuring its atomic radius with extremely finite accuracy. Potassium (K+) channels select K+ over Na+ by a factor of over one thousand despite differing in atomic radius by a mere 0.38 Å.
While all channel proteins have an inherent selectivity filter, others have additional gating.2 Gating is a response to a predetermined trigger that allows the channel protein to undergo a conformational change. This action subsequently causes another conformational change that either opens or closes the channel, allowing or disallowing its specific particle to pass. Channel proteins can be physically or chemically modulated through a number of different mechanisms.
Voltage gating
Voltage gated protein channels play a particularly important role in excitable neuronal and muscle tissues.2
Ligand gating
Ligand gated channel proteins are activated in response to the binding of a ligand.17 Typically, ligand binding occurs at an allosteric binding site independent of the channel protein’s pore. The binding of a ligand at the allosteric binding site causes a conformational change in the structure of the channel protein, subsequently causing an influx or efflux of ions. Release of the ligand allows the channel protein to return to its original shape. Structurally, ligand gated channel proteins generally differ from other channels due to the presence of an additional protein domain that serves as the allosteric binding site.2
The prototypical example of ligand gating is the nicotinic acetylcholine receptor located on the postsynaptic side of the neuromuscular junction.18
Other gating
Channel proteins may be gated in less common instances by methods such as light activation, mechanical activation, or secondary messanger activation.2Light activated protein channels contain a photoswitch through which a photon causes a conformational change in the channel protein causing it to open or close. Only one such protein channel exists naturally.19 Mechanically activated protein channels open or close in response to a mechanical stimulus and are vital to the touch, hearing, and balance sensations in human.20 Ligand-gated protein channels are typically linked to second messanger gating.2 Second messenger gating functions stepwise in that a neurotransmitter binds to a channel protein receptor which, in turn, reveals an active site to which the conformation-changing ligand binds.
Active Transport
Active transport, simply put, is the movement of particles through a transport protein from low concentration to high concentration at the expense of metabolic energy.21 The most common energy source used by cells is adenosine triphosphate or ATP, though other sources such as light energy or the energy stored in an electrochemical gradient are also utilized.2 In the case of ATP, energy is chemically harvested through hydrolysis.22 ATP hydrolysis in turn causes a conformational change in the transport protein which allows mechanical movement of the particle in question.2 Active transport systems are, therefore, energy-coupling devices as chemical and mechanical processes are linked to achieve particle movement. Active transport is classified as either Primary Active Transport or Secondary Active Transport. Figure 4 (displayed above) displays a ribbon structure of a commonly depicted ABC Vitamin B12importer active transport protein.
Primary Active Transport
Primary active transport uses the energy found in ATP, photons, and electrochemical gradients directly in the transport of molecules from low concentration to high concentration across the cellular membrane.23
Using ATP
The enzyme-catalyzed hydrolysis reaction removing a phosphate from ATP, thereby forming ADP, causes a conformational change in the transport protein allowing particles to influx or efflux.23 Enzymes catalyzing ATP-driven primary active transport are called ATPases.2
_____________________________________________________________________________________
Figure 5. Primary active transport, with the use of ATP, is depicted above progressing left to right and top to bottom.
The most universal example of ATP hydrolysis driving primary active transport in cells is the sodium-potassium pump.2 The sodium-potassium pump is responsible for controlling both sodium and potassium concentrations inside the cell. The sodium-potassium pump is extremely important in maintaining the cell’s resting potential.
Figure 6. Ribbon structure of a sodium-potassium ATPase pump.
Using Electrochemical Gradient Energy
An electrochemical gradient has two components: 1) an electrical component caused by charge difference on either side of the cellular membrane and 2) a chemical component resulting from differing concentrations of ions across the cellular membrane.24 The electrochemical gradient is generated by the presence of a proton (H+) gradient. A proton gradient is an interconvertible form of energy that can ultimately be used by the transport protein to move particles across the cellular membrane.2
A quintessential example of electrochemical gradient energy in primary active transport is the mitochondrial electron transport chain (ETC).25 The ETC uses the energy produced from the reduction of NADH to NAD+ to create a proton gradient by pumping protons into the inner mitochondrial space.
Using Photon Energy
The energy stored in a photon, the basic unit of light, is used to generate a proton gradient through a process similar to that found in electrochemical gradients.24 The stepwise passing of electrons in an electron transport chain reduces a molecule like NADH and ultimately generates a proton gradient.
Plant photosynthesis is an example of primary active transport using photon energy.2 Chlorophyll absorbs a photon of light and consequently loses an electron which it passes pheophytin causing a subsequent electron transport chain.26 This ETC ultimately ends in the reduction of NADH to NAD+ creating a proton gradient across the chloroplast membrane.
Secondary Active Transport
Secondary active transport achieves an identical result as primary active transport in that particles are moved from low concentration to high concentration at the expense of energy.2 Secondary active transport, however, functions independent of direct ATP coupling. Rather, the electrochemical energy generated from pumping ions out of the cell is used. Secondary active transport is classified as either symporter of antiporter.
Symports
Symport secondary active transport uses a downhill movement of one particle to transport another particle against its concentration gradient.27 Symports move both particles in the same direction through a transmembrane transport protein.
A common symport example is SGLT1, a glucose symport. SGLT1 tranports one glucose molecule into the cell for every two sodium ions transported into the cell.28 The SGLT1 symport is located throughout the body, particularly in the nephron of the kidney.
Antiports
Antiport secondary active transport moves two or more different particles across the cellular membrane in opposite directions.27 Antiport secondary active transport moves one particle down its concentration gradient and uses the energy generated from that process to move another particle up its concentration gradient.
The sodium-calcium exchanger found throughout humans in excitable cells is a simple and common example of an antiport. Three sodium ions travel down their concentration gradient in exchange for one calcium ion.29
Contributors:
• Garret Powell (Truman State University)
• Jordan Kaminski (Truman State University) | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Proteins/Case_Studies%3A_Proteins/Membrane_Transport.txt |
The formation of disulfide bonds has a direct application in producing curls in hair by the permanent wave process. Hair keratin consists of many protein alpha-helices. Three alpha-helices are interwoven into a left-handed coil called a protofibril. Eleven protofibrils are bonded and coiled together to make a microfibril. Hundreds of these microfibrils are cemented into an irregular bundle called a macrofibril. These in turn are mixed with dead and living cells to make a complete strand of hair.
Introduction
Although it may seem incredible, in order for hair to grow 6 inches in one year, 9-1/2 turns of a -helix must be produced every second. The alpha-helices are extensively cross-linked with disulfide bonds from cysteine. These bonds enable keratin to have a somewhat elastic nature. If the alpha -helices stretch unevenly past each other, the disulfide cross-links return them to the original position when the tension is released.
Disulfide Bonds
Disulfide bonds are formed by oxidation of the sulfhydryl groups on cysteine. Different protein chains or loops within a single chain are held together by the strong covalent disulfide bonds. The alpha-helices in the hair strands are bonded by disulfide links. In the permanent wave process, a basic reducing substance (usually ammonium thioglycolate) is first added to reduce and rupture some of the disulfide cross-links.
Waves
When the hair gets wet, water molecules intrude into the keratin strands. The sheer numbers of water molecules are able to disrupt some of the hydrogen bonds which also help to keep the alpha-helices aligned. The helices are able to slip past each other and will retain a new shape in the hair drying process as new hydrogen bonds are formed. The hair strands are able for a short time to maintain the new curl in the hair.
For a permanent wave, we will continue the discussion from the use of the reducing agent. The hair is put on rollers or curlers. Since the alpha-helices are no longer tightly cross-linked to each other, the alpha-helices can shift positions in relation to each other. An oxidizing agent, usually a dilute solution of hydrogen peroxide, (also called the neutralizer) is added to reform the disulfide bonds in their new positions. The permanent will hold these new disulfide bond positions until the hair grows out, since new hair growth is of course not treated.
Sickle Cell Anemia
The incorrect amino acid sequence in a protein may lead to fatal consequences. For example, the inherited disease, sickle cell anemia, results from a single incorrect amino acid at the 6th position of the beta - protein chain out of 146. Hemoglobin consists of four protein chains - two beta and two alpha.
See the graphic on the left for the sequences. This one alteration of the sequence of amino acids in hemoglobin changes its molecular geometry and hence its ability to carry oxygen and its solubility characteristics. The red blood cells change into a sickled shape instead of the normal round shape, become trapped in the small blood capillaries, and cause a great deal of pain.
Quiz:Which type of hemoglobin is apparently more polar and soluble in water?
Which type of hemoglobin is more non-polar and insoluble? | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Proteins/Case_Studies%3A_Proteins/Permanent_Hair_Wave.txt |
Thumbnail: Ball-and-stick model of the glutathione molecule, \(C_{10}H_{17}N_3O_6S\). (Public Domain; Ben Mills).
Peptides and Proteins
This page looks briefly at the hydrolysis of proteins into their constituent amino acids using hydrochloric acid.
The chemistry of the reaction
If you have already studied the hydrolysis of amides under acidic conditions, you will find that this is basically the same reaction. That's not surprising because what biologists and biochemists call a peptide link (in proteins, for example) is what chemists call an amide link. With an amide like ethanamide, the carbon-nitrogen bond in the amide group is broken and you get a carboxylic acid formed:
$CH_3CONH_2 + H_2O + H^+ \rightarrow CH_3COOH + NH_4^+$
Now imagine doing the same thing with a simple dipeptide made of any two amino acids.
Instead of ammonium ions, you get positive ions made from the -NH2 groups reacting with hydrogen ions.
You need the extra hydrogen ion in the equation (compared with the amide equation) to react with the -NH2 group on the left-hand end of the dipeptide - the one not involved in the peptide link.
If you scale this up to a polypeptide (a protein chain), each of the peptide links will be broken in exactly the same way. That means that you will end up with a mixture of the amino acids that made up the protein - although in the form of their positive ions because of the presence of the hydrogen ions from the hydrochloric acid.
Doing the reaction
There are two ways of carrying out this reaction - an old, slow method, and a new, fast one.
The old slow way
The protein is heated with 6 M hydrochloric acid for about 24 hours at 110°C. (6M hydrochloric acid is slightly more than semi-concentrated.)
The new fast way
Protein samples are placed in tubes in a sealed container containing 6 M hydrochloric acid in an atmosphere of nitrogen. The whole container is then placed in a microwave oven for about 5 - 30 minutes (depending on the protein) with temperatures up to 200°C. The hydrochloric acid vaporizes, comes into contact with the protein samples and hydrolyses them. This method is used to hydrolyse small samples of protein during protein analysis.
Peptide Bonds
The formation of peptides is nothing more than the application of the amide synthesis reaction. By convention, the amide bond in the peptides should be made in the order that the amino acids are written. The amine end (N terminal) of an amino acid is always on the left, while the acid end (C terminal) is on the right. The reaction of glycine with alanine to form the dipeptide glyclalanine is written as shown in the graphic on the left. Oxygen (red) from the acid and hydrogens (red) on the amine form a water molecule. The carboxyl oxygen (green) and the amine nitrogen (green) join to form the amide bond.
If the order of listing the amino acids is reversed, a different dipeptide is formed such as alaninylglycine.
Exercise \(1\)
Write the reactions for:
1. ala + gly ---> Answer graphic
2. phe + ser ----> Answer graphic
Salt Formation Contrast
The salt formation reaction is simply the transfer of the -H (positive ion) from the acid to the amine and the attraction of the positive and negative chagres. The acid group becomes negative, and the amine nitrogen becomes positive because of the positive hydrogen ion. No water is formed and there is only one product, the salt.
Backbone Peptide or Protein Structure
The structure of a peptide can be written fairly easily without showing the complete amide synthesis reaction by learning the structure of the "backbone" for peptides and proteins. The peptide backbone consists of repeating units of "N-H 2, CH, C double bond O; N-H 2, CH, C double bond O; etc. After the backbone is written, go back and write the specific structure for the side chains as represented by the "R" as gly-ala-leu for this example. The amine end (N terminal) of an amino acid is always on the left (gly), while the acid end (C terminal) is on the right (leu).
Exercise \(2\)
Write the tripeptide structure for val-ser-cys. First write the "backbone" and then add the specific side chains. Answer graphic
Exercise \(3\)
Write the structure for the tripeptide:
1. a ) glu-cys-gly ---> Answer graphic
2. b) phe-tyr-asn ---> Answer graphic
Glutathione
Glutathione is an important tripeptide present in significant concentrations in all tissues. It contains glutamic acid, cysteine, and glycine. What is unusual about the structure of glutathione (bottom figure below? Compare it to a "normal" tri peptide of glu-cys-gly (top graphic). Instead of the usual backbone, the carboxyl acid "side chain" is part of the backbone peptide structure. The normal carboxyl group is the so called side chain in this case.
The function of glutathione is to protect cells from oxidizing agents which might otherwise damage them. The oxidizing agents react with the -SH group of cysteine of the glutathione instead of doing damage elsewhere. Many foreign chemicals get attached to glutathione, which is really acting as a detoxifying agent.
Contributors
Charles Ophardt (Professor Emeritus, Elmhurst College); Virtual Chembook
Proteins and Amino Acids
Proteins, from the Greek proteios, meaning first, are a class of organic compounds which are present in and vital to every living cell. In the form of skin, hair, callus, cartilage, muscles, tendons and ligaments, proteins hold together, protect, and provide structure to the body of a multi-celled organism. In the form of enzymes, hormones, antibodies, and globulins, they catalyze, regulate, and protect the body chemistry. In the form of hemoglobin, myoglobin and various lipoproteins, they effect the transport of oxygen and other substances within an organism.
Contributors and Attributions
William Reusch, Professor Emeritus (Michigan State U.), Virtual Textbook of Organic Chemistry | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Proteins/Peptides_and_Proteins/Hydrolysis_of_Proteins.txt |
Secondary structure refers to the shape of a folding protein due exclusively to hydrogen bonding between its backbone amide and carbonyl groups. Secondary structure does not include bonding between the R-groups of amino acids, hydrophobic interactions, or other interactions associated with tertiary structure. The two most commonly encountered secondary structures of a polypeptide chain are α-helices and beta-pleated sheets. These structures are the first major steps in the folding of a polypeptide chain, and they establish important topological motifs that dictate subsequent tertiary structure and the ultimate function of the protein.
• Protein Folding
• Secondary Structure: α-Helices
An α-helix is a right-handed coil of amino-acid residues on a polypeptide chain, typically ranging between 4 and 40 residues. This coil is held together by hydrogen bonds between the oxygen of C=O on top coil and the hydrogen of N-H on the bottom coil.
• Secondary Structure: β-Pleated Sheet
This structure occurs when two (or more, e.g. ψ-loop) segments of a polypeptide chain overlap one another and form a row of hydrogen bonds with each other. This can happen in a parallel arrangement or in anti-parallel arrangement. Parallel and anti-parallel arrangement is the direct consequence of the directionality of the polypeptide chain.
• Secondary Structure: α-Pleated Sheet
A similar structure to the beta-pleated sheet is the α-pleated sheet. This structure is energetically less favorable than the beta-pleated sheet, and is fairly uncommon in proteins. An α-pleated sheet is characterized by the alignment of its carbonyl and amino groups; the carbonyl groups are all aligned in one direction, while all the N-H groups are aligned in the opposite direction.
• The Structure of Proteins
This page explains how amino acids combine to make proteins and what is meant by the primary, secondary and tertiary structures of proteins. Quaternary structure isn't covered. It only applies to proteins consisting of more than one polypeptide chain.
Thumbnail: Structure of human hemoglobin. The proteins α and β subunits are in red and blue, and the iron-containing heme groups in green. (CC BY-SA 3.0; Zephyris).
Protein Structure
Introduction and Protein Structure
Proteins have several layers of structure each of which is important in the process of protein folding. The first most basic level of this structure is the sequence of amino acids themselves.1 The sequencing is important because it will determine the types of interactions seen in the protein as it is folding. A novel sequence-based method based on the assumption that protein-protein interactions are more related to amino acids at the surface than those at the core.2 This study shows that not only is the amino acids that are in a protein important but also the order in which they are sequenced. The interactions of the amino acids will determine what the secondary and tertiary structure of the protein will be.
The next layer in protein structure is the secondary structure. The secondary structure includes architectural structures that extend in one dimension.1 Secondary structure includes α-Helixes and β-sheets ( Figure \(1\)). The α-helices, the most common secondary structure in proteins, the peptide –CO–NH–groups in the backbone form chains held together by NH ̄OC hydrogen bonds.”3 The α-helices form the backbone of proteins and help to aid in the folding process. The β-sheets form in two distinct ways. They are able to form in both parallel β-pleated sheets and anti parallel β-pleated sheets.1 When the α-helix or β-sheet is formed, the excluded volumes generated by the backbone and side chains overlap, leading to an increase in the total volume available to the translational displacement of water molecules.4 This is important because it leads to a more thermodynamically stable conformation and leads to less strain on the protein as a whole and thus are aided by the conformation.
The tertiary structure is the next layer in protein structure. This takes the α-Helixes and β-sheets and allows them to fold into a three dimensional structure.1 Most proteins take on a globular structure once folded. The description of globular protein structures as an ensemble of contiguous closed loops or tightened end fragments reveals fold elements crucial for the formation of stable structures and for navigating the very process of protein folding.5 The globular proteins generally have a hydrophobic core surrounded by a hydrophilic outer layer. These interactions are important because they lead to the global structure and help create channels and binding sites for enzymes.
The last layer of protein structure is the quaternary structure. The folding transition and the functional transitions between useful states are encoded in the linear sequence of amino acids, and a long- term goal of structural biology is to be able to predict both the structure and function of molecules from the information in the sequence.6 The Subunit organization is the last level of structure in protein molecules.1 The organization of the subunits is important because that determines the types of interactions that can form and dictates its use in the body.
Protein Folding
Proteins are folded and held together by several forms of molecular interactions. The molecular interactions include the thermodynamic stability of the complex, the hydrophobic interactions and the disulfide bonds formed in the proteins. The figure below (Figure \(2\)) is an example of protein folding.
The biggest factor in a proteins ability to fold is the thermodynamics of the structure. The interaction scheme includes the short-range propensity to form extended conformations, residue-dependent long-range contact potentials, and orientation-dependent hydrogen bonds.7 The thermodynamics are a main stabilizing force within a protein because if it is not in the lowest energy conformation it will continue to move and adjust until it finds its most stable state. The use of energy diagrams and maps are key in finding out when the protein is in the most stable form possible.
The next type of interaction in protein folding is the hydrophobic interactions within the protein. The framework model and the hydrophobic collapse model represent two canonical descriptions of the protein folding process. The first places primary reliance on the short-range interactions of secondary structure and the second assigns greater importance to the long-range interactions of tertiary structure.6 These hydrophobic interactions have an impact not just on the primary structure but then lead to changes seen in the secondary and tertiary structure as well. Globular proteins acquire distinct compact native con- formations in water as a result of the hydrophobic effect.7 When a protein has been folded in the correct way it usually exists with the hydrophobic core as a result of being hydrated by waters in the system around it which is important because it creates a charged core to the protein and can lead to the creation of channels within the protein. The hydrophobic interactions are found to affect time correlation functions in the vicinity of the native state even though they have no impact on same time characteristics of the structure fluctuations around the native state.7 The hydrophobic interactions are shown to have an impact on the protein even after it has found the most stable conformation in how the proteins can interact with each other as well as folding themselves.
Another type of interaction seen when the protein is folding is the disulfide linkages that form in the protein (Figure \(3\)). The disulfide bond, a sulfur- sulfur chemical bond that results from an oxidative process that links nonadjacent (in most cases) cysteine’s of a protein.9 These are a major way that proteins get into their folded form. The types of disulfide bonds are cysteine-cysteine linkage is a stable part of their final folded structure and those in which pairs of cysteines alternate between the reduced and oxidized states.9 The more common is the linkages that cause the protein to fold together and link back on itself compared to the cysteines that are changing oxidation states because the bonds between cysteines once created are fairly stable.
Misfunctions
Proteins can miss function for several reasons. When a protein is miss folded it can lead to denaturation of the protein. Denaturation is the loss of protein structure and function.1 The miss folding does not always lead to complete lack of function but only partial loss of functionality. The miss functioning of proteins can sometimes lead to diseases in the human body.
Alzheimer's Disease
Alzheimer's Disease (AD) is a neurological degenerative disease that affects around 5 million Americans, including nearly half of those who are age 85 or older.10 The predominant risk factors of AD are age, family history, and heredity. Alzheimer’s disease typically results in memory loss, confusion of time and place, misplacing places, and changes in mood and behavior.11 AD results in dense plaques in the brain that are comprised of fibrillar β-amyloid proteins with a well-orders β-sheet secondary structure.12 These plaques visually look like voids in the brain matter (see figure 5) and are directly connected to the deterioration of thought processes. It has been determined that AD is a protein misfolding disease, where the misfolded protein is directly related to the formation of these plaques in the brain.13
It is yet to be fully understood what exactly causes this protein misfolding to begin, but several theories point to oxidative stress in the brain to be the initiating factor. This oxidation results in damage to the phospholipids in the brain, which has been found to result in a faster accumulation of amyloid β-proteins.14
Cystic Fibrosis
Cystic Fibrosis (CF) is a chronic disease that affects 30,000 Americans. The typical affects of CF is a production of thick, sticky mucus that clogs the lungs and leads to life-threatening lung infection, and obstructs the pancreas preventing proper food processing.15 CF is caused by protein misfolding. This misfolding then results in some change in the protein known as cystic fibrosis transmembrane conductance regulator (CFTR), which can result in this potentially fatal disease.16 In approximately 70% of CF cases, a deletion of phenylalanine at position 508 in the CFTR is deleted. This deletion of Phe508 seems to be directly connected to the formation of CF.17 The protein misfolding that results in CF occurs prior to birth, but it is not entirely clear as to why.
Resources
1. Garrett, R.H., and Grisham, C.M. Biochemistry fourth edition; Brooks/Cole. Australia, 2010. p. 93-95, 135, 143, 160.
2. Chang, D.T., Syu Y., Lin, P., Predicting the protein-protein interactions using primary structures with predicted protein surface. BCM Bioinformatics. Jan 2010
3. Bodis, P., Schwartz, E., Koepf, M. Cornelissen, J. Rowan A.E., Roeland J. Nolte, M. and Woutersen S. Vibrational self-trapping in beta-sheet structures observed with femtosecond nonlinear infrared spectroscopy. The Journal Of Chemical Physics 131, 2009
4. Yasuda, S., Yoshidome, T. Oshima, H. Kodama R., Harano, Y. and Kinoshita, M. Effects of side-chain packing on the formation of secondary structures in protein folding. The Journal of Chemical Physics 132, 2010
5. Papandreoul, N., Berezovsky I.N., Lopes, A., Eliopoulos, E., and Chomilier, J. Universal positions in globular proteins from observation to simulation. Eur. J. Biochem. 271, 4762–4768 (2004)
6. Hausratha, A.C. A Kinetic theory of tertiary contact formation coupled to the helix-coil transition in polypeptides. The Journal of Chemical Physics. 2006
7. Cieplaka M. and Niewieczerza S. Hydrodynamic interactions in protein folding. Journal of Chemical Physics. 130 2009
8. Gront, D. Kolinskia, A. and Skolnick, J. A new combination of replica exchange Monte Carlo and histogram analysis for protein folding and thermodynamics. Journal of Chemical Physics. Vol. 115 2001
9. Kadokura, H., Katzen F. and Beckwith, J. Protein disulfide bond formation in prokatyotes. Annual Review Biochem 2003.
10. Alzheimer's Disease. Centers for Disease Control and Prevention. 2010, http://www.cdc.gov/aging/aginginfo/alzheimers.htm.
11. Alzheimer's Association. 2011, www.alz.org.
12. Glenner, G. G.; Wong, C. W. Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun.1984, 120, 885-890.
13. Hashimoto, M.; Rockenstein, E.; Crews, L.; Mashliah, E. Role of Protein Aggregation in Mitochondrial Dysfunction and Neurodegeneration in Alzheimer’s and Parkinson’s Diseases. Neuromolecular Med. 2003, 4, 21-36
14. The Cystic Fibrosis Foundation. 2011, http://www.cff.org/AboutCF.
15. Cheung, J. C. and Deber, C. M. Misfolding of the Cystic Fibrosis Transmembrane Conductance Regulator and Disease. Biochemistry 2008, 47, 1465-1473.
16. Koppaka, V.; Axelsen, P. Accelerated Accumulation of Amyloid β Proteins on Oxidatively Damaged Lipid Membranes. Biochemistry 2000, 39, 10011-10016.
17. Riordan, J.M. et al. Identification of the cysticfibrosisgene: cloning and characterization of complementary DNA. Science 1989, 245, 1066-1073. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Proteins/Protein_Structure/Protein_Folding.txt |
An α-helix is a right-handed coil of amino-acid residues on a polypeptide chain, typically ranging between 4 and 40 residues. This coil is held together by hydrogen bonds between the oxygen of C=O on top coil and the hydrogen of N-H on the bottom coil. Such a hydrogen bond is formed exactly every 4 amino acid residues, and every complete turn of the helix is only 3.6 amino acid residues. This regular pattern gives the α-helix very definite features with regards to the thickness of the coil and the length of each complete turn along the helix axis.
The structural integrity of an α-helix is in part dependent on correct steric configuration. Amino acids whose R-groups are too large (tryptophan, tyrosine) or too small (glycine) destabilize α-helices. Proline also destabilizes α-helices because of its irregular geometry; its R-group bonds back to the nitrogen of the amide group, which causes steric hindrance. In addition, the lack of a hydrogen on Proline's nitrogen prevents it from participating in hydrogen bonding.
Another factor affecting α-helix stability is the total dipole moment of the entire helix due to individual dipoles of the C=O groups involved in hydrogen bonding. Stable α-helices typically end with a charged amino acid to neutralize the dipole moment.
Secondary Structure: -Pleated Sheet
This structure occurs when two (or more, e.g. ψ-loop) segments of a polypeptide chain overlap one another and form a row of hydrogen bonds with each other. This can happen in a parallel arrangement:
Or in anti-parallel arrangement:
Parallel and anti-parallel arrangement is the direct consequence of the directionality of the polypeptide chain. In anti-parallel arrangement, the C-terminus end of one segment is on the same side as the N-terminus end of the other segment. In parallel arrangement, the C-terminus end and the N-terminus end are on the same sides for both segments. The "pleat" occurs because of the alternating planes of the peptide bonds between amino acids; the aligned amino and carbonyl group of each opposite segment alternate their orientation from facing towards each other to facing opposite directions.
The parallel arrangement is less stable because the geometry of the individual amino acid molecules forces the hydrogen bonds to occur at an angle, making them longer and thus weaker. Contrarily, in the anti-parallel arrangement the hydrogen bonds are aligned directly opposite each other, making for stronger and more stable bonds.
Commonly, an anti-parallel beta-pleated sheet forms when a polypeptide chain sharply reverses direction. This can occur in the presence of two consecutive proline residues, which create an angled kink in the polypeptide chain and bend it back upon itself. This is not necessary for distant segments of a polypeptide chain to form beta-pleated sheets, but for proximal segments it is a definite requirement. For short distances, the two segments of a beta-pleated sheet are separated by 4+2n amino acid residues, with 4 being the minimum number of residues.
The Structure of Proteins
This page explains how amino acids combine to make proteins and what is meant by the primary, secondary and tertiary structures of proteins. Quaternary structure isn't covered. It only applies to proteins consisting of more than one polypeptide chain.
The primary structure of proteins
Drawing the amino acids
In chemistry, if you were to draw the structure of a general 2-amino acid, you would probably draw it like this:
However, for drawing the structures of proteins, we usually twist it so that the "R" group sticks out at the side. It is much easier to see what is happening if you do that.
That means that the two simplest amino acids, glycine and alanine, would be shown as:
Peptides and polypeptides
Glycine and alanine can combine together with the elimination of a molecule of water to produce a dipeptide. It is possible for this to happen in one of two different ways - so you might get two different dipeptides.
Either:
Or:
In each case, the linkage shown in blue in the structure of the dipeptide is known as a peptide link. In chemistry, this would also be known as an amide link, but since we are now in the realms of biochemistry and biology, we'll use their terms.
If you joined three amino acids together, you would get a tripeptide. If you joined lots and lots together (as in a protein chain), you get a polypeptide.
A protein chain will have somewhere in the range of 50 to 2000 amino acid residues. You have to use this term because strictly speaking a peptide chain isn't made up of amino acids. When the amino acids combine together, a water molecule is lost. The peptide chain is made up from what is left after the water is lost - in other words, is made up of amino acid residues.
By convention, when you are drawing peptide chains, the -NH2 group which hasn't been converted into a peptide link is written at the left-hand end. The unchanged -COOH group is written at the right-hand end.
The end of the peptide chain with the -NH2 group is known as the N-terminal, and the end with the -COOH group is the C-terminal.
A protein chain (with the N-terminal on the left) will therefore look like this:
The "R" groups come from the 20 amino acids which occur in proteins. The peptide chain is known as the backbone, and the "R" groups are known as side chains.
The primary structure of proteins
Now there's a problem! The term "primary structure" is used in two different ways. At its simplest, the term is used to describe the order of the amino acids joined together to make the protein. In other words, if you replaced the "R" groups in the last diagram by real groups you would have the primary structure of a particular protein. This primary structure is usually shown using abbreviations for the amino acid residues. These abbreviations commonly consist of three letters or one letter. Using three letter abbreviations, a bit of a protein chain might be represented by, for example:
If you look carefully, you will spot the abbreviations for glycine (Gly) and alanine (Ala) amongst the others.
If you followed the protein chain all the way to its left-hand end, you would find an amino acid residue with an unattached -NH2 group. The N-terminal is always written on the left of a diagram for a protein's primary structure - whether you draw it in full or use these abbreviations.
The wider definition of primary structure includes all the features of a protein which are a result of covalent bonds. Obviously, all the peptide links are made of covalent bonds, so that isn't a problem.
But there is an additional feature in proteins which is also covalently bound. It involves the amino acid cysteine.
If two cysteine side chains end up next to each other because of folding in the peptide chain, they can react to form a sulfur bridge. This is another covalent link and so some people count it as a part of the primary structure of the protein.
Because of the way sulphur bridges affect the way the protein folds, other people count this as a part of the tertiary structure (see below). This is obviously a potential source of confusion!
The secondary structure of proteins
Within the long protein chains there are regions in which the chains are organised into regular structures known as alpha-helices (alpha-helixes) and beta-pleated sheets. These are the secondary structures in proteins. These secondary structures are held together by hydrogen bonds. These form as shown in the diagram between one of the lone pairs on an oxygen atom and the hydrogen attached to a nitrogen atom:
Although the hydrogen bonds are always between C=O and H-N groups, the exact pattern of them is different in an alpha-helix and a beta-pleated sheet. When you get to them below, take some time to make sure you see how the two different arrangements works.
The alpha-helix
In an alpha-helix, the protein chain is coiled like a loosely-coiled spring. The "alpha" means that if you look down the length of the spring, the coiling is happening in a clockwise direction as it goes away from you. The next diagram shows how the alpha-helix is held together by hydrogen bonds. This is a very simplified diagram, missing out lots of atoms. We'll talk it through in some detail after you have had a look at it.
What's wrong with the diagram? Two things:
First of all, only the atoms on the parts of the coils facing you are shown. If you try to show all the atoms, the whole thing gets so complicated that it is virtually impossible to understand what is going on.
Secondly, I have made no attempt whatsoever to get the bond angles right. I have deliberately drawn all of the bonds in the backbone of the chain as if they lie along the spiral. In truth they stick out all over the place. Again, if you draw it properly it is virtually impossible to see the spiral.
So, what do you need to notice?
Notice that all the "R" groups are sticking out sideways from the main helix.
Notice the regular arrangement of the hydrogen bonds. All the N-H groups are pointing upwards, and all the C=O groups pointing downwards. Each of them is involved in a hydrogen bond.
And finally, although you can't see it from this incomplete diagram, each complete turn of the spiral has 3.6 (approximately) amino acid residues in it.
If you had a whole number of amino acid residues per turn, each group would have an identical group underneath it on the turn below. Hydrogen bonding can't happen under those circumstances.
Each turn has 3 complete amino acid residues and two atoms from the next one. That means that each turn is offset from the ones above and below, such that the N-H and C=O groups are brought into line with each other.
Beta-pleated sheets
In a beta-pleated sheet, the chains are folded so that they lie alongside each other. The next diagram shows what is known as an "anti-parallel" sheet. All that means is that next-door chains are heading in opposite directions. Given the way this particular folding happens, that would seem to be inevitable.
It isn't, in fact, inevitable! It is possible to have some much more complicated folding so that next-door chains are actually heading in the same direction. The folded chains are again held together by hydrogen bonds involving exactly the same groups as in the alpha-helix.
The tertiary structure of proteins
The tertiary structure of a protein is a description of the way the whole chain (including the secondary structures) folds itself into its final 3-dimensional shape. This is often simplified into models like the following one for the enzyme dihydrofolate reductase. Enzymes are, of course, based on proteins.
The model shows the alpha-helices in the secondary structure as coils of "ribbon". The beta-pleated sheets are shown as flat bits of ribbon ending in an arrow head. The bits of the protein chain which are just random coils and loops are shown as bits of "string". The color coding in the model helps you to track your way around the structure - going through the spectrum from dark blue to end up at red. You will also notice that this particular model has two other molecules locked into it (shown as ordinary molecular models). These are the two molecules whose reaction this enzyme catalyses.
What holds a protein into its tertiary structure?
The tertiary structure of a protein is held together by interactions between the the side chains - the "R" groups. There are several ways this can happen.
Ionic interactions
Some amino acids (such as aspartic acid and glutamic acid) contain an extra -COOH group. Some amino acids (such as lysine) contain an extra -NH2 group.
You can get a transfer of a hydrogen ion from the -COOH to the -NH2 group to form zwitterions just as in simple amino acids.
You could obviously get an ionic bond between the negative and the positive group if the chains folded in such a way that they were close to each other.
Hydrogen bonds
Notice that we are now talking about hydrogen bonds between side groups - not between groups actually in the backbone of the chain.
Lots of amino acids contain groups in the side chains which have a hydrogen atom attached to either an oxygen or a nitrogen atom. This is a classic situation where hydrogen bonding can occur.
For example, the amino acid serine contains an -OH group in the side chain. You could have a hydrogen bond set up between two serine residues in different parts of a folded chain.
You could easily imagine similar hydrogen bonding involving -OH groups, or -COOH groups, or -CONH2 groups, or -NH2 groups in various combinations - although you would have to be careful to remember that a -COOH group and an -NH2 group would form a zwitterion and produce stronger ionic bonding instead of hydrogen bonds.
van der Waals dispersion forces
Several amino acids have quite large hydrocarbon groups in their side chains. A few examples are shown below. Temporary fluctuating dipoles in one of these groups could induce opposite dipoles in another group on a nearby folded chain.
The dispersion forces set up would be enough to hold the folded structure together.
Sulfur bridges
Sulfur bridges which form between two cysteine residues have already been discussed under primary structures. Wherever you choose to place them doesn't affect how they are formed! | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Proteins/Protein_Structure/Secondary_Structure%3A_-Helices.txt |
Nonprotein components of certain enzymes are called cofactors. If the cofactor is organic, then it is called a coenzyme. Coenzymes are relatively small molecules compared to the protein part of the enzyme and many of the coenzymes are derived from vitamins. The coenzymes make up a part of the active site, since without the coenzyme, the enzyme will not function.
• Flavin Adenine Dinucleotide (FAD)
The structure shown on the left is for FAD and is similar to NAD+ in that it contains a vitamin-riboflavin, adenine, ribose, and phosphates. As shown it is the diphosphate, but is also used as the monophosphate (FMN).
• Nicotinamide Adenine Dinucleotide (NAD)
Nicotinamide is from the niacin vitamin. The NAD+ coenzyme is involved with many types of oxidation reactions where alcohols are converted to ketones or aldehydes. It is also involved in the first enzyme complex 1 of the electron transport chain.
• Vitamin A: β-Carotene
β-carotene is the molecule that gives carrots, sweet potatoes, squash, and other yellow or orange vegetables their orange color. It is part of a family of chemicals called the carotenoids, which are found in many fruit and vegetables, as well as some animal products such as egg yolks. Carotenoids were first isolated in the early 19th century, and have been synthesized for use as food colorings since the 1950s.
• Vitamin B₁₂: Cobalamin
Cobalamin, or Vitamin B12, is the largest and the most complex out of all the types of Vitamins. The discovery of Cobalamin was made as scientists were seeking to find a cure for pernicious anemia, an anemic disease caused by an absence of intrinsic factor in the stomach.
Vitamins Cofactors and Coenzymes
The structure shown on the left is for FAD and is similar to NAD+ in that it contains a vitamin-riboflavin, adenine, ribose, and phosphates. As shown it is the diphosphate, but is also used as the monophosphate (FMN).
Introduction
In the form of FMN it is involved in the first enzyme complex 1 of the electron transport chain. A FMN (flavin adenine mononucleotide) as an oxidizing agent is used to react with NADH for the second step in the electron transport chain. The simplified reaction is:
NADH + H+ + FMN → FMNH2 + NAD+
Red.Ag. Ox.Ag.
Note the fact that the two hydrogens and 2e- are "passed along" from NADH to FFMN. Also note that NAD+ as a product is back to its original state as an oxidizing agent ready to begin the cycle again. The FMN has now been converted to the reducing agent and is the starting point for the third step.
Coenzyme Q or Ubiquinone
Ubiquinone: As its name suggests, is very widely distributed in nature. There are some differences in the length of the isoprene unit (in bracket on left) side chain in various species. All the natural forms of CoQ are insoluble in water, but soluble in membrane lipids where they function as a mobile electron carrier in the electron transport chain. The long hydrocarbon chain gives the non-polar property to the molecule.
CoQ acts as a bridge between enzyme complex 1 and 3 or between complex 2 and 3. Electrons are transferred from NADH along with two hydrogens to the double bond oxygens in the benzene ring. These in turn convert to alcohol groups. The electrons are then passed along to the cytochromes in enzyme complex 3.
Coenzyme A
Although not used in the electron transport chain, Coenzyme A is a major cofactor which is used to transfer a two carbon unit commonly referred to as the acetyl group. The structure has many common features with NAD+ and FAD in that it has the diphosphate, ribose, and adenine. In addition it has a vitamin called pantothenic acid, and finally terminated by a thiol group. The thiol (-SH) is the sulfur analog of an alcohol (-OH). The acetyl group (CH3C=O) is attached to the sulfur of the CoA through a thiol ester type bond. Acetyl CoA is important in the breakdown of fatty acids and is a starting point in the citric acid cycle.
Nicotinamide Adenine Dinucleotide (NAD)
Nicotinamide is from the niacin vitamin. The NAD+ coenzyme is involved with many types of oxidation reactions where alcohols are converted to ketones or aldehydes. It is also involved in the first enzyme complex 1 of the electron transport chain.The structure for the coenzyme, NAD+, Nicotinamide Adenine Dinucleotide is shown in Figure $1$.
Role of NAD+
One role of $NAD^+$ is to initiate the electron transport chain by the reaction with an organic metabolite (intermediate in metabolic reactions). This is an oxidation reaction where 2 hydrogen atoms (or 2 hydrogen ions and 2 electrons) are removed from the organic metabolite. (The organic metabolites are usually from the citric acid cycle and the oxidation of fatty acids--details in following pages.) The reaction can be represented simply where M = any metabolite.
$MH_2 + NAD^+ \rightarrow NADH + H^+ + M: + \text{energy}$
One hydrogen is removed with 2 electrons as a hydride ion ($H^-$) while the other is removed as the positive ion ($H+$). Usually the metabolite is some type of alcohol which is oxidized to a ketone.
Figure $1$
Alcohol Dehydrogenase
The NAD+ is represented as cyan in Figure $2$. The alcohol is represented by the space filling red, gray, and white atoms. The reaction is to convert the alcohol, ethanol, into ethanal, an aldehyde.
$CH_3CH_2OH + NAD^+ \rightarrow CH_3CH=O + NADH + H^+$
This is an oxidation reaction and results in the removal of two hydrogen ions and two electrons which are added to the NAD+, converting it to NADH and H+. This is the first reaction in the metabolism of alcohol. The active site of ADH has two binding regions. The coenzyme binding site, where NAD+ binds, and the substrate binding site, where the alcohol binds. Most of the binding site for the NAD+ is hydrophobic as represented in green. Three key amino acids involved in the catalytic oxidation of alcohols to aldehydes and ketones. They are ser-48, phe 140, and phe 93.
Figure $2$: Active site of Alcohol Dehydrogenase | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Vitamins_Cofactors_and_Coenzymes/Flavin_Adenine_Dinucleotide_%28FAD%29.txt |
β-carotene is the molecule that gives carrots, sweet potatoes, squash, and other yellow or orange vegetables their orange color. It is part of a family of chemicals called the carotenoids, which are found in many fruit and vegetables, as well as some animal products such as egg yolks. Carotenoids were first isolated in the early 19th century, and have been synthesized for use as food colorings since the 1950s. Biologically, β-carotene is most important as the precursor of vitamin A in the human diet. It also has anti-oxidant properties and may help in preventing cancer and other diseases.
Introduction
The long chain of alternating double bonds (conjugated) is responsible for the orange color of beta-carotene. The conjugated chain in carotenoids means that they absorb in the visible region - green/blue part of the spectrum. So β-carotene appears orange, because the red/yellow colors are reflected back to us.
Vitamin A
Vitamin A has several functions in the body. The most well known is its role in vision - hence carrots "make you able to see in the dark". The retinol is oxidized to its aldehyde, retinal, which complexes with a molecule in the eye called opsin. When a photon of light hits the complex, the retinal changes from the 11-cis form to the all-trans form, initiating a chain of events which results in the transmission of an impulse up the optic nerve. A more detailed explanation is in Photochemical Events.
Other roles of vitamin A are much less well understood. It is known to be involved in the synthesis of certain glycoproteins, and that deficiency leads to abnormal bone development, disorders of the reproductive system, xerophthalmia (a drying condition of the cornea of the eye) and ultimately death.
Vitamin A is required for healthy skin and mucus membranes, and for night vision. Its absence from diet leads to a loss in weight and failure of growth in young animals, to the eye diseases; xerophthalmia, and night blindness, and to a general susceptibility to infections. It is thought to help prevent the development of cancer. Good sources of carotene, such as green vegetables are good potential sources of vitamin A. Vitamin A is also synthetically manufactured by extraction from fish-liver oil and by synthesis from beta-ionone.
Vitamin A is structurally related to β-carotene. β-Carotene is converted into vitamin A in the liver. Two molecules of vitamin A are formed from on molecule of beta carotene.
Oxidation: If you compare the two molecules, it is clear that vitamin A (retinol) is very closely related to half of the beta-carotene molecule. One way in which beta-carotene can be converted to vitamin A is to break it apart at the center and is thought to be most important biologically. The breakdown of beta-carotene occurs in the walls of the small intestine (intestinal mucosa) and is catalyzed by the enzyme β-carotene dioxygenase to form retinal.
Reduction Reaction: The retinal reduced to retinol by retinaldehyde reductase in the intestines. This is the reduction of an aldehyde by the addition of hydrogen atoms to make the alcohol, retinol.
Esterification Reaction: The absorption of retinol from the alimentary tract is favored by the simultaneous absorption of fat or oil, especially if these are unsaturated. Retinol is esterified to palmitic acid and delivered to the blood via chylomicrons. Finally the retinol formed is stored in the liver as retinyl esters. This is why cod liver oil used to be taken as a vitamin A supplement. It is also why you should never eat polar bear liver if you run out of food in the Arctic; vitamin A is toxic in excess and a modest portion of polar bear liver contains more than two years supply! Beta-carotene, on the other hand, is a safe source of vitamin A. The efficiency of conversion of beta-carotene to retinol depends on the level in the diet. If you eat more beta-carotene, less is converted, and the rest is stored in fat reserves in the body. So too much beta-carotene can make you turn yellow, but will not kill you with hypervitaminosis. | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Vitamins_Cofactors_and_Coenzymes/Vitamin_A.txt |
Cobalamin, or Vitamin B12, is the largest and the most complex out of all the types of Vitamins. The discovery of Cobalamin was made as scientists were seeking to find a cure for pernicious anemia, an anemic disease caused by an absence of intrinsic factor in the stomach. Cobalamin was studied, purified, and collected into small red crystals, and its crystallize structure was determined during an X ray analysis experiment conducted by Scientist Hodkin. A molecule structure of Cobalamin is simple, yet contains a lot of different varieties and complexes as shown in Figure \(1\). The examination of the vitamin’s molecular structure helps scientists to have a better understanding of how the body utilizes Vitamin B12 into building red blood cells and preventing pernicious anemia syndromes.
The metalloenzyme structure of Cobalamin presents a corrin ring with Cobalt, the only metal in the molecule, positioned right in the center of the structure by four coordinated bonds of nitrogen from four pyrrole groups. These four subunit groups are separated evenly on the same plane, directly across from each other. They are also connected to each other by a C-CH3 methylene link on the other sides, by a C-H on one side and by two pyrroles directly coming together. Together, they form a perfect corrin ring as shown in figure 2. The fifth ligand connected to Cobalt is a nitrogen coming from the 5,6-dimethhylbenzimidazole. It presents itself as an axial running straight down from the cobalt right under the corrin ring. This benzimidazole is also connected to a five carbon sugar, which eventually attaches itself to a phosphate group, and then straps back to the rest of the structure. Since the axial is stretched all the way down, the bonding between the Cobalt and the 5,6-dimethylbenzimidazole is weak and can sometimes be replaced by related molecules such as a 5-hydrozyl-benzimidazole, an adenine, or any other similar group. In the sixth position above the Corrin ring, the active site of Cobalt can directly connect to several different types of ligands. It can connect to CN to form a Cyanocobalami, to a Methyl group to form a methylcobalamin, to a 5’-deoxy adenosy group to form an adenosylcobalamin, and OH, Hydroxycobalamin. Cobalt is always ready to oxidize from 1+ change into 2+ and 3+ in order to match up with these R groups that are connected to it. For example, Hydroxocobalamin contains cobalt that has a 3+ charge while Methyladenosyl contains a cobalt that has a 1+ Charge.
The point group configuration of Cobalamin is C4v. In order to determine this symmetry, one must see that the structure is able to rotate itself four times and will eventually arrive back to its original position. Furthermore, there are no sigma h plane and no perpendicular C2 axe. However, since there are sigma v planes that cut the molecules into even parts, it is clear to determine that the structure of Cobalamin is a C4v. With Cobalt being the center metal of the molecule, Cobalamin carried a distorted octahedral configuration. The axial that connects Cobalt to the 5,6 dimethyl benzimidazole is stretched all the way down to the bottom. Its distance is several times longer than the distance from the Cobalt and the attached R group above it. This sometimes can also be referred to as a tetragonal structure. The whole shape overall is similar to an octahedral, but the two axial groups are different and separated into uneven distances. Since there is only one metalloenzyme center in the system, the point group and configuration just mentioned is also assigned to the structure as a whole. Since the metallocoenzyme structure is stretched out, it is quite weakly coordinated and can be break apart or replaced with other groups as mentioned above.
Scientists have shown that both IR and Raman Spectroscopy were used to determine the structure of the molecule. This is determined by observing the character tables of point group C4v, the point group symmetry of Cobalamin. On the IR side, one can see that there are groups such as drz, (x, y), (rz, ry). On the other hand, on the Raman side, there are groups such as x square +y square, z square, x square – y square, xy, xz, yz. The Raman side indicated that there were stretching modes in the molecule and relates back to the stretching of the 5,6 dimethyl benzimidazole axial that connected directly below the Cobalt metal. The stretching can be seen in Figure 3.
Cobalamin enzymes can catalyze a few different types of reactions. One of them is the reaction of Intramolecular rearrangements. During this rearrangement coenzyme is exchanged to the two groups attached to adjacent carbon atoms. Another reaction involves transferring the methyl group in certain methylation reactions, such as the conversion of homocysteine to methionine, biosysnthesis of choline and thymine etc. These interactions can bring beneficial values to the biological bodies.
Cobalamin has many beneficial effects in regard to biological existences. They play a role to maintain healthy body system and help to aid the production of the body’s genetic materials. Cyanocobalamin, one type of cobalamin, works to generate the forming of red blood cells and heal many different damages in the nervous system. Cobalamin also serves as a vital role in the metabolism of fatty acids essential for the maintainence of myelin. Studies have shown that people with Vitamin B12 deficiency will reveal irregular destruction of the myeline shealth, which leads to parlysis and death. Some of the other symptoms of the lack of cobalamin are poor growth, megaloblastic bone marrow, Gi tract changes, Leucoopenia and hyper-segmented nutrophills, degenerative changes in spinal cord and nervous system and excretion of methyl malonic acid and homocystin in urine.
Throughout the years, Vitamin B12 has shown to be essential for the functioning of the nervous system and the production of red blood cell. A study conducted by researchers at the National Institutes of Health, Trinity College Dublin, suggested that a deficiency in Vitamin B12 might increase the risk of neural tubes defect in children (Miller). Therefore, by studying the structure and function of Cobalamin, scientists can experiment and form Vitamin B12 in their laboratories and serve the community as a whole.
Vitamin B: Cobalamin
Introduction
Cobalamin or Vitamin B12, a water soluble vitamin that has known functions for improving brain and nerve cells, and the production of adequate blood cells.1 It is commonly found in meats, poultry, dairy products, eggs and seafood. However, organisms such as bacteria and algae are also known to produce the active form of vitamin B12 through fermentation. The structure of cobalamin is unique with the central atom; cobalt, that has potential for metalloenzyme active sites. In particular, coenzyme B12 or adenosylcobalamin (AdoCbl) is an essential for several enzymes such as methylmaonly-CoA mutase, diol dehydratase, and ethanolamine ammonia lyase.2
Cobalamin is an important biologically active, though small, enzyme involved in several configurational changes on its active site. As the name suggests, Cobalamin incorporates several structural elements surrounding a cobalt atom as the metalloenzyme active site. The vitamin-B12 configuration of Cobalamin that is of importance to biological life, primarily in the function of mechanisms in the liver, is that of Cyanocobalamin, one of the rare cases where a cyanide group is presently ingested by living organisms where otherwise would be toxic. Four main forms of Cobalamin exist and are interchanged in the body as the B12 performs various functions. The Cyanocobalamin used as the B12 vitamin supplement however is not naturally occurring, and must be generated then ingested in order to configurationally lose its cyanide group for a methyl group. Various functions in cell metabolism are involved with the transfer of the removable ligand group on the Cobalamin active site, most importantly is the transfer of methyl groups thus acting as a catalyst between configurational changes in many enzymes.
Figure 1. Adenosylcobalamin
Symmetry
Cobalamin has a formula C63H88CoN14o14P and molecular mass 1355.37 g/mol. This is a fairly large molecule, thus, analyzing the structure confirmation can determine the point group. The structure contains sigma vertical planes and has no sigma horizontal plane. The central atom, Cobalt, has an R attached that makes the molecule unique in several enzymatic catalyses. The point group of Cobalamin is assigned as C4v.
Mechanism
Figure 2. Minimal mechanism of diol dehydratase reaction.
The mechanism of diol dehydrates is known to catalyzes the conversion of 1,2-diols to the corresponding aldehydes. Figure 2 shows a mechanism for this enzymatic dehydration that involves the hydrogen atom abstraction from C1 (1,2-diol) and the migration of an OH group from C2 to C1 of 1,2-propanediol. The adenosyl (AdoCH2) radical that is generated by the hemolytic cleavage of the Co-C covalent bond in AdoCbl plays an essential role in this OH group migration and thus effectively promotes this chemically difficult reaction.2 Therefore, the OH group on C2 migrates to C1 leading to a formation of a 1,1-diol radical, which leads to the formation of the 1-1-diol and the regeneration of AdoCH2 radical.
The crystal structure of diol dehydratase with cyanocobalamin and adeninlypentylcobalamin have shown that both the OH groups of substrate coordinate directly to K+ ion at the active site, which implies the participation of K+ ion in the OH group migration.3 Kamachi and colleagues performed density functional theory to reveal the catalytic roles of K+ ion in the diol dehydratase reaction. As a result, the course of a reaction the substrate and the radical intermediates are always bound to K+ ion until the release of product aldehyde from the active site and that OH group proceed with the aid of K+. Therefore, the role of K+ ion have suggest that it is the most important role in the reaction to fix the substrate and the intermediates in a proper position in order to ensure the hydrogen abstraction and recombination.
Figure 3. Optimized structure of Diol Dehydratase with 13,500 atoms
Figure 2 shows the optimized structure of the enzyme in the QM region. K+ ion is corresponding to the five oxygen atoms originated from the side chain of Gln141, Glu170, Glu221, Gln296, and the carbonyl group of Ser352.2 The sixth and the seventh coordination positions are occupied by O1 and O2 of the substrates (S)-1,2-propanediol (PDO); the S-enantiomer is preferred in the binding by enzyme.2 The ribose moiety of 5’-deoxyadenosyl radical and the side chain of His143 are also involved in the QM region.
The interaction of the migrating OH group with the imidazolum ion of His143 has been considered to be essential for the stabilization of the transition state for the OH migration. The Ribosyl rotation for the radical transfer from AdoCbl to substrate can essentially promote the Co-C cleavage upon binding to apodiol dehydratase, where adeninylpropylcobalamin (AdePeCbl) and other longer chain homologues cannot.2 The presence of the adenine-binding site in dio dehydratase was recently determined by the crystal structure analysis of the diol hydratase-AdePeCbl complex.8 The crystal structure shows that the adenine moiety of this analogue is trapped by hydrogen-bonding network with a water molecule and surrounding amion acid residues, Ser224, Ser229, Ser301, and Gly261.2 For this reason, the adenine-binding pocket fixes the adenine ring to allow tight binding of adenylpentyl group to the Co atom at a distance of 1.89 A, which is the main reason for the catalytic inactivity of the analogue.
[NEED TO RELOAD THIS IMAGE PROPERLY]
Figure 4. Active site structure of diol dehydratase.
Figure 4, part A shows an X-ray structure of the diol dehydratase-AdePeCbl complex. Part B shows Diol dehydratase-AdoCbl model complex produced by replacing the pentyl moiety of A with ribose. Part C shows the optimized structure of the diol dehydratase- AdoCbl complex model after the rotation of the ribose moiety.4 However, there is still argument whether the K+ ion in the active site remains with the substrate and radical intermediates through the reaction. Further studies could contribute to the understanding of hydrogen bonds to the active site residues, hydrogen abstraction and the steroselective hydrogen recombination.
Where R = -OH, Hydroxycobalamin, -CN, Cyanocobalamin, -Me, Methylcobalamin, -Ado, 5-deoxyadensosine. The Cyanocobalamin is generated in-situ by bacteria in the gastrointestinal systems of many mammals, or by the carbonization of Hydroxycobalamin created by other types of bacteria when exposed to a charcoal environment. While the B12 ingested may be the Cyanocobalamin, the cyanide group is removed in the body when absorbed and is decomposed in the process of removal. Once in the body, the B12 structure is used in the transport of methyl groups in DNA construction as well as 5-deoxyadensosine in mitochondrial energy production in cells. However important this B12 interaction is in the human body, humans don't naturally utilize nor produce any of this B12, rather the functional use of Folic Acid in the body is merely replaced by Cobalamin. As a result, many health effects of both deficiency of B12 or folic acid can be rectified by the addition of the other if need be. Deficiency of either B12 or folic acid can result in several neurological disorders and lack of motivation or onset of depression, which can possibly be related to the available energy production in cells being altered or slowed down due to this deficiency. However, too much Cobalamin in the human blood stream can potentially lead to several serious diseases, many effects of which are still being researched and less understood due to its homogenous behavior alongside other compounds already present in the human body, such as folic acid. Several of these diseases are both the cause of overutilization of Cobalamin, and the resulting effect of which, including several types of leukemia, resulting in the high levels of Cobalamin being stored in tissues. This large amount of stored Cobalamin being involved in the high presence of haptocorrin, from the corrin portion of the cobalamin structure, leading to several, some life threatening, liver diseases. Even though
The active site of the cobalt metal in Cobalamin possesses an octahedral configuration, forming primary sigma bonds with the transfer ligand, R, and the amine ring in the Corrin ring structure, while secondary pi-type bonding occurs in the three planar imidazole-type rings of the Corrin structure, and with the imidazole-type rings below axis, as shown above. This structural configuration allows a unique electron configuration along with nearby pi-system stabilization making the cobalt atom active site a preferred and semi-stable target for transferring the hydroxyl, methyl, cyano, and 5-deoxyadensosine in the various functions of its enzyme catalysis action. If only the three planar and one sub-axial ligand bonds from the nitrogens in the imidazole-like ring structures are taken as identical, and the sigma bonding amine group and 'R' active site group are taken as separate, the cobalt and its immediate ligand environment can be seen to possibly possess a Cs type symmetry with one mirror plane running through the R and amine ligands.
Though the above Cs symmetry applies if only the simple immediate ligand environment is considered, because of the complexity in the structure of the actual surrounding Corrin ring and Nucleotide loop in the plane and below the active site of the imidazole-type structures can't in reality be taken as identical once the structure is extended beyond a couple bond lengths away. Though this may be the case, the immediate electron density of the supposed identical imidazole-type ring groups connected to the cobalt metal may be considered near identical enough to promote the rotational configuration of the 'R' group attached to be related to the amine ligand link instead. For the R groups being -OH, -CN, or Me this has no effect whatsoever on the bonding rotation as these ligands are considered symmetric along the attachment site, however the 5-deoxyadensosine link used extensively in the energy production in cells will be effected by this symmetry of the active site, and could potentially be one reason this Cobalamin performs well in the transfer of these groups.
Cobalamin is an important compound used in the human body which is used in various configurations for specific tasks in enzymatic catalysis in subgroup transfer between systems, though not naturally utilized, it is more of a replacement for Folic Acid in the human body for these same functions, making it an important vitamin in sufficient, but not over extensive, quantities.
Reference
1. “Definition of Vitamin B12” Web May 22, 2010. <http://nutrition.about.com/od/nutrientglossary/g/vitaminb12.htm>
2. Kamachi, T.; Toraya, T,; Yoshizawa, K. J. Am. Chem. Soc. 2004, 126, 16207-16216
3. Masuda, J.; Shibata, N.; Morimoto, Y.’ Toraya.; Yasuoka, N. Structure 2000, 8, 775.
4. Eda, M.; Kamachi, T.; Yoshisawa, K.; Toraya, T. Bull. Chem. Soc. Jpn 2002, 75, 1469.
5. “Figure 1” Web May 22,2010. <http://en.Wikipedia.org/wiki/Vitamin_B12>
6. A. A. M. Ermens, L. T. Vlasveld, J. Lindemans, Significance of elevated cobalamin (vitamin B12) levels in bloodClinical Biochemistry, Volume 36, Issue 8, November 2003, Pages 585-590
7. Harry P. C. Hogenkamp, Douglas A. Collins, David Live, Linda M. Benson, Stephen Naylor, Synthesis and characterization of nido-carborane-cobalamin conjugates. Nuclear Medicine and Biology, Volume 27, Issue 1, January 2000, Pages 89-92
8. Tilak Chandra, Kenneth L. Brown, Vitamin B12 and α-ribonucleosides Tetrahedron, Volume 64, Issue 1, 1 January 2008, Pages 9-38
9. Lucio Randaccio, Silvano Geremia, Jochen Wuerges, Crystallography of vitamin B12 proteins, Journal of Organometallic Chemistry, Volume 692, Issue 6, 15 February 2007, Pages 1198-1215
Contributors and Attributions
• Kris Tapper (UCD) | textbooks/chem/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Vitamins_Cofactors_and_Coenzymes/Vitamin_B%3A_Cobalamin/Cobalamin_1.txt |
1.1. Environmental toxicology
If you want to re-use this chapter, for e.g. in your electronic learning environment, feel free to copy this url: maken.wikiwijs.nl/120137/1__Introduction
Author: Ad Ragas
Reviewers: Kees van Gestel, Nico van Straalen
Learning objectives
You should be able to
• characterize the field of environmental toxicology;
• explain what type of knowledge from environmental chemistry, toxicology and ecology is relevant for environmental toxicology;
• explain the difference between environmental toxicology and ecotoxicology.
Keywords: Environmental toxicology, environmental chemistry, toxicology, ecology, ecotoxicology
Environmental toxicology is the science that studies the fate and effects of potentially hazardous chemicals in the environment. It is a multidisciplinary field assimilating and building upon knowledge, concepts and techniques from other disciplines, such as toxicology, analytical chemistry, biochemistry, genetics, ecology and pathology. Environmental toxicology emerged in response to the growing awareness in the second part of the 20th century that chemicals emitted to the environment can trigger hazardous effects in organisms living in this environment, including humans. Section 1.3 gives a brief summary of the history of environmental toxicology.
One way to depict the field of environmental toxicology is by a triangle consisting of chemicals, the environment and organisms (Figure 1). The triangle illustrates that the fate and potential hazardous effects of chemicals emitted to the environment are determined by the interactions between these chemicals, the environment and organisms. The fate of substances in the environment is the topic of environmental chemistry, the effects of substances on living organisms is studied by toxicology, and the implications of these effects on higher levels of biological organization are analyzed by the field of ecology.
Another term widely used to refer to this field of study is ecotoxicology. The main distinction is the inclusion of human health as an endpoint in environmental toxicology, whereas ecotoxicology is restricted to ecological endpoints. Since the current book includes human health as an assessment endpoint for environmental contaminants, the term environmental toxicology is preferred over ecotoxicology.
Environmental chemists study the fate of chemicals in the environment, e.g. their distribution over different environmental compartments and how this distribution is influenced by the physicochemical properties of a chemical and the characteristics of the environment. They aim to understand the pathways and processes involved in the environmental fate of a chemical after it has been emitted to the environment, including processes such as advection, deposition and (bio)degradation. Within the context of environmental toxicology, the ultimate aim is to produce a reliable assessment of the exposure of organisms, an aim which is often complicated by the enormous heterogeneity of the environment.
Environmental chemists use a variety of tools to analyze and assess the fate of chemicals in the environment. Two fundamental tools are analytical measurements and mathematical modelling. Measurements are essential to acquire new knowledge and insight into the behavior of chemicals in the environment., e.g. measurements on emissions, environmental concentrations and specific processes such as biodegradation. These measurements are analyzed to discover patterns, e.g. between substance properties and environmental characteristics. Once revealed, such patterns can be integrated into a comprehensive mathematical model to predict the fate of and exposure to substances in the environment. If sufficiently validated, these models can subsequently be used by risk assessors to assess the exposure of organisms to chemicals, reducing the need for expensive measurements.
Chapter 2 focuses on the types of chemicals occurring in the environment, their sources and the concentrations found at contaminated sites. In Chapter 3, focus will be on the fate and transport of these chemicals, including aspects of bioavailability and bioaccumulation in organisms.
Toxicologists study the effects of chemicals on organisms, often at the individual level. Fundamental toxicologists aim to understand the mechanisms involved in the toxicity of a compound, whereas more applied toxicologists are primarily interested in the relationship between exposure and effect, often with the aim of identifying an exposure level that can be considered safe. Within this context, the dose concept as introduced by Parcelsus at the start of the 16th century is essential (see Section 1.3), i.e. the likelihood of adverse effects depends on the dose organisms are being exposed to.
The processes taking place after exposure of an organism to a toxicant are often divided into toxicokinetic and toxicodynamic processes. Toxicokinetic processes are those that describe the fate of the toxicant in the organism, including processes such as absorption, distribution, metabolism and excretion (ADME). These toxicokinetic or ADME processes are sometimes collectively referred to as "What the body does to the substance" and determine the exposure level at the site of toxic action, or internal dose. Toxicodynamic processes are those that describe the evolution of an adverse effect from the moment that the toxicant, or one of its metabolites, interacts with a molecular receptor in the body. This interaction is often referred to as the primary lesion or molecular initiating event (MIE). Toxicodynamic processes are sometimes collectively referred to as "What the substance does to the body" and the chain of events leading to an adverse outcome as the adverse outcome pathway (AOP).
The toxicity of a compound thus depends on toxicokinetic as well as toxicodynamic processes. Traditionally, this toxicity is being determined by exposing whole organisms in the laboratory to the substance of interest, and subsequently monitoring the health status of these organisms. However, as a result of the growing societal pressure to reduce animal testing, as well as the increased mechanistic understanding and improved molecular techniques, this so-called "black box approach" is more and more being replaced by a combination of in vitro toxicity testing and "in silico" predictive approaches. Physiologically-based toxicokinetic (PBTK) models are increasingly used to model the fate of chemicals in the body, resulting in a prediction of the internal exposure. In vitro tests and advanced molecular techniques at the gene (genomics) or protein (proteomics) level may subsequently be used to determine whether these internal exposure levels will trigger adverse effects, although many challenges remain in the prediction of adverse effects based on in vitro test and omics information. Chapter 4 focuses on dose-response relationships, modes of action, species differences in sensitivity and resistance against toxicants.
Ecologists study the interactions between organisms and their environment. Ecology is an important pillar of environmental toxicology, because ecological knowledge is needed to translate effects at the individual level to the ecosystem level; an important endpoint of ecological risk assessments. Such a translation requires specific knowledge, e.g. on life cycles of organisms, natural factors regulating their populations, genetic variability within populations, spatial distribution patterns, and the role organisms play in processes like nutrient cycling and decomposition. Effects considered relevant at the individual level, such as a tumor risk, may turn out to be irrelevant at the population or ecosystem level. Similarly, subtle effects at the individual level may turn out to be highly relevant at the ecosystem level, e.g. behavioral changes after environmental exposure to antidepressants which may affect the population dynamics of fish species. In recent years, there is an increasing interest for the role of the landscape configuration, distribution patterns and their dynamics in environmental toxicology. The spatial configuration of the landscape, the distribution of species and the timing of exposure events turn out to be important determinants of ecosystem effects. The ecological aspects of environmental toxicology will be discussed in Chapter 5.
1.1. Question 1
What is the difference between environmental toxicology and ecotoxicology?
1.1. Question 2
Indicate from the following terms whether these belong to the environmental chemistry, toxicology or ecology?
(Bio)degradation
Fate and exposure model
Dose
Toxicodynamics
Adverse outcome pathway
Population dynamics
Landscape configuration
1.1. Question 3
Give an example how subtle effects which may remain undetected in a toxicity test can be relevant at the population or ecosystem level. | textbooks/chem/Environmental_Chemistry/Environmental_Toxicology_(van_Gestel_et_al.)/01%3A_Environmental_Toxicology/1.01%3A_Environmental_toxicology.txt |
1.2. DPSIR
Author: Ad Ragas
Reviewers: Frank van Belleghem
Learning objectives
You should be able to:
• list and describe the five categories of DPSIR;
• structure a simple environmental problem using the DPSIR framework;
• describe the position and role of environmental toxicology within the DPSIR framework;
• indicate the most important advantages and disadvantages of the DPSIR framework.
Keywords: Drivers, pressures, state variables, impacts, responses
On the one hand, environmental toxicology is rooted in more fundamental scientific disciplines like biology and chemistry where curiosity is an important driver for gathering new knowledge. On the other hand, environmental toxicology is a problem-oriented discipline. As such, it is part of the broader field of environmental sciences which analyses the interactions between society and its physical environment in order to promote sustainability. Within this context, knowledge about the interactions of substances with the biotic and abiotic environment is being generated with the ultimate aim to prevent and address potential pollution problems in society. To be able to contribute optimally, an environmental toxicologist should know how pollution problems are structured and what the role of environmental toxicologists is in analysing, preventing and solving such problems. A widely used framework for structuring environmental problems is DPSIR. DPSIR stands for Drivers, Pressures, State, Impacts and Responses (Figure 1). The aim of the current section is to explain the DPSIR framework.
Communication tool
Communication is essential when analysing and addressing societal issues such as environmental pollution. As an environmental toxicologist, you will have to communicate with fellow scientists to develop a common understanding of the pollution problem, and with policy makers and stakeholders (e.g., producers of chemicals and consumers that are being exposed to chemicals) to explain the scientific state of the art. It is likely that you will use terms like "cause", "source" and "effects". However, not everybody will use and perceive these terms in the same way. Some people may argue that a farmer is the main cause of pesticide pollution, whereas others may argue that it is the pesticide manufacturer, or even the increasing world population. Likewise, some people may perceive the concentration of pesticides in water as an effect of pesticide use, whereas others may refer to the extinction of species when talking about effects. These differences may result in miscommunication, complicating scientific analysis and the search for appropriate solutions.
The DPSIR framework is a tool that helps preventing such communication problems. It provides a common and flexible frame of reference to structure environmental issues by describing these in terms of drivers, pressures, state (variables), impacts and responses (Figure 1). Flexibility is an important characteristic of the framework, enabling adaptation to the problem at hand. The DPSIR framework should not be considered a panacea or used as a mould that rigidly fits all environmental issues. Its main strength is that it stimulates communication between scientists, policy makers and other actors and thereby supports the development of a common understanding.
The framework
The DPSIR framework essentially is a cause-and-effect chain that aims to capture the main processes involved in an environmental issue; from its origin to the changes it triggers in the environment and in society. These processes are organized in five main categories, i.e.:
• are the human needs underlying the human activities that ultimately result in adverse effects. An example is the human need for food resulting in the use of pesticides such as neonicotinoids.
• are human activities initiated to fulfil human needs and resulting in changes in the physical environment that ultimately lead to - often unforeseen - adverse consequences for the environment or certain groups of society that are perceived as problematic, either now or in the future. An example is the use of neonicotinoids in agriculture.
• refers to the status of the physical environment. The state of the environment is often quantified using observable changes in environment parameters, e.g., the concentration of neonicotinoids in water, air, soil and biota.
• are any changes in the physical environment or society that are a consequence of the environmental pressures and that are perceived as problematic by society or some groups in society. An example is the increasing bee mortality that is, at least partly, attributed to the use of neonicotinoids. Or the human health effects of pesticides.
• are all initiatives developed by society to address the issue. These can range from gathering knowledge to developing policy plans and taking measures to mitigate effects or reduce emissions. Examples include the introduction of a risk-based admission procedure for neonicotinoids, the introduction of more efficient spraying techniques, and the development of environmentally friendly pest control techniques.
In principle, any environmental issue can be captured in a DPSIR. But it is important to realize that the labelling of processes as either drivers, pressures, state (variables), impacts or responses is likely to differ between people since the categories are broadly defined and the level of detail in the processes considered may vary. For example, some people may argue that "agriculture" should be classified as a driver, whereas others may argue it is a pressure. Yet other people may deal with this issue by adapting the DPSIR framework, i.e. by adding a new category called "human activities" that is placed in-between the drivers and the pressures. Another typical issue is the labelling of consecutive changes in the physical environment such as rising CO2 levels, increases in temperature and changes in species abundance. These changes can be labelled as changes in consecutive state variables, i.e. state variables of 1st, 2nd and 3rd order. The idea is that 1st order changes trigger 2nd order changes, e.g. rising CO2 levels triggering a rise in temperature, and 2nd order changes trigger 3rd order changes, in this case a shift in species abundance. The change in species abundance may also be labelled as an impact, provided this change is perceived as problematic by (groups in) society. The category "impacts" is closely related to the protection goals of risk assessment (see the Section Ecosystem services and protection goals). If there is consensus in society that an impact should be prevented, it becomes a protection goal. All these examples illustrate that the DPSIR framework should be applied in a flexible way and that communication is essential.
Environmental toxicology mainly focuses on the Pressures, State and Impacts blocks of the DPSIR chain. The use of chemicals by society, e.g. in agriculture or in consumer products, and their emission to the environment belongs to the Pressure block. The fate of chemicals in the environment and their accumulation in organisms belongs to the State block. And the adverse effects triggered in ecosystems and humans belong to the Impact block. An important step in risk assessment of chemicals (Chapter 6) is the derivation of safe exposure levels such as the Predicted No Effect Concentration (PNEC) for ecosystems or the Acceptable Daily Intake (ADI) for humans. In terms of DPSIR, this boils down to defining an acceptable impact level (e.g. a zero effect level or a 1 in a million tumor risk) and translating this into a corresponding state parameter (e.g. the chemical concentration in air or water). Fate modelling (Section on Modelling exposure) aims to predict soil, water, air and organisms (all State parameters) based on emission data (a Pressure parameter).
The DPSIR framework has been criticized because it tries to capture all processes in cause-and-effect relationships, resulting in a bias towards the physical dimension of environmental issues, e.g. human activities, emissions, physical effects and mitigations measures. The societal dimension is less easily captured, e.g. knowledge generation, governance structures, resources needed to implement measures, awareness and societal framing of the problem (Svarstad et al., 2008). Although the DPSIR framework can been adapted to accommodate some of these aspects (e.g., see Figure 2), it should be acknowledged that it has its limitations. Several alternative frameworks have been developed, and some of these better capture the societal dimension (Gari et al., 2015; Elliott et al., 2017). Nevertheless, DPSIR can be a useful framework to contextualize the problems that are addressed in environmental toxicology. It nicely shows why knowledge on the fate and impact of chemicals (state and impacts) is needed to address pollution issues and that the use of this knowledge is always subject to valuation, i.e. it depends on how society values the adverse effects triggered by the pollution. DPSIR is also widely used by national and international institutes such as the European Environment Agency (EEA), the United States Environmental Protection Agency (US-EPA) and the Organisation for Economic Cooperation and Development (OECD). The DPSIR framework is sometimes also used as a first step in modelling, especially its physical dimension. Once relevant processes have been identified, these are then described quantitatively resulting in models that can be used to predict environmental concentrations or ecological effects of substances based on knowledge about human activities or emissions.
References
Gari, S.R., Newton, A., Icely, J.D. (2015). A review of the application and evolution of the DPSIR framework with an emphasis on coastal social-ecological systems. Ocean & Coastal Management 103, 63-77.
Svarstad, H., Petersen, L.K., Rothman, D., Siepel, H., Wätzold, F. (2008). Discursive biases of the environmental research framework DPSIR. Land Use Policy 25, 116-125.
Elliott, M., Burdon, D., Atkins, J.P., Borja, A., Cormier, R., de Jonge, V.N., Turner, R.K. (2017). "And DPSIR begat DAPSI(W)R(M)!" - A unifying framework for marine environmental management. Marine Pollution Bulletin 118, 27-40.
1.2. Question 1
Indicate whether the following phenomena should be labelled as drivers, pressures, state (variables), impacts and responses.
1. The number of fish in a water body
2. The pesticide concentration in a water body
3. The development of a new spaying technique to reduce pesticide emissions
4. The need for food
5. Crop cultivation
6. Spraying pesticides
1.2. Question 2
Pharmaceuticals are being used to protect the health of humans, farm animals and pets. After use, part of these pharmaceuticals may reach the environment where they may trigger adverse effects in ecosystems. In theory, humans may also be affected, e.g. after swimming in polluted surface water or consumption of polluted drinking water. Besides direct toxic effects, antibiotics may also result in the emergence of antibiotic resistance, which threatens human health.
List the most important drivers, pressures, state (variables), impacts and responses for the issue of "pharmaceuticals in the environment".
1.2. Question 3
On which blocks of the DPSIR framework do you focus when you work as an environmental toxicologist? | textbooks/chem/Environmental_Chemistry/Environmental_Toxicology_(van_Gestel_et_al.)/01%3A_Environmental_Toxicology/1.02%3A_DPSIR.txt |
1.3. Short history
Author: Ansje Löhr
Reviewers: Ad Ragas, Kees van Gestel, Nico van Straalen
Learning Objective:
You should be able to
• summarize the history of environmental toxicology
• describe the increasing awareness over time of environmental and health risks
Keywords: Paracelsus; Rachel Carson (Silent Spring); Awareness; SETAC; standards
History
From earliest times, man has been confronted with the poisonous properties of certain plants and animals. Poisonous substances are indeed common in nature. People who still live in close contact with nature generally possess an extensive empirical knowledge of poisonous animals and plants. Poisons were, and still are, used by these people for a wide range of applications (catching fish, poisoning arrowheads, in magic rituals and medicines). The first Egyptian medical documentation (written in the Ebers Papyrus) dates from 1550 BC and demonstrates that the ancient Egyptians had an extensive knowledge of the toxic and curative properties of natural products. A good deal is known about the information regarding toxic substances possessed by the Greeks and the Romans. They were very interested in poisons and used them to carry out executions. Socrates, for example, was executed using an extract of hemlock (Conium maculatum). It was also not unusual to use a poison to murder political opponents. Poisons were ideal for that purpose, since it was usually impossible to establish the cause of death by examining the victim. To do so would have required advanced chemical analysis, which was not available at that time.
Early European literature also includes a considerable number of writings on toxins, including the so-called herbals, such as the Dutch "Herbarium of Kruidtboeck" by Petrus Nylandt dating from 1673. Poisoning sometimes assumed the character of a true environmental disaster. One example is poisoning by the fungus Claviceps purpurea, which occurs as a parasite in grain, particularly in rye (spurred rye) and causes the condition known as ergotism. In the past, this type of epidemic has killed thousands of people, who ingested the fungus with their bread. There are detailed accounts of such calamities. For example, in the year 992 an estimated 40,000 people died of ergotism in France and Spain. People were not aware of the fact that death was caused by eating contaminated bread. It was not until much later that it came to be understood that large-scale cultivation of grain involved this kind of risk.
Paracelsus
It was pointed out centuries ago that workers in the mining industry, who came into contact with a variety of metals and other elements, tended to develop specific diseases. The symptoms regularly observed as a result of contact with arsenic and mercury in the mining industry were described in detail by the famous Swiss physician Paracelsus (Figure 1) in his 1567 treatise "Von der Bergsucht und anderen Nergkrankheiten" (miners sickness and other diseases of mining). During the emergence of the scientific renaissance of the 16th century, Paracelsus (1493 - 1541) drew attention to the dose-dependency of the toxic effect of substances. In the words of Paracelsus, "all Ding sind Gifft … allein die Dosis macht das ein Ding kein Gifft is" (everything is a poison … it is only the dose that makes it not a poison). This principle is just as valid today. At the same time, it is one of the most neglected principles in the public understanding of toxicology.
A work from the same period "De Re Metallica" by Gergius Agricola (Georg Bauer, 1556), deals with the health aspects of working with metals. Agricola even advised preventive aspects, such as wearing protective clothing (masks) and using ventilation.
Scrotum cancer in chimney sweepers: carcinogenicity of occupational exposure
Another example of the rising awareness of the effects of poisons on human health came with the suggestion, by Percival Pott in 1775, that the high frequency of scrotum cancer among British chimney sweepers was due to exposure to soot. He was the first to describe occupational cancer.
A part of the essay by Percival Pott "The fate of these people seems singularly hard; in their early infancy, they are most frequently treated with great brutality, and almost starved with cold and hunger; they are thrust up narrow, and sometimes hot chimnies, where they are bruised, burned, and almost suffocated; and when they get to puberty, become peculiarly liable to a most noisome, painful, and fatal disease." See the rest of the original text of his essay here.
Soot consists of polycyclic aromatic hydrocarbons (PAHs) and their derivatives. The exposure to soot came with concurrent exposure to a number of carcinogens such as cadmium and chromium. From the 1487 cases of scrotal cancer reported, 6.9 % occurred in chimney sweepers. Scrotal and other skin cancers among chimney sweepers were at the same time also reported from several other countries.
Peppered moth in polluted areas
Changes in the environment due to environmental pollution led to interesting insights in the potential of species to adapt for survival and the role of natural selection in it. A famous example of such micro-evolution is the peppered moth, Biston betularia, that is generally a mottled light color with black speckles. This pattern gives them good camouflage against lichen-covered tree trunks while resting during the day. During the industrial revolution, the massive increase in the burning of coal resulted in the emission of dark smoke turning the light trees in the surrounding areas dark. As a consequence, the dark, melanic form of the peppered moth took over in industrial parts of the United Kingdom during the 1800s. The melanic forms used to be quite rare, but their dark color served as a protective camouflage from bird populations in the polluted areas. This allowed them to become dominant in areas with soot-covered trunks. Two British biologists, Cedric Clarke and Phillip Sheppard, discovered this when they pinned dead moths of the two types on dark and light backgrounds to study their predation by birds. The dark moths had an advantage in the dark forests, a result of natural selection. In areas where air pollution has decreased the melanic form became less abundant again.
Video on peppered moths
Awareness in the 1950s and 1960s
After the second world war, synthetic chemical production became widespread. However, there was limited awareness of the environmental and health risks. In the 1950s, Environmental Toxicology came to light as a result of increasing concern about the impact of toxic chemicals on the environment. This led toxicology to expand from the study of the toxic impacts of chemicals on man to that of toxic impacts on the environment. An important person in raising this awareness was Rachel Carson. Her book "Silent Spring", published in 1962, in which she warned of the dangers of chemical pesticides, triggered widespread public concern on the dangers of improper pesticide use.
First have a look at an historical clip on the use of dichlorodiphenyltrichloroethane, commonly known as DDT, that was developed in the 1940s as the first modern synthetic insecticide.
Silent Spring - Rachel Carson
DDT is very persistent and tends to concentrate when moving through the food chain. As a consequence, the use of DDT led to very high levels, especially in organisms high in the food chain. Bioaccumulation in birds appeared to cause eggshell thinning and reproductive failure. Because of the increasing evidence of DDT's declining benefits and its environmental and toxicological effects, the United States Department of Agriculture, the federal agency responsible for regulating pesticide use, began regulatory actions in the late 1950s and 1960s to prohibit many of its uses. By the 1980s, the use of DDT was also banned from most Western countries.
Large environmental disasters
As a result of large environmental disasters, awareness amongst the general public increased. An enormous industrial pesticide disaster occurred in 1984 in Bhopal, India, when more than 40 ton of the highly toxic methyl isocyanate (MIC) gas leaked from a pesticide plant into the towns located near the plant. Almost 4000 people were killed immediately and 500,000 people were exposed to the poisonous substance causing many additional deaths because of gas-related diseases. The plant was actually initially only allowed to import MIC but was producing it on a large scale by the time of the disaster and safety procedures were far below (international) standards for environmental safety. The disaster made it very clear that this should be changed to avoid other large-scale industrial disasters.
The Sandoz agrochemical spill close to Basel in Switzerland in 1986 was the result of a fire in a storehouse. The emission of large amounts of pesticide caused severe ecological damage to the Rhine river and massive mortality of benthic organisms and fish, particularly eels and salmonids.
At the time of these incidents, environmental standards for chemicals were still largely lacking. The incidents triggered scientists to do more research on the adverse environmental impacts of chemicals. Public pressure to control chemical pollution increased and policy makers introduced instruments to better control the pollution, e.g. environmental permitting, discharge limits and environmental quality standards.
Our Common Future
In 1987, the World Commission on Environment and Development released the report "Our Common Future", also known as the Brundtland Report. This report placed environmental issues firmly on the political agenda, defining sustainable development as "a development that meets the needs of the present without compromising the ability of future generations to meet their own needs". Another influential book was "Our stolen future" written by Theo Colborn and colleagues in 1996. It raised awareness of the endocrine disrupting effects of chemicals released into the environment and threatening (human) reproduction by emphasizing it not only concerns feminization of fish or other organisms in the environment, but also the human species.
Please watch the video "Developments in Environmental Toxicology - Interview with two pioneers" included at the start of the Introduction of this book.
SETAC
Before the 1980s, no forum existed for interdisciplinary communication among environmental scientists -biologists, chemists, toxicologists- as well as managers and others interested in environmental issues. The Society of Environmental Toxicology and Chemistry (SETAC) was founded in North America in 1979 to fill this void. In 1991, the European branch started its activities and later SETAC also established branches in other geographical units, like South America, Africa and South-East Asia. SETAC publishes two journals: Environmental Toxicology and Chemistry (ET&C) and Integrated Environmental Assessment and Management (IEAM). SETAC also is active in providing training, e.g. a variety of online courses where you can acquire skills and insights in the latest developments in the field of environmental toxicology. Based on the growth in the society's membership, the meeting attendance and their publications, a forum like SETAC was clearly needed. Read more on SETAC, their publications and how you can get involved here.
Where SETAC focuses on environmental toxicology, international toxicological societies have also been established like EUROTOX in Europe and the Society of Toxicology (SOT) in North America. In addition to SETAC, EUROTOX and SOT, many national toxicological societies and ecotoxicological counterparts or branches became active since the 1970s, showing that environmental toxicology has become a mature field of science. One element indicative of this maturation, also is that the different societies have developed programmes for the certification of toxicologists.
References
Carson, R. (1962). Silent Spring. Houghton Mifflin Company.
Colborn, T., Dumanoski, D., Peterson Myers, J. (1996). Our Stolen Future: Are We Threatening Our Fertility, Intelligence, and Survival? A Scientific Detective Story. New York: Dutton. 306 p.
World Commission on Environment and Development (1987). Our Common Future. Oxford: Oxford University Press. p.27.
1.3. Question 1
Paracelsus is famous for the dose-dependency of the toxic effect of substances. What is meant by dose-dependency?
1.3. Question 2
What is the difference between toxicology and environmental toxicology?
1.3. Question 3
How was sustainable development defined in "Our Common Future" also known as the Brundtlandt report? | textbooks/chem/Environmental_Chemistry/Environmental_Toxicology_(van_Gestel_et_al.)/01%3A_Environmental_Toxicology/1.03%3A_Short_history.txt |
2.1. Introduction
Authors: John Parsons, Steven Droge
Reviewer: Kees van Gestel
Leaning objectives:
You should be able to:
• mention the main groups of environmental pollutants
• comprehend the molecular structures of the most important organic pollutants
• mention the most important functional groups determining the environmental properties of organic pollutants
Keywords: natural toxicants, molecular structures, pollutant classes, anthropogenic pollutants
Introduction
Environmental toxicology deals with the negative effects of the exposure to chemicals we regard as pollutants (or contaminants/toxicants). Environmental toxicants receive a lot of media attention, but many critical details are getting lost or are easily forgotten. The clip "You, Me, DDT" shows the discovery of the grandson of the works of his grandfather, the Swiss inventor of the insecticide DDT, Paul Hermann Müller, who received the Nobel Prize for Medicine for that in 1948 (see also Section 1.3). The clip "Stop the POPs" interviews (seemingly) common people about one of the most heavily regulated group of pollutants.
Organisms, including humans, have always been exposed to chemicals in the environment and rely on many of these chemicals as nutrients. Volcanoes, flooding of acid sulfur lakes, and forest fires have caused widespread contamination episodes. Organisms are also in many cases directly or indirectly involved in the fate and distribution of undesirable chemicals in the environment. Many naturally occurring chemicals are toxicants already (see also Section 1.3), think for example about:
• local arsenic or mercury hotspots in the Earth's crust, contaminating water pumps or rice irrigation fields;
• plant-based defence chemicals such as alkaloids, morphine in poppy seeds, juglone from black walnut trees);
• fungal toxins, such as mycotoxins threatening grain storage depots after harvests;
• bacterial toxins, such as the botulinum toxin, a neurotoxic protein produced by the bacterium Clostridium botulinum which is the most acutely lethal toxin known at ~10 ng/kg body weight when inhaled;
• phycotoxins, produced by algae, in mass algal blooms or those that may end up at dangerous levels in shell food;
• zootoxins in animals, such as venom of snakes and defensive toxins on the skin of amphibians.
Human activities have had an enormous impact on the increased exposure to natural chemicals as a result of, for example, the mining and use of metals, salts and fossil fuels from geological resources. This is for example the case for many metals, nutrients such as nitrate, and organic chemicals present in fossil fuels. Additionally, the industrial synthesis and use of organic chemicals, and the disposal of wastes, have resulted in a wide variety of hazardous chemicals that had either never existed before, or at least not in the levels or chemical form that occur nowadays in our heavily polluted global system. These are typically organic chemicals that are referred to as anthropogenic (`due to humans in nature´) or xenobiotic (`foreign to organisms´) chemicals. In this chapter we aim to clarify the key properties and functionalities of the most common groups of pollutants as a result from human activities, and provide some background on how we can group them and understand their behaviour in the environment.
In the field of environmental toxicology, we are most often concerned about the effects of two distinct types of contaminants: metals and organic chemicals. In some cases other chemicals, such as radioactive elements, may also be important while we could also consider the ecological effects of highly elevated nutrient concentrations (eutrophication) as a form of environmental toxicology.
Metals
Metals and metalloids (elements intermediate between metals and non-metals) comprise the majority of the known elements. They are mined from minerals and used in an enormous variety of applications either in their elemental form or as chemicals with inorganic or organic elements or ions. Many metals occur as cations, but many processes influence the dissolved form of metals. Aluminium for example for example is only present under very acidic conditions as dissolved cation (Al3+), while at neutral pH the metal speciates into for example certain hydroxides (Al(OH3)0). Mercury as free ion is present at (Hg2+) but due to microbial transformation the highly toxic product methylmercury (CH3Hg+, or MeHg+) is formed. Mining and processing of metals together with disposal of metal-containing wastes are the main contributors to metal pollution although sometimes metals are introduced deliberately into the environment as biocides. The widely used pesticide copper sulfate in e.g. grape districts is an example (LINK on comparison to glyphosate here). More information on metals considered to be environmental pollutants is given in Section 2.2.1.
Organic chemicals
Organic chemicals are manufactured to be used in a wide variety of applications. These range from chemicals used as pesticides to industrial intermediates, fossil fuel related hydrocarbons, additives used to treat textiles and polymers, such as flame retardants and plasticisers, and household chemicals such as detergents, pharmaceuticals and cosmetics.
Organic chemicals that we regard as environmental pollutants include a huge variety of different structures and have a wide variety of properties that influence environmental distribution and toxicity. With such a wide variety of chemicals to deal with, it is useful to classify them into groups. Depending on our interest, we can base this classification on different aspects, for example on their chemical structure, their physical and chemical properties, the applications the chemicals are used in, or their effects on biological systems. These aspects are of course closely related to their chemical structure as this is the basis of the properties and effects of chemicals. An overview of different ways of classifying environmental contaminant (sometimes referred to as ecotoxicants) is shown in Tables 1A, 1B, and 1C.
Table 1A. Grouping options of organic contaminants with specific chemical structures
Term
Characteristics
Examples
Hydrocarbons
More CHx units: higher hydrophobicity/lipophilicity, and lower aqueous solubility
hexane
Polycyclic aromatic hydrocarbons
Combustion products. Flat structure
naphthalene, B[a]P
Halogenated hydrocarbons
H substituted by fluor, chlorine, bromide, iodine. Often relatively persistent
PCB, DDT, PBDE
Dioxins and furans
Combustion/industrial products, one or two oxygen atoms between two aromatic rings. Highly toxic.
TCDD, TCDF
Organometallics
Organic chemicals containing metals, used e.g. in anti-fouling paints
tributyltin
Organophosphate pesticides
Phosphate esters, often connecting two lipophilic groups. Act on nervous system
chlorpyrifos
Pyrethroids
Usually synthetic pesticides based on natural pyrethrum extracts
fenvalerate
Neonicotinoids
Synthetic insecticides with aromatic nitrogen, related to the alkaloid nicotine
imidacloprid
… Endless varieties / combinations and ...
...too many characteristics to list
...
Table 1B. Grouping options of organic contaminants with specific properties
Term
Characteristics
Examples
Persistent organic pollutants (POPs)
Bioaccumulative, end up even in remote Arctic systems
PCBs, PFOS
Persistent mobile organic chemicals (PMOCs)
Difficult to remove during drinking water production
PFBA, metformin
Ionogenic organic chemicals (IOCs)
Acids or bases, predominantly ionized under environmental pH
Prozac, MDMA, LAS
Substances of unknown or variable composition, complex reaction products or of biological materials (UVCB)
Multicomponent compositions of often analogue structures with wide ranging properties.
Oil based lubricants
Plastics
Chains of repetitive monomer structures. Wide ranging size/dimensions.
Polyethylene,
silicone, teflon
Nanoparticles (NP)
Mostly manufactured particles with >50% having dimensions ranging 1 - 100 nm.
Titanium dioxide (TiO2), fullerene
Table 1C. Grouping options of organic contaminants with specific usage
Term
Characteristics
Examples
Pesticides
Herbicides
Insecticides
Fungicides
Rodenticides
Biocides
Toxic to pests
Toxic to plants
Toxic to insects
Toxic to fungi
Toxic to rodents
Toxic to many species
DDT
atrazine, glyphosate
Chlorpyrifos, parathion
Phenyl mercury acetate
Hydrogen cyanide
Benzalkonium
Pharmaceuticals
Specifically bioactive chemicals with often (un)known side effects. Many bases.
diclofenac (pain killer), iodixanol (radio-contrast), carbemazepine, prozac
Drugs of abuse
Often opioid based but also synthetic designer drugs with similar activity. Many are are ionogenic bases.
cannabinoids, opioids,
amphetamine, LSD
Veterinary Pharmaceuticals
Can include relatively complex (ionogenic) structures
antibiotics, antifungals, steroids, non-steroidal anti-inflammatories
Industrial Chemicals
Produced in large volumes by chemical industry for a wide array of products and processes
phenol
Fuel products
Flammable chemicals
kerosene
Refrigerants and propellants
Small chemicals with specific boiling points
freon-22
Cosmetics/personal care products
Wide varieties of specific ingredients of formulations that render specific properties of a product
sunscreen, parabenes
Detergents and surfactants
Long hydrophobic hydrocarbon tails and polar/ionic headgroups
Sodium lauryl sulfate (SLS), benzalkonium
Food and Feed Additives
To preserve flavor or enhance its taste, appearance, or other qualities
"E-numbers", acetic acid = E260 in EU, additive 260 in other countries
Chapter 2 mostly discusses groups of chemicals in separate modules according to the specific environmental properties in Table 1B (Section 2.2) and specific applications in Table 1C (Section 2.3), according to which certain regulations apply in most cases. The property classifications can be based on (often interrelated) properties such as solubility (in water), hydrophobicity (tendency to leave the water), surface activity (tendency to accumulate at surfaces of two phases, such as for "surfactants"), polarity, neutral or ionic chemicals and reactivity. Other classifications very important for environmental toxicology are based on environmental behaviour or effects, such as persistency ("P" increasing problems with increased emissions), bioaccumulation potential ("B", up-concentration in food chains), or type of specific toxic effects ("T"). The influence of specific chemical structures such as in Table 1A is further clarified in the current introductory chapter in order to better understand the basic chemical terminology.
Structures of organic chemicals and functional groups
• Hydrocarbons and polycyclic aromatic hydrocarbons
As the name suggest, hydrocarbons contain only carbon and hydrogen atoms and can therefore be considered to be the simplest group of organic molecules. Nevertheless, this group covers a wide variety of aliphatic, cycloaliphatic and aromatic structures (see Figure 1 for some examples) and also a wide range of properties. What this group shares is a low solubility in water with the larger molecules being extremely insoluble and accumulating strongly in organic media such as soil organic matter.
As a result of the ability of carbon to form strong bonds with itself and other atoms to form structures containing long chains or rings of carbon atoms there is a huge and increasing number (millions) of organic chemicals known. Chemicals containing only carbon and hydrogen are known as hydrocarbons. Aliphatic molecules consist of chains of carbon atoms as either straight or branched chains. Molecules containing multiple carbon-carbon bonds (C=C) are known as unsaturated molecules and can be converted to saturated molecules by addition of hydrogen.
Cyclic alkanes consist of rings or carbon atoms. These may also be unsaturated and a special class of these is known as aromatic hydrocarbons, for example benzene in Figure 1. The specific electronic structure in aromatic molecules such as benzene makes them much more stable than other hydrocarbons. Multiple aromatic rings linked together make perfectly flat molecules, such as pyrene in Figure 1, that can be polarized to some extent because of the shared electron rings. In larger sheets, these polycyclic aromatic molecules also make up the basic graphite structure in pencils, and also typically represent the strongly adsorbing surfaces of black carbon phases such as soot and activated carbon.
The structures of organic chemicals help to determine their properties as behaviour in the environment. At least as important in this regard, however, is the presence of functional groups. These are additional atoms or chemical groups that are present in the molecule that have characteristic chemical effects such as increasing or decreasing solubility in water, giving the chemical acidic or basic properties or other forms of chemical reactivity. The common functional groups are shown in Table 2.
Table 2. Common Functional Groups, where R are carbon backbone or hydrogen units
• Halogenated hydrocarbons: first generation pesticides
The first organic chemical recognised as an environmental pollutant was the insecticide DDT (see clip 1 at the start of this chapter). It later became clear that other organochlorine pesticides such as lindane and dieldrin (Table 3) were also widely distributed in the environment. This was also the case for polychlorinated biphenyls (PCBs) and other organochlorinated industrial chemicals. These chemicals all share a number of undesirable properties such as environmental persistence, very low solubility in water and high level of accumulation in biota to potentially toxic levels. Many organochlorines can be viewed as hydrocarbons in which hydrogen atoms have been replaced by chlorine. This makes them even less soluble than the corresponding hydrocarbon due to the large size of chlorine atoms. In addition, chlorination also makes the molecules more chemically stable and therefore contributes to their environmental persistence. Other organochlorines contain additional functional groups, such as the ether bridges in PCDDs and PCDFs (better known as dioxins and dibenzofurans) and ester groups in the 2,4-D and 2,4,5-T herbicides. Many organochlorines were applied very successfully in huge quantities as pesticides for decades before their negative effects such as persistence and accumulation in biota became apparent. It is therefore no coincidence that the initial set of Persistent Organic Pollutants (POPs) identified in the Stockholm Treaty (see below) as chemicals that should be banned were all organochlorines, as shown in Table 3.
As well as chlorine, other halogens such as bromine and fluorine are used in important groups of environmental contaminants. Organobromines are best known as flame retardants and have been applied in large quantities to improve the fire safety of plastics and textiles. They share many of the same undesirable properties of organochlorines and several classes have now been taken out of production. Organofluorines are another important class of halogenated chemicals, and part of the well-known group of ozone depleting CFCs (Section 2.3.6). In particular, per-and polyfluoralkyl substances are widely used as fire-stable surfactants in fire-fighting foams, as grease and water resistant coatings and in the production of fluoropolymers such as Teflon. Organofluorines are much more water soluble and much less bioaccumulative than organochlorines and organobromines but are extremely persistent in the environment.
The recognition of these organochlorines as harmful environmental contaminants eventually resulted in measures to restrict their manufacture and use in the Stockholm Convention on Persistent Organic Pollutants signed in 2001 to eliminate or restrict the production and use of persistent organic pollutants (POPs). This initial list of POPs has been subsequently augmented with other harmful halogenated organic pollutants up to a total of 29 chemicals, which are either to be eliminated, restricted, or required measured to reduce unintentional releases. POPs are further discussed in section 2.2.4.
Table 3. Key persistent organic pollutants, also named POPs - the Dirty Dozen
Additional POPs to eliminate include: chlordecone, lindane (hexachlorocyclohexane), pentachlorobenzene, endosulfan, chlorinated naphthalenes, hexachlorobutadiene, tetrabromodiphenylether, and pentabromodiphenylether decabromodiphenyl ether (BDEs).
• Alternatives for the organochlorine pesticides: effective functional groups
Since the signing of the Stockholm Convention, organochlorine pesticides have been replaced in most countries by more modern pesticide types such as the organophosphorus and carbamate insecticides. These compounds are less persistent in the environment, but still could pose elevated risks to environments surrounding the agricultural sites, and increased levels on food produced on these agricultural sites. The very toxic organophosphorus neurotoxicant parathion has been in use since the 1940s, and has the typical two lipophilic side chains on two esters (ethyl units), as well as a polar unit. Parathion has caused hundreds of fatal and non-fatal intoxications worldwide and as a result it is banned or restricted in 23 countries. The relatively comparable organophosphate structure of diazinon has been widely used for general-purpose gardening and indoor pest control since the 1970s, but residential use was banned in the U.S. in 2004. In Californian agriculture however, 35000 kg diazinon was used in 2012. The carbamate based insecticide carbaryl is toxic to target insects, and also non-target insects such as bees, but is detoxified and eliminated rapidly in vertebrates, and not secreted in milk. Although illegal in 7 countries, carbaryl is the third-most-used insecticide in the U.S., approved for more than 100 crops. In 2012, 52000 kg carbaryl was used in California, while this was 3 times more in 2000. Neonicotinoid insecticides, with the typical aromatic ring containing nitrogen, form a third generation of pesticide structures. Imidacloprid is currently the most widely used insecticide worldwide, but as of 2018 banned in the EU, along with two other neonicotinoids clothianidin and thiamethoxam.
• Relatively simple and (very) complex pollutants
As well as the pesticides discussed above, many other chemicals are brought into the environment inadvertently during their manufacture, distribution and use and the range of chemicals recognised as problematic environmental contaminants has expanded enormously. These include fossil fuel-related hydrocarbons, surfactants, pigments, biocides and chemicals used as pharmaceuticals and personal care products (PPCPs). Figure 1 in Boxall et al. (2012) gives an illustrative overview of the major routes by which PPCPs, but also many other anthropogenic contaminants other than pesticides, are released into the environment. Particularly wastewater treatment systems form the main entry point for many industrial and household products.
The wide variety of contaminant structures does not mean that most chemicals have become increasingly more complex. For risk assessment, molecular properties such as water solubility, volatility and lipophilicity are often estimated based on quantitative structure-property relationships (Section 3.4.3). With increasingly complex structures, such property-estimations based on the molecular structure become more uncertain.
The antibiotic erythromycin for example (Figure 3), is a very complex chemical structure (C37H67NO13) that has 13 functional units along with a 14 member ring. In addition, the tertiary nitrogen group is an amine base group that can give the molecule a positive charge upon protonation, depending on the environmental pH. Erythromycin is on the World Health Organization's List of Essential Medicines (the most effective and safe medicines needed in a health system), and therefore widely used. Continuous emissions in waste streams pose a potential threat to many ecosystems, but many environmentally and toxicologically relevant properties are scarcely studied, and poorly estimated.
There are also many contaminants or toxicants with a seemingly simple structure. Many surfactants are simple linear long chain hydrocarbons with a polar or charged headgroup (Figure 4). The illicit drug amphetamine has only a benzene ring and an amine unit, the illicit drug GHB only an alcohol and a carboxylic acid, the herbicide glyphosate only 16 atoms. Still, these 4 chemical examples also have acidic or basic units that often result in predominantly charged organic molecules, which also strongly influences their environmental and toxicological behaviour (see sections on PMOCs and Ionogenic Organic Compounds). In case of glyphosate, the chemical has 4 differently charged forms depending on the pH of the environment. At common pH of 7-9, glyphosate has all charged groups predominantly ionized, making it very difficult to derive calculations on environmental properties. | textbooks/chem/Environmental_Chemistry/Environmental_Toxicology_(van_Gestel_et_al.)/02%3A_Environmental_Chemistry_Chemicals/2.01%3A_Introduction.txt |
2.2. Pollutants with specific properties
2.2.1. Metals and metalloids
Author: Kees van Gestel
Reviewers: John Parsons, Jose Alvarez Rogel
Learning objectives:
You should be able to:
• describe the difference between metals and metalloids
• describe a classification using different binding affinities of metals to macromolecules and infer on its importance for their toxicity and/or bioaccumulation
• mention important sources of metal pollution
Keywords: Heavy metals, Metalloids, Rare earth elements, Essential elements
Introduction
The majority of the elements in the periodic table consists of metals: Figure 1.
The distinction between metals and heavy metals (density relative to water < or >5 g cm-3) is not very meaningful for such a heterogeneous group of elements with rather different biological and chemical properties. The rare earth elements (REEs), lanthanides and actinides, have, for example, a high density or specific weight but are usually not considered heavy metals because of their rather different chemical behaviour. Metalloids have both metallic and non-metallic properties or are nonmetallic elements that can combine with a metal to produce an alloy. Figure 1 shows the periodic table of elements, indicating the groups of (heavy) metals, metalloids and rare earth elements.
Also indicated in Figure 1 are the elements that are known to be essential to life and include besides C, H, O and N, the major essential elements Ca, P, K, Mg, Na, Cl and S, the trace elements Fe, I, Cu, Mn, Zn, Co, Mo, Se, Cr, Ni, V, Si, As and B (the latter only for plants) and some elements that may support physiological functions at ultra-trace levels (Li, Al, F and Sn) (Walker et al., 2012).
Chemical and physical properties
Except for mercury, most pure metals are solid at room temperature. In general, metals are good electrical and thermal conductors having high luster and malleability. Upon heating, metals readily emit electrons. These descriptors of metals, however, are not very helpful when having to deal with elements that do not exist prominently in the pure elemental state, but rather are present as metal compounds, complexes, and ions at fairly low environmental concentrations.
More useful are characteristics that influence metal transport between environmental compartments and their interaction with abiotic and biotic components of the environment. The speciation, the chemical form in which an element occurs (e.g., oxidized, free ion or complexed to inorganic or organic molecules), determines its transport and interaction in the environment (see Section on Metal Speciation). Chemical bonding is determined by outer orbital electron behavior, with metals tending to lose electrons when reacting with nonmetals. In many normal biological reactions, metals are cofactors within coenzymes (e.g. in vitamins) and can act as electron acceptors and donors during oxidation and reduction reactions (Newman, 2015).
Nieboer and Richardson (1980) proposed a classification, based on the equilibrium constant for the formation of metal complexes. They distinguished:
• Class A-metals: acting as hard Lewis acids (electron acceptors) with high affinity for oxygen-containing groups in macromolecules, such as carboxyl and alcohol groups. Al, Ba, Be, Ca, K, Li, Mg, Na and Sr belong to this group;
• Class B-metals: acting as soft Lewis acids with high affinity for nitrogen- and sulphur-containing groups in macromolecules, such as amino and sulphydryl groups. This group includes Ag, Au, Bi, Hg, Pd, Pt and Tl.
In addition, an intermediate or borderline group is defined, in which the type A or B characteristics are less pronounced. As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Sb, Sn, Ti, V, and Zn belong to this group.
This classification of metals is highly relevant for the transport across cell membranes, the intercellular storage in granules and the induction of metal-binding proteins as well as for their behaviour in the environment in general.
Occurrence
(Heavy) metals and rare earth elements are diffusely distributed over the Earth, but at some places certain elemental combinations are highly concentrated (in metal ores). Despite this diffuse distribution, differences in background metal concentrations in soils can be large, depending on the type and origin of rock or sediment (Table 1).
Table 1. Background concentrations (mg/kg dry weight) of (heavy) metals and metalloids in crust material and median and maximum concentrations in different top soils across the world. Derived from Kabata-Pendias and Mukherjee (2007) and Alloway (2013).
In general, volcanic rock (e.g. basalt) contains high and sedimented rock (e.g. limestone) low metal levels. But there is no relation between metal concentrations in the Earth's crust and the elemental requirements of organisms.
Emissions of metals
Upon weathering of stone formations and ores, elements are released and enter local, regional and global biogeochemical cycles. Depending on their water solubility and on soil properties and vegetation, metals may be transported through the environment and deposited or precipitated at places close to or far away from their source.
Volcanoes take account of the largest natural input of metals to the environment but the concentrations of these metals in the soil are rarely elevated to toxic levels due to the massive dilution which takes place in the atmosphere. Permanently active volcanoes may be an important local source of (metal) pollution.
A special case is arsenic, that may occur as a natural element of soils. At some places, As levels are fairly high, particularly in ground water. High As-groundwater areas are found in Argentina, Chile, Mexico, China and Hungary, and also in Bangladesh, India (West Bengal), Cambodia, Laos and Vietnam. In the latter countries, especially in the Bengal Basin, millions of wells have been dug to provide safe drinking water. Irrigation pumping leads to an inflow of oxygen and organic carbon, which causes a mobilisation of arsenic normally bound to ferric oxyhydroxides in these soils. As a result in many wells dissolved As concentrations are exceeding the World Health Organisation (WHO) guideline value of 10 µg/L for drinking water.
Important anthropogenic sources of metals in the environment include:
• Metal mining, which may also lead to an enormous physical disturbance of the environment (destruction of ecosystems).
• Metal smelting.
• Use of metals in domestic and industrial products, and the discharge of domestic waste and sewage.
• Metal-containing pesticides, e.g. 'Bordeaux Mixture (copper sulphate with lime (Ca(OH)2), used as a fungicide in viniculture, hop-growing and fruit-culture, and metal-containing fungicides, such as organo-tin compounds.
• The use of metals and especially of REEs in microelectronics.
• Energy-producing industries burning coal and oil, and producing metal-containing fly ash.
• Transport of energy and traffic making use of electricity, giving rise to corrosion of electric wires and pylons.
• Non-metal industries, e.g. leather (chromium) and cement production (thallium).
• , using Tetra Ethyl Lead (TEL) as anti-knocking agent in petrol (nowadays banned in most countries) and the use of catalysts in cars (platinum, palladium).
Anthropogenic releases of many metals, such as Pb, Zn, Cd and Cu, are estimated to be between one and three orders of magnitude higher than natural fluxes (Depledge et al. 1998). An estimated amount of up to 50,000 tonnes of mercury are released naturally per year as a result of degassing from the Earth's crust, but human activities account for even larger emissions (Walker et al. 2012).
References
Alloway, B.J. (2013). Heavy Metals in Soils. Trace Metals and Metalloids in Soils and their Bioavailability. Third Edition. Environmental Pollution, Volume 22, Springer, Dordrecht.
Depledge, M.H., Weeks, J.M., Bjerregaard, P. (1998). Heavy metals. In: Calow, P. (Ed.). Handbook of Ecotoxicology. Blackwell Science, Oxford, pp. 543-569.
Kabata-Pendias, A., Mukherjee, A.B. (2007). Trace Elements from Soil to Human. Springer Verlag, Berlin.
Newman, M.C. (2015). Fundamentals of Ecotoxicology. The Science of Pollution. Fourth Edition. CRC Press, Taylor & Francis Group. Boca Raton.
Nieboer, E., Richardson, D.H.S. (1990). The replacement of the nodescript term 'Heavy metals' by a biologically and chemically significant classification of metal ions. Environmental Pollution (Ser. B) 1, 3-26.
Walker, C.H., Hopkin, S.P., Sibly, R.M., Peakall, D.B. (2012). Principles of Ecotoxicology, Fourth Edition. CRC Press Taylor & Francis Group, London.
2.2. Question 1
Why can the term heavy metal not be used when referring to different elements considered metals?
2.2. Question 2
Why are some elements indicated as essential elements?
2.3. Question 3
Why is the classification of Nieboer and Richardson relevant for the biological interactions of metals with living organisms?
2.4. Question 4
Name at least 5 sources of metal emission to the environment.
2.5. Question 5
Besides leading to the emission of metals, metal mining may another important environmental effect. Which one?
in preparation
2.2.3. Industrial Chemicals
Authors: Steven Droge
Reviewer: Michael McLachlan
Leaning objectives:
You should be able to
• discuss a history perspective on key chemical legislations around the world
• look up registration dossiers yourself to obtain relevant ecotoxicological information
• realize that complete dossiers are most urgent for high production tonnage substances and the most hazardous substances
• understand why for some groups of chemicals already specific regulations were in place apart from common industrial substances.
Keywords: Chemical industry, tonnage, hazardous chemicals, REACH, regulation
Introduction
The chemical industry produces a wide variety of chemicals that find use in industrial process and as ingredients in day-to-day products for consumers. Instead of chemicals, 'substances' may be a more carefully worded description as it also includes complex mixtures, polymers and nanoparticles. Many substances are produced by globally distributed companies in very high volumes, ranging for example from 100 - 10,000 tonnes (1 tonne = 1000 kg) per year. Worldwide, governments have tried to control and assess chemical safety, as nicely summarized on the ChemHAT website. Australia for example, has the Industrial Chemicals (Notification and Assessment) Act 1989 (2013 version). Just like elsewhere in the world, in the European Union (EU) a variety of regulatory institutes at all levels of government used to perform safety assessments regarding the use of substances in products, and how these are emitted into waste streams. This changed dramatically in 2007.
On June 1st, 2007 (Figure 1), a new EU regulation went into force called REACH ). This law reversed the role of governments in chemical safety assessment, because it placed the burden of proof on companies that manufacture a chemical, import a chemical into the EU, or apply chemicals in their products. Within REACH companies must identify and manage the risks linked to the chemicals they manufacture and market in the EU. REACH stands for Registration, Evaluation, Authorisation and Restriction of Chemicals. China soon followed with the analogous "China REACH" in 2010, and then came South Korea in 2015 with "K-REACH". The main focus in this module is on EU-REACH as the leading and well documented example. Other legislation regulating industrial chemicals can often be easily found online, e.g. via the ChemHAT link above.
In REACH, each chemical is registered only once. Accordingly, companies must work together to prepare one dossier that demonstrates to the European Chemical Agency (ECHA) how chemicals can be safely used, and they must communicate the risk management measures to the users. ECHA, or any Member State, authorizes the dossiers, and can start a "restriction procedure" when they are concerned that a certain substance poses an unacceptable risk to human health or the environment. If the risks cannot be managed, authorities can restrict the use of substances in different ways. In the long run, the most hazardous substances should be substituted with less dangerous ones.
So which chemicals have been registered in the past decade (2008-2018) in REACH?
In principle, REACH applies to all chemical 'substances' in the EU zone. This includes metals, such as "iron" and "chromium", organic chemicals such as "methanol" and "fatty acids" and "ethyl-4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)piperidine-1-carboxylate (see Box 1)", and (nano)particles like "zink oxide" and "silicon dioxide", and polymers. Discover for example the registration dossier link in Box 1.
Box 1. Examples from the REACH dossiers
The REACH registration data base can be searched via LINK. Accept the disclaimer, and you are ready to search for chemicals based on name, CAS number, substance data, or use and exposure data.
Search for example for the name "ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)piperidine-1-carboxylate" and you find the link to the dossier of this substance with CAS 79794-75-5 as compiled by the registrant. This complex chemical name is better known as the antihistamine drug Loratadine, but this name does not show up in the dossier search!
Click on the name to get basic information on the compound. The hazard classification reads: "Warning! According to the classification provided by companies to ECHA in REACH registrations this substance is very toxic to aquatic life, is very toxic to aquatic life with long lasting effects, is suspected of causing cancer, causes serious eye irritation, is suspected of causing genetic defects, causes skin irritation, may cause an allergic skin reaction and may cause respiratory irritation." This compound is "PBT" labeled based on limited available data (classifying as a combination of Persistent / Bioaccumulative / Toxic). However, the section [About this substance] reads: "for industrial use resulting in the manufacture of another substance (use of intermediates)." As an intermediate in a restricted process, many parts of the dossier did not have to be completed for REACH. As a medicinal product Loratadine is strictly regulated elsewhere. Scroll down to the REACH link for the registration dossier (.../21649) to find out more the different entries for this chemical.
If we do a search for [" Bisphenol "], we get a long list of optional chemicals, for example Bisphenol A (CAS 80-05-7) but also for example Bisphenol S if you scroll down further (CAS 80-09-1). If we look at the dossier of the first Bisphenol A entry, with tonnage "100 000 - 1 000 000 tonnes per annum", you can find a long list of REACH information packages besides the dossier, as this chemical is hotly debated. The dossier for Bisphenol A was evaluated in 2013, and also this is available (look for the pdf in the Dossier evaluation status). In this compliance check, the registrant is requested to submit additional rat and mouse toxicity data, along with statements of reasons. There is for example also a link to the [Restriction list (annex XVII)], which leads to a pdf called 66.pdf, which states an adopted restriction for this chemical within the REACH framework and the previous legislation, Directive 76/769/EEC: "Shall not be placed on the market in thermal paper in a concentration equal to or greater than 0,02 % by weight after 2 January 2020".
Find your own chemical of interest to discover more on the transparancy of the chemical information on which risk assessment is based.
However, some groups of chemicals are (partly) exempt from REACH because they are covered by other legislation in the EU:
• Active substances used in plant protection products (Section 2.3.1) and biocidal products (Section 2.3.2) are considered as already having been registered and assessed by institutes separate from ECHA. Biocides such as disinfectants and pest control products are per definition hazardous chemicals, but they are also very useful in many ways. The very strict and elaborate biocide laws aim to verify that the potential risk of harm associated with the intended emission scenarios is in balance with expected benefits.
• Food and feedstuff additives (Section 2.3.9) have different legislation and authorisation laws to demonstrate (following a scientific evaluation) that the additive has no harmful effects on human and animal health or on the environment ( developed since 1988, schematic graph, Regulation (EC) No 1331/2008)
• Medicinal products (Sections 2.3.3 and 2.3.4) have different legislation and authorisation laws to guarantee high standards of quality and safety of medicinal products, while promoting the good functioning of the internal market with measures that encourage innovation and competiveness (starting with Directive 65/65 in 1965, an overview since, a pdf of the 2001 EU legislation 2001/83/EC)
• "Waste" is not part of the REACH domain, but a product recovered from waste is not.
A detailed overview of European chemical safety guidelines related to chemicals with different application types is presented in Figure 2 in Van Wezel et al. (2017).
Following pre-registration of the 145,297 chemicals most likely to require regulation, REACH came into force in 2008 in a stepwise process with different deadlines for different groups of chemicals. The first dossiers were to be completed by 2010 for the highest produced volume chemicals (>1000 tonnes/y) and the most hazardous chemicals (CMRs >1 tonne/y, and chemicals with known very high aquatic toxicity >100 tonnes/y). These groups potentially pose the greatest risk because of either their high emissions or their inherent toxicity. In 2013, registration dossiers for chemicals with a lower tonnage (100-1000 tonnes/y) were to be completed. By May 31 2018, all chemicals with a quantity of 1-100 tonnes/y chemicals on the EU market should have been registered. New chemicals will all be subject to the REACH procedures.
In 2018, 21.787 substances had been registered under REACH. A total of 14.262 companies were involved. In comparison, 15.500 substances were registered in 2016 (i.e., 6287 chemicals were added in the two following years). In 2018, 48% of all substance registrations had been done in Germany. For 24% of the registered substances a dossier was already available prior to REACH, 70% are "old chemicals" for which no registration had been done before REACH was initiated, and only 6% are newly developed substances that needed to be registered before manufacture or import could start.
There are multiple benefits of REACH regulation of industrial chemicals. Most data on chemicals entered in the registration process is publically available, creating transparency and improving customer awareness. If registered chemicals are classified as Substance of Very High Concern (SVHC) based on the chemical information in these dossiers and after agreement from research panels, alternatives that passed the same regulation can be suggested instead.
The necessity to add data on potential toxicity for so many chemicals has been combined with a strong focus on, and further development of, animal friendly testing methods. Read-across from related chemicals, weight of evidence approaches, and calculations based on chemical structures (QSAR) allow much experimental testing to be circumvented. In vitro studies are also used, but a 2017 REACH document (REACH alternatives to animal testing 2017, which followed up 2011 and 2014 reports) reports that 5.795 in vitro studies were used overall to determine endpoints for REACH, compared to 9,287 in vivo studies (ratio of 0.6). Clearly, many new animal tests have been performed under REACH to complete the dossiers on industrial chemicals. Prenatal developmental and repeated dose toxicity testing as well as extended one generation reproductive toxicity studies remain difficult to circumvent without animal use. However, the safe use of industrial chemicals must be ensured and demonstrated.
References:
Van Wezel, A.P., Ter Laak, T.L., Fischer, A., Bäuerlein, P.S., Munthee, J., Posthuma, L. (2017). Mitigation options for chemicals of emerging concern in surface waters; operationalising solutions-focused risk assessment . Environmental Science: Water Research 3, 403-414.
2.2.3. Question 1
Name three important advantages of the set-up of an international chemical legislation such as REACH.
2.2.3. Question 2
Name three important challenges, or even disadvantages, of the set-up of an international chemical legislation such as REACH.
2.2.3. Question 3
The total amount of chemical substances for which CAS numbers have been appointed, was 68,062,538 on June 28, 2019. The CAS REGISTRY is updated daily with thousands of new substances. Provide some reasons why only 21,787 substances have been registered for REACH in 2018.
2.2.4. POPs
(draft)
Authors: Jacob de Boer
Reviewer:
Leaning objectives:
You should be able to
• understand how POPs are defined
• recognize chemical structures of POPs
• gain knowledge on the purpose of the Stockholm Convention on POPs
Keywords: Persistence, bioaccumulation, long range transport, toxicity, analysis
Introduction
Chemicals are generally produced because they have a useful purpose. These purposes can vary widely, such as to protect crops by killing harmful insects or fungi, to protect materials against catching fire, to act as a medicine, to enable a proper packing of food materials, etc. Unfortunately, the properties which make a chemical attractive to use, often have a downside when it comes to environmental behavior and/or human health. A number of synthetic chemicals have properties that make them persistent organic pollutants (POPs). POPs are xenobiotic (foreign to the biosphere) chemicals that are persistent, bioaccumulative and toxic ('PBT') in low doses. In addition, they are transported over long distances. Criteria for these properties, which are used to define a chemical as a POP, were set by the United Nations (UN) Stockholm Convention, which was adopted in 2001 and entered into force in 2004 (Fiedler et al., 2019). These criteria are summarized in Table 1 (http://chm.pops.int). The objective of the Stockholm Convention is defined in article 1: "Mindful of the precautionary approach, to protect human health and the environment from the harmful impacts of persistent organic pollutants". Initially, 12 chemicals (aldrin, chlordane, dieldrin, DDT, endrin, heptachlor, hexachlorobenzene (HCB), mirex, polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and toxaphene) were listed as POPs. Gradually the list was extended with new POPs, which appeared to fulfil the criteria. For some of the new chemicals exceptions were made for limited use, in case no suitable alternatives are available. For example, in the battle against malaria DDT can still be used to a limited extent for in-house spraying in Africa (Van den Berg, 2009). Until now all POPs are chemicals that contain carbon and halogen atoms. Some POPs, such as the PCDDs and PCDFs (together often short-named as dioxins) are not intentionally produced. They are formed and released unintentionally during thermal processes. PCDDs and PCDFs tended to be released by waste incinerators (Karasek and Dickson, 1987). The combination of elevated temperatures and the presence of chlorine from e.g. polyvinylchloride (PVC) led to the formation of the extremely toxic PCDDs and PCDFs. Stack emissions could contaminate entire areas around the incinerators with consequences for the quality of cow milk or local crop. Dioxins were first discovered after the Seveso (Italy) disaster (1976) when high quantities of dioxins were released after an explosion in a trichlorophenol factory (Mocarelli et al., 1991). Meanwhile, in many countries incinerators have been improved by changing the processes and installing appropriate filters.
Table 1. Stockholm Convention criteria for persistence, bioaccumulation, toxicity and long range transport of POPs.
Persistence
i
Evidence that the half-life of the chemical in water is greater than two months, or that its half-life in soil is greater than six months, or that its half-life in sediment is greater than six months; or
ii
Evidence that the chemical is otherwise sufficiently persistent to justify its
consideration within the scope of this Convention
Bioaccumulation
i
Evidence that the bio-concentration factor or bio-accumulation factor in aquatic species for the chemical is greater than 5,000 or, in the absence of such data, that the log Kow is greater than 5
ii
Evidence that a chemical presents other reasons for concern, such as high bioaccumulation in other species, high toxicity or ecotoxicity; or
iii
Monitoring data in biota indicating that the bio-accumulation potential of the
chemical is sufficient to justify its consideration within the scope of this Convention
Long range transport potential
i
Measured levels of the chemical in locations distant from the sources of its release
that are of potential concern
ii
Monitoring data showing that long-range environmental transport of the chemical,
with the potential for transfer to a receiving environment, may have occurred via air,
water or migratory species; or
iii
Environmental fate properties and/or model results that demonstrate that the chemical has a potential for long-range environmental transport through air, water or migratory species, with the potential for transfer to a receiving environment in locations distant from the sources of its release. For a chemical that migrates significantly through the air, its half-life in air should be greater than two days
Adverse effects
i
Evidence of adverse effects to human health or to the environment that justifies consideration of the chemical within the scope of this Convention; or
ii
Toxicity or ecotoxicity data that indicate the potential for damage to human health or
to the environment
Structures and use
Whereas all initial POPs were chlorinated chemicals and mainly pesticides, POPs that were added at a later stage also included brominated and fluorinated compounds and chemicals with a more industrial application. Brominated diphenylethers (PBDEs) and hexabromocyclododecane (HBCD) belong to the group of brominated flame retardants. These chemicals are being produced in high quantities. Many national legislations require the use of flame retardants in many materials, such as electric and electronic systems (TV, cell phones, computers), furniture and building materials. Although the PBDEs and HBCD have been banned in most countries, other brominated flame retardants are still being produced in annually growing volumes.
Perfluorinated alkyl substances (PFASs) have many applications. Examples are Teflon production, use in fire-fighting foams, ski wax, as dirt and water repellant on outdoor clothes and carpets and many more. They are different from most other POPs because they are both lipophilic and hydrophilic due to a polar group present in most of the molecules. Examples of structures of a few POPs are given in Figure 1.
Persistence and bioaccumulation
The carbon-halogen bond is so strong that any type of degradation is unlikely to occur or will only occur on the long term and to a minor extent. Due to the size of the halogen atom, the strength of the halogen-carbon bond decreases from C-F to C-Cl, C-Br and C-I. In addition, these halogenated chemicals are lipophilic and, therefore, easily migrate to lipids, such as in living organisms. Because fish is a primary target, POPs enter the food chain in this way and biomagnification can occur (De Boer et al., 1998). High levels of POPs are, consequently, found in marine mammals (seals, whales, polar bears) and also in humans (Meironyte, 1999). Women may transfer a part of their POP load again to their children, the highest quantities to their firstborns.
Long range transport
Chemicals that migrate significantly through the air with a half-life in air greater than two days qualify for the POP criterion of long range transport. Many chemicals are indeed transported by air, often in different stages. Chemicals are emitted from a stack or evaporate from the soil in relatively warm areas and travel in the atmosphere toward cooler areas, condensing out again when the temperature drops. This process, repeated in 'hops', can carry them thousands of kilometers within days. This is called the 'grasshopper effect' (Gouin et al., 2004). It results in colder climate zones, in particular countries around the North Pole, receiving relatively high amounts of POPs.
Adverse environmental and health effects
There is very little doubt on the toxicity of POPs. Of course, the dose is always determining if a compound is causing an effect in the environment or in humans. POPs, however, are very toxic at very low doses. The Seveso disaster showed the high toxicity of dioxins for humans. Polybrominated biphenyls (PBBs) caused a high mortality in cattle when they were inadvertently fed with these chemicals (Fries and Kimbrough, 2008). Evidence of toxicity is often coming from laboratory studies with animals (in vivo) and more recently from in vitro studies. These studies are in particular important for the assessment of chronic toxicity. Many POPs are carcinogenic or act as endocrine disruptor.
Analysis
The analysis of POPs in environmental or human matrices is relatively complicated and costly. The compounds need to be isolated from the matrix by extraction. Subsequently, the extracts need to be cleaned from interfering compounds such as fat from organisms or sulphur in case of sediment or soil samples. Finally, due to the required sensitivity and selectivity, expensive instrumentation such as gas or liquid chromatography combined with mass spectrometry is needed for their analysis (Muir and Sverko, 2006). UN Environment is investing in large capacity building programs to train laboratories in developing countries in this type of analysis. According to the Stockholm Convention, countries shall manage stockpiles and wastes containing POPs in a manner protective of human health and the environment. POPs in wastes are not allowed to be reused or recycled. A global monitoring program has been installed to assess the effectiveness of the Convention.
Future
Much has to be done to achieve the original goals of eliminating the production and use of POPs and gradually reduce their spreading into the environment. A global treaty such as the Stockholm Convention with 182 countries involved is in a continuous challenge with procedures and political realities in countries, which hamper the achievement of perceived simple goals such as to eliminate the use of PCBs in 2025. The goals are, however, extremely important, as POPs are a global threat for current and future generations.
References
De Boer, J., Wester, P.G., Klamer, J.C., Lewis, W.E., Boon, J.P. (1998). Brominated flame retardants in sperm whales and other marine mammals - a new threat to ocean life? Nature 394, 28-29.
Fiedler, H., Kallenborn, R., de Boer, J., Sydnes, L.K. (2019). United Nations Environment Programme (UNEP): The Stockholm Convention - A Tool for the global regulation of persistent organic pollutants (POPs). Chem. Intern. 41, 4-11.
Fries, G.F., Kimbrough, R.D. (2008). The PBB episode in Michigan: An overall appraisal. CRC Critical Rev. Toxicol. 16, 105-156.
Gouin, T., Mackay, D., Jones, K.C., Harner, T., Meijer, S.N. (2004). Evidence for the "grasshopper" effect and fractionation during long-range atmospheric transport of organic contaminants. Environ. Pollut. 128, 139-148.
Karasek, F.W., Dickson, L.C. (1987). Model studies of polychlorinated dibenzo-p-dioxin formation during municipal refuse incineration. Science 237, 754-756.
Meironyte, D., Noren, K., Bergman, Å. (1999). Analysis of polybrominated diphenyl ethers in Swedish human milk. A time-related trend study, 1972-1997. J. Toxicol. Environ. Health Part A 58, 329-341.
Mocarelli, P., Needham, L.L., Marocchi, A., Patterson Jr., D.G., Brambilla, P., Gerthoux, P.M. (1991). Serum concentrations of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin and test results from selected residents of Seveso, Italy. J. Toxicol. Environ. Health 32, 357-366.
Muir, D.C.G., Sverko, E. (2006). Analytical methods for PCBs and organochlorine pesticides in environmental monitoring and surveillance: a critical appraisal. Anal. Bioanal. Chem. 386, 769-789.
Van den Berg H. (2009). Global status of DDT and its alternatives for use in vector control to prevent disease. Environ. Health Perspect. 117:1656-63.
2.2.4. Question 1
Which criteria are used by the Stockholm Convention to define chemicals as POPs?
2.2.4. Question 2
What are the objectives of the Stockholm Convention on POPs?
2.2.4. Question 3
How can dioxins be formed?
2.2.4. Question 4
Why is the analysis of POPs in environmental and human matrices expensive?
2.2.5. Persistent Mobile Organic Chemicals (PMOCs)
Authors: Pim de Voogt
Reviewers: John Parsons, Hans Peter Arp
Leaning objectives:
You should be able to:
• define a substance's persistence
• define a partition coefficient
• understand the relationship between KOW, DOW, KD and mobility
• understand the relationship between a substance's mobility and persistence on the one hand and its potential for human exposure on the other
Keywords: Mobility, persistence, PMT
Introduction
Ecosystems and humans are protected against exposure to hazardous substances in several ways. These include treating our wastewater so that substances are prevented from entering receiving surface waters, and purification of source waters intended for drinking water production.
Currently, a majority of the drinking water produced in Europe is either not treated or treated by conventional technologies. The latter remove substances by degradation (physical, microbiological) or by sorption. However, chemicals that are difficult to break down and that can pass through soil layers, water catchments and riverbanks and cross natural and technological barriers may eventually reach the tap water. Typically, these chemicals are persistent and mobile.
Polarity
When the electrons in a molecule are unevenly divided over its surface, this results in an asymmetric distribution of charge, with positive and negative regions. Such molecules have electric dipoles (see Figure 1) and are polar, in contrast to molecules where the charge is evenly distributed thus resulting in the molecule being neutral or apolar. The ultimate form of polarity is when a permanent charge is present in a compound. Such chemicals are called ionogenic. We distinguish between cations (having a permanent positive charge, e.g. protonated bases, and quaternary amines) and anions (negatively charged ions, e.g. dissociated acids, and organosulfates). Ionic charges in molecules can be pH dependent (e.g. acids and bases). Most, and in particular small, polar and ionic chemicals are water soluble, in other words they have a strong affinity to water (often referred to as hydrophilic). Because water is one of the most polar liquids possible (a strong negative charge on the oxygen and two strong positive charges on each hydrogen), this means that for very polar organic molecules solvation by water is more favorable energetically then sorption to solid particles.
Chemicals that are nonpolar are inherently poorly water soluble and therefore tend to escape from the water compartment, resulting in their evaporation, or sorption to sediments and soils, or uptake and accumulation in organisms. It is therefore relatively easy to remove them from water during water treatment. In contrast, mobile organic chemicals, especially those that do not breakdown easily, pose a more serious threat for (drinking) water quality because they are much more difficult to remove. It should be noted that mobility and polarity can be thought of as a gradient, rather than a distinct category, with water being the most polar molecule, a large aliphatic wax being the most non-polar molecule, and all other organic molecules falling somewhere in the spectrum between.
In a recent study contaminants were analysed in Dutch water samples covering the journey from WWTP effluent to surface water to groundwater and then to drinking water. While the concentration level of total organic contaminants decreased by about 2 orders of magnitude from the WWTP effluents to the groundwater used for drinking water production, the hydrophilic contaminants (using chromatographic retention time as an indicator for hydrophilicity) in the WWTP effluents remained in the water throughout its passage to groundwater and into the drinking water (see Figure 2).
Mobility and persistence
The mobility of chemicals in aquatic ecosystems is determined by their distribution between water and solid particles. The more the substance has an affinity for the solid phase the less mobile it will be. The distribution coefficient is known as KD, which expresses the ratio between the concentrations in the solid phase (soil, sediment, suspended particles), CS, and the dissolved phase at equilibrium, CW, i.e. KD = CS/CW. For neutral non-polar chemicals the distribution is almost entirely determined by the amount of organic carbon in the solid phase, fOC, and hence their distribution is usually expressed by KOC, the organic carbon-normalized KD (i.e. KOC = KD/ fOC). Unfortunately, there are relatively few reliable KD or KOC data available, in particular for polar chemicals. Instead, KOW is often used as a proxy for KOC. The n-octanol/water partition coefficient: KOW, is the equilibrium distribution coefficient of a chemical between n-octanol and water, KOW = Coctanol/Cwater. It's logarithmic value is often used as a proxy to express the polarity of a compound: a high log KOW means that the compound favors being in the octanol phase rather than in water, which is typically the case for a nonpolar compound. For ionizable chemicals we need to account for the pH dependency of KOW: at low pH an organic acid will become protonated (this in turn depends on its pKa value) and thus less polar. DOW is the pH-dependent KOW. It can be assumed that ions, whether cationic or anionic, will no longer dissolve into octanol but rather be retained in the water, because ions have much higher affinity for water than for octanol.
Accounting for this, for organic acids, the pH dependency of the DOW can be expressed as:
Therefore, as pH increases above the pKa, the smaller the DOW will get in the case of organic acids. In the case of basis, the opposite is true; the more the pH dips below the pKa of an organic base, the more cations form and the lower the Dow becomes.
However, one has to keep in mind that the assumption that the (log) KOW or DOW value inversely correlates with a compound's aquatic mobility is, certainly, very simplistic. The behavior of an ionic solute will obviously also be determined by interactions i) with sites other than organic carbon, e.g. ionizable or ionic sites on soil and sediment particles, and ii) with other ions in solution.
The persistence of a compound is assessed in experimental tests by monitoring the rate of disappearance of the compound from the most relevant compartment. This is often done using standardized test protocols. In the European REACH legislation of chemicals, criteria have been established to qualify chemicals as persistent (P) and "very persistent"(vP) based on the outcomes of such tests. Table 1 presents the P and vP criteria used. Unfortunately, good-quality experimental data on half-lives are rare and obtaining such data requires both time-consuming and expensive testing.
Currently there is no certified definition of a compound's mobility (M). Several possible compound properties have been proposed to characterize mobility, including a compound's aqueous solubility or its KOC value. If (experimental) KOC values are not available, DOW values can be used as a proxy.
Table 1. P and vP criteria identical to Annex XIII to the REACH regulation (source: ECHA chapter R.11. Version 3.0, June 2017)
Persistent (P) in any of the following situations
Very persistent (vP) in any of the following situations
Freshwater
Half-life > 40 days
Half-life > 60 days
Marine water
Half-life > 60 days
Half-life > 60 days
Freshwater sediment
Half-life > 120 days
Half-life > 180 days
Marine sediment
Half-life > 180 days
Half-life > 180 days
Soil
Half-life > 120 days
Half-life > 180 days
Table 2. Proposed cut off values of compound properties proposed by the German Environmental Agency (UBA) to define substance mobility*
Mobile (M) if compound is P or vP and any of the following situations
very Mobile (vM) if compound is P or vP and any of the following situations
Lowest experimental log KOC
(at pH 4-9)
≤4.0
≤3.0
Log DOW
(at pH 4-9)
≤4.0 if no experimental
Log KOC data available
≤3.0 if no experimental
Log KOC data available
* note that the proposed criteria may change by the time of publication
Regulation and gaps in knowledge
The majority of chemicals for which international guidelines exist or that are identified as priority pollutants in existing regulations (e.g. EU Water Framework Directive and REACH), are nonpolar with log DOW values mostly above two (see Figure 3b). The German Ministry of Environment (UBA) has recently proposed to develop regulation for chemicals with P, M and toxic (T) properties (PMT substances) analogous to the existing PBT criteria used for regulation of chemicals in the EU. UBA proposed to use cut-off values of the KOC or DOW (if KOC data are not available) to define Mobile (M) or very mobile (vM) in conjunction with persistence criteria (see Table 2). Note that the KOC and DOW values have to be obtained from testing at an environmentally relevant pH range (pH 4-9).
When we consider current analytical techniques used for monitoring contaminants in the environment, it can be readily seen that the scope of techniques most often used (gas chromatography, GC, and reversed-phase liquid chromatography, RPLC) do not overlap with what is required for chemicals having log DOW values typical of the most mobile chemicals, having a log Dow below zero (see Figure 3a). Consequently, there is limited information available on the occurrence and fate of these mobile chemicals in the environment. Nevertheless, some examples of persistent and mobile chemicals have been identified. These include highly polar pesticides and their transformation products, for instance glyphosate and aminomethylphosphonic acid (AMPA), short-chain perfluorinated carboxylates and sulfonates, quaternary ammonium chemicals such as diquat and paraquat and complexing agents such as EDTA. There are, however, likely to be many more chemicals that could be classified as PMOC and we can therefore conclude that there is a gap in the knowledge and regulation of persistent mobile organic chemicals.
References
Arp, H.P.H., Brown, T.N., Berger, U., Hale, S.E. (2017). Ranking REACH registered neutral, ionizable and ionic organic chemicals based on their aquatic persistency and mobility. Environmental Science: Processes Impacts 19, 939-955.
Reemtsma, T., Berger, U., Arp, H. P. H., Gallard, H., Knepper, T. P., Neumann, M., Benito Quintana, J., de Voogt, P. (2016). Mind the gap: persistent and mobile organic chemicals - water contaminants that slip through. Environmental Science & Technology 50, 10308-10315.
Sjerps, R.M.A., Vughs, D., van Leerdam, J.A., ter Laak, T.L., van Wezel, A.P. (2016). Data-driven prioritization of chemicals for various water types using suspect screening LC-HRMS. Water Research 93, 254-264.
2.2.5. Question 1
What is the reason that PMOCs may end up in drinking water despite the application of removal processes?
2.2.5. Question 2
Explain why the average hydrophilicity of compounds present in drinking water is higher than that of surface waters or wastewater from which is has been produced.
2.2.6. Ionogenic organic chemicals
Authors: Steven Droge
Reviewer: John Parsons, Satoshi Endo
Leaning objectives:
You should be able to:
• understand that IOCs are abundantly present in many types of chemical classes, but all share common features such as dissociation of a proton from a polar moiety (acids to form anions) and association of a proton onto a polar moiety (bases to form cations).
• calculate the fraction of neutral/ionic species for each chemical at a given pKa and pH.
• make some rough predictions about pKa values from the IOC molecular structure.
Keywords: pKa, dissociation constant, speciation, drugs, surfactants, solubility
Introduction
Ionogenic organic chemicals (IOCs) are widely used in industry and daily life, but also abundantly present as chemicals of emerging concern. For environmental risk assessment purposes, IOCs may be defined as organic acids, bases, and zwitterionic chemicals that under common environmental pH conditions exist for a large part as charged (ionic) species, with only modest fraction of neutral species. The environmentally relevant pH range could be argued to lie between 4 (acidic creeks, even lower in polluted streams from volcanic regions or mine drainage systems) to 9 (sewage treatment effluents). The environmental behaviour of IOC pollutants of concern are different from neutral chemicals of concern, because the aqueous pH controls the neutral fraction of dissolved IOCs and the ionic form is highly soluble and interacts partly via electrostatic interactions with environmental susbtrates. Note that the major fraction of an IOC can also be neutral in a certain environmental system, and in that case it is often the neutral form that dominates the chemical's behavior.
IOCs are common in many different types of pollutant classes. A random subset analysis of all EU (pre-)registered industrial chemicals indicated that large fractions of the total list of >100,000 chemicals are IOCs (51% neutral; 27% acids; 14% bases; 8% zwitterions/amphoterics). In another source, It has been estimated that >60% of all prescription drugs (Section 2.3.3) are IOCs (Manallack, 2007), and even higher fractions for illicit drugs (Section 2.3.4) (Figure 1). Well known examples are basic beta-blockers (e.g. propranolol), basic antidepressants (e.g. fluoxetine and sertraline), acidic non-steroidal anti-inflammatory drugs (NSAID such as diclofenac), and basic opioids (e.g. morphine, cocaine, heroin) and basic designer drugs (e.g. MDMA). The majority of surfactants and polyfluorinated chemicals (e.g. PFOS and GenX) are IOCs (Section 2.3.8), as well as wide variety of important pesticides (e.g. zwitterionic glyphosate) (Section 2.3.1) and (natural) toxins (Section 2.1) (e.g. peptide based multi-ionic cyanobacterial toxins).
Environmental behavior of IOCs
The release into the environment is specific for each of these types of IOCs with a different use, but in many cases happens via sewage treatment systems. If sorption to sewage sludge is very strong, application of sludge onto terrestrial (agricultural) systems is a key entry in many countries. However, many IOCs are rather hydrophilic and will mainly be present in wastewater effluent released into aquatic systems. As they are hydrophilic, they are considered rather mobile which allows for rapid transport through e.g. groundwater plumes, soil aquifers, and (drinking water) filtration steps. The distinction between (mostly) neutral chemicals and IOCs is important because the ionic molecular form generally behaves very differently from the corresponding neutral molecular form. For example, in many aspects ionic molecules are non-volatile compared to the corresponding neutral molecules, while neutral molecules are more hydrophobic than the corresponding ionic molecules. As a result of their lower "hydrophobicity", ionic molecules often bind with lower affinity to soils and are therefore more mobile in the environment. The ionic forms bioaccumulate to a lower extent and can therefore be less toxic than the corresponding neutral form (though not necessarily). However, there are various important exceptions for these rules. For example, clay minerals sorb cationic IOCs fairly strongly via ion exchange mechanisms. Certain proteins (e.g. the blood serum protein albumin) tightly bind anionic chemicals because of cationic subdomains on specific (enzymatic) pockets, which allows for effective transport throughout our systems and over cell membranes.
Calculating and predicting the dissociation constant (pK
The critical chemical parameter describing the ability to ionize is the acid dissociation constant (pKa). The pKa defines at which pH 50% of the IOC is in either the neutral or ionic form by releasing an H+ from the neutral molecule acids (AH to anion A-), or accepting an H+ onto the neutral molecule base (B to cation BH+). The equilibrium between neutral acid and dissociated form can thus be defined as:
[ HA ] ↔ [H+] + [A- ] (eq.1)
where the chemical's equilibrium speciation is defined as:
(eq.2)
which gives the pKa as:
(eq.3)
and as a function of pH, the ratio of the acid and anion is defined as:
for acids and for bases (eq.4)
Although the term pKb is also used to denote the base association constant, it is conventional we consider [BH+] as acid and use 'pKa' and other relationships for bases as well. The fraction of neutral species (fN) for simple IOCs (one acidic or basic site) can be readily calculated with a derivatization of the Henderson-Hasselbalch equation:
(eq.5)
in which α = 1 for bases, and -1 for acids.
in which α = 1 for bases, and -1 for acids. Using equation 5, Figure 2 presents a typical speciation profile for an acid (shown with pKa 5, so perhaps a carboxylic acid) and a base (shown with pKa 9, so perhaps a beta-blocker drug). Following the curve of equation 5, it is interesting to see some simple rules: if the pH is 1 unit lower than the pKa, the deprotonated species fraction is present at 10%. If the pH is 2 units lower than the pKa, the deminished species fraction is present at 1%, 3 units lower would give 0.1%, etc. From this, it is easy to make a good estimate for the protonation of a strong basic drug like MDMA (pKa reported 9.9-10.4) in blood (pH 7.4): with a maximum of 3 units lower pH, up to 99.9% of the MDMA will be in the protonated form, and only 0.1% neutral. For toxicological modeling studies, e.g. in terms of permeation through the blood-brain barrier membrane, this is highly relevant.
Boxes 1-3. Extended learning: calculating the dissociation constant for multiprotic chemicals:
see end of this module
Acidic IOCs:
For example, the painkiller (or non-steroidal anti-inflammatory drug, or NSAID) diclofenac is a carboxylic acid with a pKa of 4.1. This means that at pH 4.1, 50% of the dissolved diclofenac is in the dissociated (anionic) form (so, (1 - fN) from equation 5). At pH 5.1 (1 unit higher than the pKa) this is roughly 90% (90.91% to be more precise, but simply remembering 90% helps), at pH 6.1 (2 units higher than the pKa) this is 99%. This stepwise increase in 50-90-99% with each pH unit works for all acids, and for bases the other way round. Test for yourself that at physiological pH of 7.4 (e.g. in blood) diclofenac is calculated to be 99.95% anionic.
Many carboxylic acids have a pKa in the range of 4-5, but the neighboring molecular groups can affect the pKa. Particularly electronegative atoms such as chlorine, fluorine, or oxygen may lower the pKa, as they reduce the forces holding the dissociating proton to the oxygen atom. For example, trichloroacetic acid (CCl3-COOH) has a pKa of 0.77, while acetic acid (CH3-COOH) has a pKa of 4.75. For the same reason, perfluorinated carboxylic acids have a strongly reduced acidic pKa compared to the analogous non-perfluorinated carboxylic acids.
Sulfate acids (see figure 3) are very strong acids, with a pKa <0. These acids almost always occur in their pure form as a salt, for example the common soap ingredient sodium dodecylsulfate ("SDS" or "SLS", Na.C12H25-SO4). Other common detergents are sulfonates, such as linear alkylbenzenesulfonate ("LAS", C10-14-(benzyl-SO3)), where the SO3 anionic moiety is attached to a benzene ring, which can be positioned to different carbon atoms of a long alkyl chain. Even at the lower environmental pH range of about 4 these soap chemicals are fully in the anionic form. Such very strong acids, but also many weaker acids, are thus often sold in pure form as salts with sodium, potassium, or ammonium, which causes them to have different names and CAS numbers (e.g. Na.C12H25-SO4 or K.C12H25-SO4) than the neutral form.
Many phenols have a pKa > 8, and are therefore mostly neutral under environmental pH. Electron-withdrawing groups on the aromatic ring of the phenol group, such as Cl, Br and I, can lower the dissociation constant. For example, the dinitrophenol-based pesticide dinoseb has a pKa of 4.6, and is thus mostly anionic in the aquatic environment. Note that a hydroxyl group (-OH) not connected to an aromatic ring, such as -OH of alcohols, can in most cases for risk assessment be considered permanently neutral.
To help interpret the differences in pKa between molecules, it sometimes helps to remember that in more acidic solutions, there are simply higher H+ concentrations, in a logarithmic manner on the pH scale. At pH 3, the concentration H+ protons in solution is 1 mM, while at pH 9 the H+ concentration is 1 nM (6 pH units equals 106 times lower concentrations). The affinity ("Ka") of H+ to associate with a negatively charged molecular group, is so low for strong acids that even at very high dissolved H+ concentrations (low pH) only very few AH bonds (neutral acid fraction) are actually formed. In other words, for chemicals with a low Ka, even at low pH the neutral fraction is still low. For weak acids such as phenols, already at a very low dissolved H+ concentrations (high pH) many AH bonds (neutral acid fraction) are formed. So it can be reasoned that the affinity of common acidic groups to hold on to a proton is in the order:
SO4 < SO3 < CO2 < amide ( C(=O)NH ) < phenol < hydroxyl
Basic IOCs:
For bases, it is mostly a nitrogen atom that can accept a proton to form an organic cation, because of the lone electron pair in nitrogen. Neutral nitrogen atoms have opportunities for 3 bonds. A primary amine group has the nitrogen atom bonding to only 1 carbon atom (represented here as part of a molecular fragment R), and two additional bonds with hydrogen atoms. The remaining electron pair readily accepts another proton to form a cationic molecule [ R-NH3+ ]. Neutral secondary amines have one bond to hydrogen and two bonds to carbon atoms and can accept a proton to form [ R-NH2+-R' ], whereas neutral tertiary amines have no bonds to hydrogen but only to carbon atoms and can form [ R-NH+-(R')(R'') ]. Of course, each R group may be the same (e.g. a methyl unit).
Many basic chemicals have complex functionalities that can influence the pKa of the nitrogen moiety. However, as shown in the examples of figure 5, as long as there are at least two carbon atoms between the amine and the polar molecular fragment (for example OH, but much stronger for =O), the pKa of the basic nitrogen groups in all three types of bases (primary, secondary and tertiary amines) is high, often above 9 (dissolved H+ concentration <10-9). So even at very low H+ concentrations, dissolved protons like to be associated to such amine groups. As a result, amines such as most beta-blockers and amphetamine based drugs are predominantly positively charged molecules (organic cationic amines) in the common environmental pH range of 4-9, as well as in the pH of most biotic tissues that are useful for toxicological assessments. As soon as a polar group with oxygen (e.g. ketones or hydroxyl groups) is connected to the second carbon away from the nitrogen (e.g. R-CH(OH)-CH2-NH2) the pKa is considerably lowered. Also nitrogen atoms as part of an aromatic ring, or connected to an aromatic ring, have much lower pKa's: protons have rather low affinity to bind to these N-atoms and only start doing so if the proton concentration becomes relatively high (solution becomes more acidic).
Relevance of accounting for electrostatic interactions
Most classical pollutants, such as DDT, PCBs and dioxins, are neutral hydrophobic chemicals. On the other hand, most metals are almost always cationic species (e.g. Cd2+). Consequently, their environmental distribution and biological exposure are influenced by quite distinct processes. Obviously, predominantly charged IOCs behave somewhat in between these two extremes. The charged positive or negative groups cause a strong effect of electrostatic interactions between the IOC and environmental substrates (sorption or ligand/receptor binding). While also metals speciate into different forms, pH difference between environmental compartments can strongly influence the IOCs chemical fate and effect if ionizable group is relatively weak. An important difference to metals is that the nonionic molecular part still influences the IOC's hydrophobicity even in charged state for several processes.
As will be discussed in other chapters regarding chemical processes (see Chapter 3), it needs to be taken into account for IOCs that many environmental substrates (DOC, soil organic matter, clay minerals) are mostly negatively charged in the common range of environmental pH, but also that proteins involved in biotic uptake-distribution-effects are rich in ionogenic peptides that are part of binding pockets and reactive centers.
References
Manallack, D.T. (2007). The pKa distribution of drugs: application to drug discovery. Perspectives in Medical Chemistry 1, 25-38.
Box 1. Extended learning: calculating the dissociation constant for multiprotic chemicals:
Several common inorganic acids are multiprotic: they have multiple protons that can dissociate.
Multiples species can occur at a certain pH, such as for phosphoric acid (H3PO4, H2PO4-, HPO42- , HPO42-), and carbonic acid (H2CO3, HCO3-, CO32-). It is important to realize that there are actually two micro-species of HCO3-, because two hydroxylgroups can dissociate: HO-C(=O)-OH
A polyprotc acid HnA can undergo n dissociations to form n+1 species. Each dissociation has a pKa.
But how to calculate the fraction of each species of multiprotic chemicals?
The charge of a polyprotic acid can be described as Hn-jAj-. A useful variable, v, can be defined for each general polyprotic acid:
for each dissociation reaction:
Hn-j+1A-j+1 ↔ H+ + Hn-jAj-
the dissociation constant Kj is:
The degree of dissociation of the acid (η) is equal to the ratio of the total charge (TC) to the total mol acid (TM). For a diprotic acid, a plot of η as a function of pH provides the dissociation curve:
which can be a fitted curve to experimental data.
The degree of protolysis for the jth species, αj, can be calculated from the ratio of [Hn-jAj-]/TM
You can set up such a calculation in MS Excel, with calculations of α0, α1, α2, at a range of different pH values ([H+] concentrations), for a given K1 and K2, and plot the speciation against pH.
More details are described by King et al. (1990) J. Chem. Educ. 67 (11), p. 932; DOI: 10.1021/ed067p932
Box 2. Example 1 for multiprotic chemicals: Carbonic acid
Let's try carbonic acid (bicarbonate, or H2CO3) as an example first. H2CO3 is the product of carbon dioxide dissolved in water. In pure water/seawater the hydration equilibrium constant Kh = [H2CO3]/[CO2] ≈ 1.7×10−3 / ≈ 1.2×10−3 respectively, indicating that only 0.1% of dissolved CO2 equilibrates to H2CO3. The dissolved concentration of CO2 depends on atmospheric CO2 levels according to the air-water distribution coefficient (Henry constant kH = pCO2/[CO2]= 29.76 atm/(mol/L)). Because of the relevance of CO2 in e.g. ocean acidification, and gas exchange in our lungs, it is interesting to see how H2CO3 speciates depending on pH. As in the formula HnA, n= 2 for H2CO3.
With pK1*=6.5 (in equilibrium with atmospheric CO2) and pK2=10.33, K1=10-6.5 and K2=10-10.3.
At pH 7, [H+]=10-7 , so at pH 7 with these dissociation constants
or from a series of Excel calculations at different pH:
you can copy/paste the following cells into cell A1 of a new Excel sheet, and extend the range of pH:
K1
3.16E-07
K2
5.01E-11
pH
5
[H+]
=10^-B3
a0 = H2CO3
=(B4*B4)/((B4*B4)+(B4*\$B\$1)+(\$B\$1*\$B\$2))
a1 = HCO3-
=(B4*\$B\$1)/((B4*B4)+(B4*\$B\$1)+(\$B\$1*\$B\$2))
a2 = CO32-
=(\$B\$1*\$B\$2)/((B4*B4)+(B4*\$B\$1)+(\$B\$1*\$B\$2))
ETA
=((B4*\$B\$1)+2*(\$B\$1*\$B\$2))/((B4*B4)+(B4*\$B\$1)+(\$B\$1*\$B\$2))
Box 3. Example 2 for multiprotic chemicals: Zwitterions
Many organic pH buffers are zwitterionic chemicals, that contain both an acidic and a basic group. Norman Good and colleagues described a set of 20 of such buffers for biochemical and biological research (see for example www.interchim.fr/ft/0/062000.pdf, or www.applichem.com/fileadmin/Broschueren/BioBuffer.pdf). Examples are MES, MOPS, HEPPS (Figure A). These buffers are selected to:
• have a buffering pKa in the range of pH6-8 where most biochemical tests are performed;
• be readily soluble in water,
• be stable in test solutions, so resistant to (non)enzymatic degradation, not forming precipitates with salts
• ideally be impermeable to cell membranes so that they don't accumulate or reach active intracellular sites
• readily available, reasonably cheap.
the zwitterionic chemicals with sulfate groups are actually always have the sulfate group charged, making it highly soluble and impermeable to cell membranes, while the amine group protonates between pH6-10, depending on neighbouring functional groups. The speciation of the amine groups in MES and MOPS simply follows the single pKa calculation of equations 1-5. In HEPPS, either of the two amines is protonated, the second pKa is 3, so the doubly charged molecules only occurs at much lower pH, but can still be used as a buffer.
A zwitterionic chemical with two apparent pKa values relatively close is p-amino-benzoic acid. If chemicals have not one ionisable group, but N ionisable groups that speciation in a relevant pH range, than the amount of possible species is 2N. So the zwitterion p-amino benzoic acid has 4 species, each with a separate pKa (pH where both species are present in equal concentrations).
Let's formulate the benzyl group in p-amino benzoic acid as X, the neutral amine base as B, and the neutral carboxylic acid as AH, so that the fully neutral species is BXAH.
Compared to the carboxylic acid, we now have under the most acidic conditions (BHXAH)+, as , the neutral species BXAH and the zwitterionic intermediate (BHXA)0, as , and anionic species at most alkaline conditions (BXA)-, as . The calculation of the fraction of each species can be calculated according to similar rules as for carbonic acid if the two dissociation constants are known (K1 = 2.4, K2=4.88).
However, this does not inform us on the ratio between the zwitterionic form and the fully neutral form. To do this, the speciation constants of the 4 microspecies are required.
[BH+XAH] ↔ [BXAH] + [H+] for which the pk1 is calculated to be 2.72
K1=10^-2,72 = [BXAH]*[H+]/ [BH+XAH]
[BH+XAH]*10^-2.72 = [BXAH]*[H+]
which rearranges to [BXAH]= 10^-2.72 *[BH+XAH] /[ H+]
[BH+XAH] ↔ [BH+XA-] + [H+] for which the pk2 is calculated to be 3.93
K2=10^-3.93 = [BH+XA-]*[H+] / [BH+XAH]
[BH+XAH]*10^-3.93 = [BH+XA-]*[H+]
which rearranges to [BH+XA-] =10^-3.93*[BH+XAH]/[H+]
[BXAH] ↔ [BXA-] + [H+] for which the pk3 is calculated to be 4.74
K3= 10^-4,74 = ([BXA-] * [H+]) / [BXAH]
[BXAH]*10^-4,74 = [BXA-] * [H+])
[BH+XA-] ↔ [BXA-] + [H+] for which the pk4 is calculated to be 4.31
K4=10^-4,31 = [BXA-]*[H+] / [BH+XA-]
[BH+XA-]*10^-4,31 = [BXA-]*[H+]
So the ratio between zwitterionic form [BH+XA-] and neutral form [BXAH] equals to:
[BH+XA-] / [BXAH] = 10^-pK2 / 10^-pK1
[BH+XA-] / [BXAH] = 10^-3.93 / 10^-2.72 = 0.06: so only 6% zwitterionic vs 94% neutral species.
The macro pK1 and pK2 are then calculated as:
K1 = ([BXAH] *[H+] + [BH+XA-]*[H+] )/ [BH+AXH]
K1 = [BXAH] *[H+]/[BH+XAH] + [BH+XA-]*[H+] / [BH+XAH]
K1 = 10^-pK1 + 10^- pK2
K1 = 10^-2.72 + 10^-3.93 =10^-2.69
K1=10^-2.69
K2 = ([BXA-]) *[H+] / ( [BH+XA-] + [BXAH] )
1/K2 = [BH+XA-]/([BXA-]) *[H+] + [BXAH] /([BXA-]) *[H+]
1/K2 = 1/10^-pK3 + 1/10^-pK4
1/K2 = 1/10^-4,31 + 1/10^-4,74 = 1/10^-4.87
K2 =10^-4.87
2.2.6. Question 1
Calculcate the neutral fraction at pH7.4 for the illicit drug GHB (apply HH equation of eq 5):
2.2.6. Question 2
Connect the chemicals to their appropriate pKa values:
2.2.6. Question 3
Decide/look up whether these chemicals are predominantly neutral/anionic/cationic in the environment:
In preparation
2.2.8. Plastics
(draft)
Author: Ansje Löhr
Reviewer: John Parsons
Leaning objectives:
You should be able to:
• indicate the relevance of plastic for society
• describe the main characteristics of (micro)plastics
• describe the main ecological effects of (micro)plastics
Keywords: Plastic types, sources of plastics, primary and secondary microplastics, plastic degradation, effects of plastics
Introduction
Since its introduction in the 1950s, the amount of plastics in the environment has increased dramatically (Figure 1). A recent study by Jambeck et al. (2015) estimated that 192 coastal countries generated 275 million metric tonnes of plastic waste in 2010 of which around 8 million tons of land-based plastic waste ends up in the ocean every year. By UN Environment plastic pollution is seen as one of the largest environmental threats. If waste management does not change rapidly, another 33 billion tonnes of plastic will have accumulated around the planet by 2050. (Micro)plastics is widely recognized as a serious problem in the ocean, however, plastic pollution is also seen in terrestrial and freshwater systems.
Classification by size and morphology
Plastics are commonly divided into macroplastics and microplastics; the latter plastic particles are <5 mm in diameter (including nanoplastics). There are several ways to classify microplastics but the following two types are often used; primary microplastics and secondary microplastics. Primary microplastics have been made intentionally, like pellets or microbeads, secondary microplastics are fragmented parts of larger objects. Microplastics show a large variety in characteristics such as size, composition, weight, shape and color. These characteristics have an influence on the behaviour in the environment, like for instance, the dispersion in water and the uptake by organisms (Figure 2). Low-density particles float on water and are therefore more prone to advection than particles with a
higher density. Similarly, spheres are more likely to be taken up by organisms than fibers. The characteristics also affect the absorption of contaminants, adsorption of microbes, and potential toxicity.
Classification by chemistry
Plastic is the term used to define a sub-category of the larger class of materials called polymers, usually synthesized from fossil fuels, although biomass and plastic waste can also be used as feedstock. Polymers are very large molecules that have characteristically long chain-like molecular architecture. There are many different types of plastics but the market is dominated by 6 classes of polymers: polyethylene (PE, high and low density), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS, including expanded EPS), polyurethane (PUR) and polyethylene terephthalate (PET) (figure 3). In order to make materials flexible, transparent, durable, less flammable and long-lived, additives to polymers are used such as flame retardants (e.g. polybrominated diphenyl ethers), and plasticisers (e.g. phthalates). Some of these substances are known to be toxic to marine organisms and to humans.
Biopolymers/ bioplastics
There is a lot of discussion on bioplastics as degradable plastics that these may still persist for a long time under marine conditions. Please watch this video by dr. Peter Kershaw.
Plastic degradation
Degradation of plastics takes place as soon as the plastic loses its original integrity and properties. There is a faster breaking up phase (degradation into microparticles) and a much slower mineralization phase (polymer chains being degraded to carbon dioxide). The degradation rate of plastics is determined by its polymer type, additive composition and environmental factors. Many commonly-used polymers are extremely resistant to biodegradation. Although plastics degrade in natural environments it is argued that no polymer can be efficiently biodegraded in a landfill site. Plastics in aquatic environments can be subject to in-situ degradation, e.g. by photodegradation or mechanical fragmentation but are in general very durable. As a result, plastics that are present in our oceans will degrade at a very slow pace, (Figure 4). So the majority of plastics produced today will persist in the environment for decades and probably for centuries, if not millennia.
Plastics in the environment
• Sources and pathways:
Plastics are found in terrestrial, freshwater, estuarine, coastal and marine environments, and even in very remote areas of the world and the deep-sea. Sources and pathways of marine litter are diverse and exact quantities and routes are not fully known. But there is a surge in interest to determine the exact quantities and types of plastic litter and pathways in the environment and most of the plastic in our oceans originates from land-based sources (Figure 5) but also from sea-based sources. Most PE and PP is used in (single-use) packaging products that have a short lifetime and end up soon as waste.
Primary microplastics in terrestrial environments mostly originate from the use of sewage sludge containing microplastics from personal care or household products. In agricultural soils the application of sewage sludge from municipal wastewater treatment plants to farmland is probably a major input, based on recent MP emission estimates in industrialized countries. Plastic pollution in terrestrial systems is also linked to the use of agricultural plastics, such as polytunnels and plastic mulches. Secondary microplastics originate from varying and diverse sources, for example from mismanaged waste either accidentally or intentionally.
• Effects:
As plastics have become widespread and ubiquitous in the environment, they are present in a diversity of habitats and can impact organisms at different levels of biological organization, possibly leading to population, community and ecosystem effects. Entanglement is one of the most obvious and dramatic physical impacts of macroplastics, as it often leads to acute and chronic injury or death. In particular the higher taxa (mammals, reptiles, birds and fish) are affected, and it may be critical for the success of several endangered species. Because of similar size characteristics to food, plastics are both intentionally and unintentionally ingested by a wide range of species, such as invertebrates, fish, birds and mammals. Ingestion of the non-nutritional plastics can cause damage and/or obstruction of the digestive tract and may lead to decreased foraging due to false feelings of satiation, resulting in reduced energy reserves.
Microplastics and in particular nanoparticles that are small enough to be taken up and translocated into tissues, cells and body fluids can cause cellular toxicity and pathological changes due to particle toxicity. In addition, there are also chemical risks involved as plastics can be a source of hazardous chemicals. These chemicals can be part of the plastic itself (i.e. monomers and additives) and/or chemicals that are absorbed from the environment into the matrix or such as lead, cadmium, mercury, persistent organic pollutants (POPs) like PAHs, PCBs and dioxins. However, as this process depends on the fugacity gradients, there is a lot of uncertainty about the extent that transfer of pollutants does occur in the environment. Actually, when taking all exposure pathways into account, the transfer from (micro)plastics seems to be a minor pathway.
Watch the video on the research of Inneke Hantoro.
Finally, marine plastics may act as floating habitats for invasive species, including harmful algal blooms and pathogens, leading to spreading beyond their natural dispersal range and creating the risk of disrupting ecosystems of sensitive habitats.
2.2.9. Nanomaterials
Author: Martina Vijver
Reviewers: Kees van Gestel, Frank van Belleghem, Melanie Kah
Leaning objectives:
You should be able to:
• explain the differences between nanomaterials and soluble chemicals.
• describe nano-specific features and explain the difference between nanomaterials and particles with a larger size, as well as how they differ from molecules.
Keywords: Nanomaterials , emerging technologies, colloids, nanoscale, surface reactivity
Introduction
Engineering at the nanoscale (i.e. 10-9 m) brings the promise of radical technological development. Due to their unique properties, engineered nanomaterials (ENMs) have gained interest from industry and entered the global market. Potentials ascribed to nanotechnology are amongst all: stronger materials, more efficient carriers of energy, cleaner and more compact materials that allow for small yet complex products. Currently, nanomaterials are used in numerous products, although exact numbers are lacking. In 2014, the market was estimated to contain more than 13,000 nano-based products (Garner and Keller, 2014). There is a wide variety of products containing nanomaterials, ranging from sunscreens and paint, to textiles, medicines, electronics covering many sectors (Figure 1).
Nanomaterials:
The European Commission in 2011 adopted a new definition of 'nanomaterial' reading 'a natural, incidental or manufactured material containing particles, in an unbound state or as an agglomerate or as an aggregate and where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm-100 nm'.
Nanomaterials occur naturally, think of minuscule small fine dust, colloids in the water column, volcanic ash, carbon black and colloids known as ocean spray. In paints the features of the colloids are used to obtain the pigment colors. From the year 2000 on, an exponential growth was seen in their synthesis due to the advanced technologies and imaging techniques needed to work on a nano-scale. First generation nanotechnologies (before 2005) generally refers to nanotechnology already on the market, either as individual nanomaterials, or as nanoparticles incorporated into other materials, such as films or composites. Surface engineering has opened the doors to the development of second and third generation ENMs. Second generation nanotechnologies (2005-2010) are characterised by nanoscale elements that serve as the functional structure, such as electronics featuring individual nanowires. From 2010 onward there has been more research and development of third generation nanotechnologies, which are characterised by their multi-scale architecture (i.e. involving macro-, meso-, micro- and nano-scales together) and three-dimensionality, for applications like biosensors or drug-delivery technologies modelled on biological templates. Self-assembling bottom-up techniques have been widely developed at industrial scale, to create, manipulate and integrate nanophases into more complex nanomaterials with new or improved technological features. Post 2015, the fourth generation ENMs are anticipated to utilise 'molecular manufacturing': achieving multi-functionality and control of function at a molecular level. Nowadays, virtually any material can be made on the nanoscale.
Size does matter
Nanoscale materials have far larger surface areas than larger objects with similar masses. A simple thought experiment shows why nanoparticles have phenomenally high surface areas.
A solid cube of a material 1 cm on a side has 6 cm2 of surface area, about equal to one side of half a stick of gum. When the 1 cm3 is filled with micrometer-sized cubes - a trillion (1012) of them, each with a surface area of 6 square micrometers - the total surface area amounts to 6 m2.. As surface area per mass of a material increases, a greater proportion of the material comes into contact with surrounding materials (Figure 2). Small particles also give that there is a high proportion of surface atoms, high surface energy, spatial confinement and reduced imperfections (Figure 2). It results in the fact that ENMs are having an enlarged reactivity. compared to larger "bulk" materials. For instance, ENMs have the potency to transfer concentrated medication across the cell membranes of targeted tissues. By engineering nanomaterials, these properties can be harnessed to make valuable new products or processes.
ENMs are often designed to accomplish a particular purpose, taking advantage of the fact that materials at the nanoscale have different properties than their larger-scale counterparts.
ENMs and environmental processes
ENMs are described as a population of particles, and quantified by the particle size distribution (PSD). Nonetheless often a single value (e.g. average ± standard deviation) is reported and not the full PSD. When the particles are suspended in an exposure medium, the size distribution of the NPs is changing over time. After being emitted into aquatic environments, NPs are subject to a series of environmental processes. These processes include dissolution and aggregation (see Figure 3) and subsequent sedimentation. It is known that the behavior and fate of NPs are highly dependent on the water chemistry. In particular, environmental parameters like pH, concentration and type of salts (especially divalent cations) and natural organic matter (NOM) can strongly influence the behaviour of NPs in the environment. For example, pH can affect the aggregation and dissolution of metallic NPs by influencing the surface potential of the NPs (von der Kammer et al., 2010). The divalent cations Ca2+ and Mg2+ are able to efficiently compress the electrical double-layer of NPs and consequently enhance homo-aggregation and hetero-aggregation of NP (see Figure 2) and the cations will be related to bridging the electrostatic interactions. In surface water, aggregation processes most often lead to sedimentation and sometimes to floating aggregates (depending on the density).
Coating of ENMs will change the dynamics of these processes. As a result of these nano-specific features, ENMs form a suspension which is different from chemicals that dissolve and form a solution. These ENMs in suspension then follow different environmental fate and behaviour compared to solutions. For this reason the way the dosage of ENPs should be expressed is highly debated within the nano-safety community. Should this be on a mass basis, as is the case for molecules of conventional chemicals (e.g. mg/L), or is the particle number the preferred dose metric such as in colloidal science (e.g. number of particles/L, or relative surface-volume ratio) or multi-mixed dosimetry expression. How to express the dose for nanomaterials is a quest still debated within the scientific community (Verschoor et al., 2019).
Classification of NMs
Although we learned from the text above that changing the form of a nanomaterial can produce a material with new properties (i.e. a new nanomaterial); often a group of materials developed is named after the main chemical component of the ENMs (e.g. nanoTiO2) that is available in different (nano)forms. Approaches to group ENMs have been presented below:
• Classification by dimensionality / shape / morphology:
Shape-based classification is related to defining nanomaterials, and has been synopsized in the ISO terminology.
• Classification by composition / chemistry:
This approach groups nanomaterials based on their chemical properties.
• Classification by complexity / functionality:
The nanomaterials that are in routine use in products currently are likely to be displaced by nanomaterials designed to have multiple functionalities, so called 2nd-4th generation nano-materials.
• Classification by biointerface:
A proposal relates to the hypothesis that nanomaterials acquire a biological identity upon contact with biofluids and living entities. Systems biology approaches will help identify the key impacts and nanoparticle interaction networks.
References
Garner, K.L., Keller, A.A. (2014). Emerging patterns for engineered nanomaterials in the environment: a review of fate and toxicity studies. Journal of Nanoparticle Research 16: 2503.
Nowack, B., Bucheli, T.D. (2007). Occurrence, behavior and effects of nanoparticles in the environment. Environmental Pollution 150, 5-22.
Verschoor, A.J., Harper, S., Delmaar, C.J.E., Park, M.V.D.Z., Sips, A.J.A.M., Vijver, M.G., Peijnenburg, W.J.G.M. (2019). Systematic selection of a dose metric for metal-based nanoparticles. NanoImpact 13, 70-75.
Von der Kammer, F., Ottofuelling, S., Hofmann, T. (2010). Assessment of the physico-chemical behavior of titanium dioxide nanoparticles in aquatic environments using multi-dimensional parameter testing. Environmental Pollution 158, 3472-3481.
2.2.9. Question 1
What gave us (as mankind) the capacity to virtually synthesize every material at the nanoscale?
2.2.9. Question 2
Explain the statement 'size does matter' for reactivity, use within your explanation the relationship between particle size and the surface area.
2.2.9. Question 3
Give an example of a nano-specific property - which is not size - that enhances reactivity of a material. | textbooks/chem/Environmental_Chemistry/Environmental_Toxicology_(van_Gestel_et_al.)/02%3A_Environmental_Chemistry_Chemicals/2.02%3A_Pollutants_with_specific_properties.txt |
2.3. Pollutants with specific use
2.3.1. Crop Protection Products
Author: Kees van Gestel
Reviewers: Steven Droge, Peter Dohmen
Leaning objectives:
You should be able to:
• describe the role of crop protection products in agriculture
• mention different types of pesticides and their different target groups.
• distinguish and mention important chemical groups of pesticides
- related to the chemistry
- related to the mode of action
• describe major components included in a commercial formulation of a crop protection product beside the active substance(s).
Keywords: Insecticides, Herbicides, Fungicides, Active substances, Formulations
Introduction
Crop protection products are used in agriculture. The principle target of agriculture is the provision of food. For this purpose, agriculture aims to reduce the competition by other (non-crop) plants and the loss of crop due to herbivores or diseases. An important tool to achieve this is the use of chemicals, such as crop protection products (CPP). Accordingly, CPPs are intentionally introduced into the environment and represent one of the largest sources of xenobiotic chemicals in the environment. These chemicals are by definition effective against the target organism, often already at fairly low doses, but may also be toxic to non-target organisms including humans. The use of pesticides, also named Crop Protection Products (CPP) or often also Plant Protection Products (PPP, the latter term may be misleading for herbicides which are intended to reduce all but the crop plants), is therefore strictly regulated in most countries. The main pesticides used in the largest volumes world-wide are herbicide all s, insecticides, and fungicides. As shown in Table 1, pesticides are used against a large number of diseases and plagues.
Table 1. Classification of pesticides according to what they are supposed to control
Pesticide type
Target
acaricides
against mites and spiders (incl. miticides)
algicides
against algae
althelmintics (vermicides)
against parasites
antibiotics
against bacteria and viruses (incl. bactericides)
bactericides
against bacteria
fungicides
against fungi
herbicides
against weeds
insecticides
against insects
miticides
against mites
molluscicides
against slugs and snails
nematicides
against nematodes
plant growth regulators
retard or accelerate the growth of plants
repellents
drive pests (e.g. insects, birds) away
rodenticides
against rodents
Formulations
A pesticidal product usually consists of one or more active substances, that are brought onto the market in a commercial formulation (spray powder, granulate, liquid product etc.). The formulation is used to facilitate practical handling and application of the chemical, but also to enhance its effect or its safety of use. The active substance may, for instance, be a solid chemical, while application requires it to be sprayed. Or the active substance degrades fast under the influence of sunlight and therefore has to be encapsulated. One of the most used types of formulation is a concentrated emulsion, which may be sprayed directly after dilution with water. In this formulation, the active substance is dissolved in an oily matrix and a detergent is added as emulsifier to make the oil miscible with water. In this way, the active substance becomes quickly available after spraying. In so-called slow-release formulations, the active substance is encapsulated in permeable microcapsules, from which it is slowly released. Another component of a formulation can be a synergist, which increases the efficacy of the active substance, for instance by blocking enzymes that metabolize the active substance. Here an overview of main formulation constituents:
• Solvents: to ease handling and application of the active substances, they are usually dissolved. For a highly water soluble compound this solvent may just be water; however, most compounds have low water solubility and they are thus dissolved in organic solvents.
• Emulsifier, detergents, dispersants: used to provide a homogeneous mixture of the active substance in the aqueous spray solution.
• Carrier: solid formulations, such as wettable granules (WG). All wettable powder (WP) formulations often use inert materials such as clay (kaolinite) as carrier.
• Wetting agent: they help providing a homogeneous film on the plant surface.
• Adjuvant: may help to increase uptake of the active substance into the plant.
• Minor constituents:
• Antifreeze agent: to keep the formulation stable also in cold storage conditions.
• Antifoam agent: some of the formulants may result in foaming during application, which is not wanted.
• Preservative, biocide: to prevent biological degradation of the active substance or the formulants during storage.
• There are numerous additional specific additional constituents for specific purposes such as colours, detterents, stickers, etc
Four types of nomenclature are used in case of pesticides:
1. The trade name, e.g. Calypso®, which is given by the manufacturer. The same active substance is often sold under more than one different trade names (accordingly, the use of trade names only is not a sufficient description of the test substance in scientific literature).
2. The code name, which is the "common" name of the active substance. Calypso® 480 SC, for example, is a concentrated suspension containing 480 g/L of the active substance thiacloprid.
3. The chemical name of the active substance. Thiacloprid is [3-[(6-chloropyridin-3-yl)methyl]-1,3-thiazolidin-2-ylidene]cyanamide.
4. The name of the chemical group to which the active substance belongs, in case of thiacloprid: neonicotinoids.
Chemical classes
Pesticides represent quite a number of different groups of chemicals. Pesticides include inorganic chemicals (like copper used as a fungicide), organic synthetic chemicals, and biologicals (organic natural compounds). Pesticides from the same chemical group may be used against different pest organisms, like the organotin compounds (see below). Some chemicals have a broad mode of action: many soil disinfectants, such as metam-sodium, kill nematodes, fungi, soil insects and weeds. Other pesticides are more selective, like neonicotinoids acting only on insects, or very selective, like the insect-growth regulator fenoxycarb, which is used against leaf-rollers without affecting its natural enemies. Selectivity of a pesticide also indicates to what extent non-target species may be affected upon its application (side-effects). Integrated pest management (IPM) aims at an as sustainable as possible crop protection system by combining biological agents (predators of the pest organism) using chemicals having a selective mode of action. Such systems are nowadays receiving increasing interest in different agricultural crops.
Some groups of pesticides that were used or still are widely used are presented in more detail. Their modes of action are discussed in Chapter 4.
• Chlorinated hydrocarbons
Best known representative of this group is DDT (dichloro diphenyl trichloroethane; Figure 1), which was discovered in 1939 by the Swiss entomologist Paul Hermann Müller and seemed to be an ideal pesticide: it was effective, cheap and easy to produce and remained active for a long period of time. As a remedy against Malaria and other insect borne diseases, it has saved millions of human lives. However, the high persistency of DDT, its strong bioaccumulation and its effects on bird populations have triggered the search for alternatives and its ban in most Western countries. But in some developing countries, because of a lack of suitable alternatives for an effective control of malaria, DDT is still in use to kill malaria mosquitos.
Other representatives of chlorinated hydrocarbons are lindane, also called gamma-hexachlorocyclohexane (Figure 1), and the cyclodienes that include the "drins" (aldrin, dieldrin, endrin, See Section 2.1) and endosulfan (Figure 1). Because of their high persistence and bioaccumulative potential, most organochlorinated pesticides have been banned.
Volatile halogenated hydrocarbons were often used as soil disinfectant. These compounds were injected into the soil, and acted as a nematicide but also killed fungi, soil insects and weeds. An example is 1,3-dichloropropene (Figure 1).
• Organophosphates
Organophosphates are esters of phosphoric acid and constitute important biological molecules such as nucleic acids (DNA) or ATP. Within the contents of pesticides this refers mainly to a group of organophosphate molecules which interfere with acetylcholinesterase. Nerve gases, produced for chemical warfare (e.g., Sarin), also belong to the organophosphates. They are much less persistent and were therefore introduced as alternatives for the chlorinated hydrocarbons. The common molecular structure of organophosphates is a tri-ester of phosphate, phosphonate, phosphorthionate, phosphorthiolate, phosphordithionate or phosphoramidate (Figure 2). With two of the three ester bonds, a methyl- or ethyl- group is bound to the P atom, while the third ester bond binds the rest group or "leaving group".
Dependent on the identity of the latter group, three sub-groups may be distinguished:
1. Aliphatic organophosphates, including malathion (Figure 3) and a number of systemic chemicals.
2. Phenyl-organophosphates, which are more stable than the aliphatic ones but also less soluble in water, like parathion (no longer allowed in Europe; Figure 3).
3. Heterocyclic organophosphates, including chemicals with an aromatic ring containing a nitrogen atom like chlorpyrifos (Figure 3).
• Carbamates
Where organophosphates are derived from phosphoric acid, carbamates are derived from carbamate (Figure 4). Their mode of action is similar to that of the organophosphates. The use of older representatives of this group, like aldicarb, carbaryl, carbofuran and propoxur, is no longer allowed in Europe, but diethofencarb (Figure 4), oxamyl and methomyl are still in use.
• Pyrethroids
A number of modern pesticides are derived from natural products. Pyrethroids are based on pyrethrum, a natural insecticide from flowers of the Persian ox-eyed daisy, Chrysanthemum roseum. Typical for the molecular structure of pyrethroids is the cyclopropane-carboxyl group (the triangular structure), which is connected with an aromatic group through an ester bond (Figure 5). Pyrethrum is rapidly degraded under the influence of sunlight. Synthetic pyrethroids, which are much more stable and therefore used on a large scale against many different insects, include cypermethrin (Figure 5), deltamethrin, lambda-cyhalothrin, fluvalinate and esfenvalerate.
Neonicotinoids
Based on the natural compound nicotine, which acts as a natural insecticide against plant herbivores, but which was banned as an insecticide due to its high human toxicity, in the 1980s a new group of more specific insecticides has been developed, the neonicotinoids (Figure 6). Several neonicotinoids (e.g., imidacloprid, thiamethoxam) are systemic. This means that they are taken up by the plant and exert their effect from inside the plant, either on the pest organism (systemic fungicides or insecticides) or on the plant itself (systemic herbicides). The systemic neonicotinoids are widely applied as seed dressing in major crops like maize and sunflower. Other compounds are mainly used in spray applications, e.g. in fruit growing (thiacloprid, acetamiprid, etc.). Although neonicotinoids are more selective and therefore preferred over the older classes of insecticides like organophosphates, carbamates and pyrethroids, in recent years they have become under debate because of their side effects on honey bees and other pollinators.
• Isothiocyanates
Isothiocyanates were used on a large scale as soil disinfectant against nematodes, fungi and weeds. The large number of chemicals with different chemical origin belonging to the isothiocyanates have in common that they form isothiocyanate in soil. A representative of this group is metam-sodium (Figure 7).
• Organotin compounds
Fentin hydroxide (Figure 8) was used as a fungicide against Phytophthora (causing potato -disease). Tributyltin compounds (TBT) were used as anti-fouling agent (algicide) on ships. TBTC (tributyltin chloride) is extremely toxic to shell-fish, such as oysters, and for this reason banned in many countries. Fenbutatin-oxide was used as an acaricide against spider mites on fruit trees (tributyltin chloride).
• Ryanoids
Also indicated as diamide insecticides, this group includes chemically distinct synthetic compounds such as chlorantraniliprole (Figure 9), flubendiamide, and cyantraniliprole, that act on the ryanodine receptor and are used against chewing and sucking insects.
• Phenoxy acetic acids
Phenoxy acetic acids are systemic herbicides, exerting their action after uptake by the leaf and translocation throughout the plant. Especially plants with broad, horizontally oriented leaves are sensitive for these herbicides. 2,4-D (Figure 10) is the best known representative of this group.
• Triazines
Triazines are heterocyclic nitrogen compounds, whose structure is characterized by an aromatic ring in which three carbon atoms have been replaced by nitrogen atoms. Triazines are usually applied to the soil before seed germination. The use of several compounds (atrazine, simazine) has been banned in Europe, while others like metribuzin and terbuthylazine (Figure 11) are still in use.
• Bipiridyls
This group contains the herbicides diquat and paraquat (Figure 12) which mainly act as contact herbicides. This means they damage the plant without being taken up. In soil, they are rapidly inactivated by strong binding to soil particles. The use of paraquat is no longer allowed in Europe, but diquat is still in use.
• Glyphosate and Glufosinate
As an alternative to the above mentioned herbicides, glyphosate and later glufosinate were developed. These are systemic broad-spectrum herbicides with a relatively simple chemical structure (Figure 13). Their low toxicity to other organisms triggered pesticide producers to introduce genetically modified crops (e.g. soybean, maize, oilseed rape, and cotton) that contain incorporated genes for resistance against these broad-spectrum herbicides. This type of resistance allows the farmer to use the herbicide without damaging the crop. For this reason, environmentalist fear an unrestricted use of these herbicides, which indeed is the case especially for glyphosate (better known under the formulation name Roundup®).
• Triazoles
Several modern fungicides are sharing a triazole group (Figure 14). These fungicides have gained importance because of problems with the resistance of fungi against other classes of fungicides. Members of this group for instance are epoxiconazole, propiconazole and tebuconazole.
• Biological pesticides
Biological pesticides are produced in living organisms as secondary metabolites to protect themselves against predators, herbivores, parasites or competition. They can be highly effective and act at low concentrations (high toxicity), but in contrast to some synthetic pesticides they are usually sufficiently biodegradable. Compounds like pyrethrum or strobilurin are produced within the plant or within the fungus and are thus protected against photolysis or other environmental degradation. Furthermore, the living organism can produce additional quantities of the secondary metabolite on demand. When used as a pesticide applied as a spray, however, the molecule needs to be modified to enhance its stability (for example against photolysis) to remain sufficiently active over a sufficient period of time. Accordingly, synthetic derivatives of these biological molecules are often more stable, less biodegradable. Examples are the Bt insecticide, which contains an endotoxin highly toxic to insects produced by the bacterium Bacillus thuringiensis, and avermectins, complex molecules synthesized by the bacterium Streptomyces avermitilis. Avermectins act as insecticides, acaricides and have anthelminthic properties. In nature, eight different forms of avermectin have been found. Ivermectin is a slightly modified structure that is synthesized and marketed commercially. Other compounds belonging to this group are milbemectin and emamectin.
Genetically modified plants containing a gene coding for the toxin produced by the bacterium Bacillus thuringiensis (or Bt) are another example of genetic modification being applied in agriculture produce insect-resistant crops.
References:
EU Pesticides Database
2.3.1. Question 1
Systemic pesticides are easily taken up by plants and internally distributed over all plant tissues. What do you expect regarding the water solubility of systemic pesticides?
1. Very Low
2. Very high
2.3.1. Question 2
Why are systemic insecticides applied as seed dressing, so dosed into the soil, dangerous for honey bees and other pollinators?
2.3.1. Question 3
A pesticide with an active substance that is hardly soluble in water is introduced to the market in a commercial formulation. What components should be present in the formulation to allow homogenous application of the pesticide by spraying?
2.3.1. Question 4
Name three important groups of insecticides and mention one typical property or characteristic.
2.3.1. Question 5
1. The figures shows three chemical structures. Indicate which of these chemicals is
• An organophosphate (circle)
• A pyrethroid (triangel)
• A neonicotinoid (square)
Aantal antwoorden: 3
2.3.2. Biocides
Author: Thomas Wagner
Reviewers: Steven Droge, Kevin Thomas
Learning objectives:
You should be able to:
• Understand the purpose of using biocides
• Distinguish different groups of biocides
• Understand the legislation concerning the production and use of biocides
• Understand the potential impact of biocides on ecosystems
Keywords: Biocides, product types, Biocidal Product Regulation (BPR), environmental impact
Introduction
European legislation describes a biocide as 'chemical substance or microorganism intended to destroy, deter, render harmless, or exert a controlling effect on any harmful organism by chemical or biological means'. The US Environmental Protection Agency (EPA), an independent agency of the U.S. federal government to protect the environment, defines biocides as 'a diverse group of poisonous substances including preservatives, insecticides, disinfectants and pesticides used for the control of organisms that are harmful to human or animal health or that cause damage to natural or manufactured products'. The definition by the EPA includes pesticides (Chapter 2.3.1). In the scientific and non-scientific literature, the distinction between biocides, pesticides and plant protection products is often vague.
Biocides are used all around us:
• The toothpaste that you used this morning contains biocides to preserve the toothpaste
• The water that you used to flush you mouth is prepared with biocides for disinfection
• The clothes that you are wearing are impregnated with biocides to prevent smells
• The food that you ate for breakfast might have contained biocides to preserve the food
• The construction materials around you have surface coatings that contain biocides to prevent biological degradation of the material
A biocide contains an 'active substance', which is the chemical that is toxic to its target organism, and often contain 'non-active co-substances', which could help in reaching desired product parameters, such as a viscosity, pH, colour, odour or increase its handling or effectiveness. The combination of active substances and non-active substances together makes up the 'biocidal product'. An example of a well-known biocidal product is TriChlor, which contains active substance chlorine that is used to disinfect swimming pools. Because it is impractical to store chlorine gas for the treatment of swimming pools, TriChlor tablets are added to the pool water. TriChlor is trichloroisocyanuric acid (Figure 1). When dissolved in water, the Cl atoms are replaced by H atoms, forming chlorine (Cl-) and cyanuric acid (Figure 2). The free chlorine is able to disinfect the swimming pool.
A biocidal product can also contain multiple biologically active substances to enhance its effectivity, such as AQUCAR™ 742 produced by DuPont. It contains glutaraldehyde (Figure 3) and quaternary ammonium compounds (Figure 4) that have a synergistic toxic effect on microorganisms that are present in oilfields and could form biofilms in the pipelines.
Product types
The biocidal products are classified into 22 different product-types by the European Chemicals Agency (ECHA) (Table 1). It is possible that an active substance can be classified in more than one product types.
Table 1. The classification of biocides in 22 product types (www.echa.europe.eu)
Main group 1: Disinfectants and general biocidal products
Product type 1 - Human hygiene biocidal products
Product type 2 - Private area and public health area disinfectants and other biocidal products
Product-type 3 - Veterinary hygiene biocidal products
Product type 4 - Food and feed area disinfectants
Product-type 5 - Drinking water disinfectants
Main group 2: Preservatives
Product-type 6 - In-can preservatives
Product-type 7 - Film preservatives
Product-type 8 - Wood preservatives
Product-type 9 - Fibre, leather, rubber and polymerised materials preservatives
Product-type 10 - Masonry preservatives
Product-type 11 - Preservatives for liquid-cooling and processing systems
Product-type 12 - Slimicides
Product-type 13 - Metalworking-fluid preservatives
Main group 3: Pest control
Product-type 14 - Rodenticides
Product-type 15 - Avidicides
Product-type 16 - Molluscicides
Product-type 17 - Piscicides
Product-type 18 - Insecticides, acaricides and products to control other arthropods
Product-type 19 - Repellents and attractants
Product-type 20 - Control of other vertebrates
Main group 4: Other biocidal products
Product-type 21 - Antifouling products
Product-type 22 - Embalming and taxidermist fluids
Legislation
In Europe, biocides are authorised for production and use by the Biocidal Products Regulation (BPR, Regulation (EU) 528/2012) of the ECHA. The BPR 'aims to improve the functioning of the biocidal product market in the EU, while ensuring a high level of protection for humans and the environment.' (echa.europe.eu/legislation). This is an alternative regulatory framework than that for the plant protection products, managed by the European Food Safety Authority (EFSA). All biocidal products go through an extensive authorisation process before they are allowed on the market. The assessment of a new active starts with the evaluation of a product by the authorities of an ECHA member state, after which the ECHA Biocidal Products Committee forms an opinion. The European Commission then makes a decision to approve or reject the new active substance based on the opinion of ECHA. This approval is granted for a maximum of 10 years and needs to be renewed after it reaches the end of the registration period. The BPR has strict criteria for new active substances, and meeting the following 'exclusion criteria' will result in the new active substance not being approved:
• Carcinogens, mutagens and reprotoxic substances categories 1A or 1B according to CLP regulation
• Endocrine disruptors
• Persistent, bioaccumulative and toxic (PBT) substances
• Very persistent and very bioaccumulative (vPvB) substances
In very special cases, new active substances will be allowed on the market when meeting this exclusion criteria, if they are important for public health and public interest and there are no alternatives available. To lower the pressure on public health and the environment, there is also a candidate list for active substances to be substituted for less harmful active substances when the old active substances meet the following criteria:
• It meets one of the exclusion criteria
• It is classified as a respiratory sensitizer
• Its toxicological reference values are significantly lower than those of the majority of approved active substances for the same product-type and use
• It meets two of the criteria to be considered as PBT
• It causes concern for human or animal health and for the environment even with very restrictive risk management measures
• It contains a significant proportion of non-active isomers or impurities
The impact of environmental release
The release of biocides in the environment can have huge consequences, since these products are designed to cause damage to living organisms. A classic example is the release of tributyltin from shipyards, harbours and on sailing routes from the antifouling paint on the hulls of ships (De Mora, 1996). Tributyltin was used in the antifouling paint from the 1950s on to prevent microorganisms from settling on the hulls of ships, which would increase the fuel costs and repair costs. However, the release of tributyltin from the paint resulted in a toxic effect on organisms at the bottom of the food chain, such as algae and invertebrates. Tributyltin then biomagnified in the food web, this way affecting larger predators, such as dolphins and sea otters. Eventually, tributyltin entered the diet of humans. The first legislation on the use of tributyltin for ships dates back to the 1980s, but it was not until the Rotterdam Convention of 2008 that the complete use of tributyltin as an active biocide in antifouling paints was banned. Biocides can also have an effect on the capability of the environment to deal with pollution. Microorganisms are responsible for cleaning polluted areas by using the pollutant as food-source. McLaughlin et al. (2016) studied the effect of the release of biocide glutaraldehyde in spilled water from hydraulic fracturing on the microbial activity and found that the microbial activity was hampered by the biocide glutaraldehyde. Hence, because of the biocide, the environment was not or slower capable to return to its original state.
References
De Mora, S.J. (1996). Tributyltin: case study of an environmental contaminant, Vol. 8, Cambridge Univ. Press
McLaughlin, M.C., Borch, T., Blotevogel, J. (2016). Spills of hydraulic fracturing chemicals on agricultural topsoil: Biodegradation, sorption and co-contaminant interactions, Environmental Science & Technology 50, 6071-6078
2.3.2. Question 1
Explain the difference between an active substance and a biocidal product.
2.3.2. Question 2
What is the purpose of using non-active co-substances in a biocidal product?
2.3.2. Question 3
A ship is transporting bananas from Costa Rica to the Netherlands. Mention 5 biocide product types that could be used on this ship, and their purpose.
2.3.2. Question 4
Mention 3 potential ways in which the environment can be damaged as the result of the (accidental) release of biocides in common daily practice.
2.3.3. Pharmaceuticals and Veterinary Pharmaceuticals
(draft)
Author: Thomas ter Laak
Reviewer: John Parsons, Steven Droge
Leaning objectives:
You should be able to:
Keywords: emission, immission, waste water treatment, disease treatment
Introduction
Pharmaceuticals are consumed by humans (human pharmaceuticals) and administered to animals (veterinary pharmaceuticals).
The active ingredients used in human and veterinary medicine partially overlap, however, the major fraction of pharmaceutically active substances in use are restricted to human consumption. In veterinary practice most of the applied pharmaceuticals are antibiotics and anti-parasitic agents, while in human medicine, pharmaceuticals to treat e.g. diabetes, pain, cardiovascular diseases, autoimmune disorders and neurological disorders make up a much larger portion of the pharmaceuticals in use. Worldwide pharmaceutical consumption has increased over the last century (several staggering numbers are summarized here). It is expected that the consumption will further increase due to a wider access to pharmaceuticals in developing countries. Additionally, demographic trends such as aging populations that is often seen in developed countries can also lead to increased consumption of pharmaceuticals, since older generations generally consume more pharmaceuticals than younger generations (van der Aa et al., 2011). The widespread and increasing use and their biological activity makes them relevant for environmental research. Below an overview is given on the emission, occurrence and fate(modeling) of pharmaceuticals in the environment.
Pharmaceuticals in the environment
Pharmaceuticals can enter the environment through various routes. Figure 1 gives and overview of emission routes of pharmaceuticals to the environment. Pharmaceutical are produced, transported to users (humans and/or animals), partially excreted by the users via urine and feces. For humans, the excrements are transport to wastewater treatment plants, septic tanks or directly emitted to soil or surface water. For animals, and especially livestock, manure contains residues of the pharmaceuticals. These pharmaceuticals end up in the environment when animals are grazing outside or when centrally collected manure is applied as fertilizer on arable land. Just like the pharmaceutical consumption, metabolism in the user (a human or an animal), the treatment and further application of communal wastewater and manure varies. Subsequently, emissions also vary between pharmaceuticals, countries and locations.
Concentrations of pharmaceuticals in the environment strongly vary, in Figure 2 concentration ranges of pharmaceuticals and some of their transformation products in the Meuse river and some tributaries are shown.
Properties of pharmaceuticals and their behavior and fate in the environment
Pharmaceuticals in use are developed for a wide array of diseases and therapeutic treatments. The chemical structures of these substances are therefore also very diverse, considering their size, structural presence of specific atoms, and physicochemical properties such as their hydrophobicity, aqueous solubility and ionization under environmentally relevant pH values, as shown for some examples in Figure 3.
As a consequence of their structural diversity their environmental distribution and fate is also very variable. This makes it difficult to generalize the environmental behavior and fate of pharmaceuticals. Nevertheless, pharmaceuticals have generally certain properties in common:
These three generic properties also make them of environmental relevance since:
Occurrence and modelling of human and veterinary pharmaceuticals in the environment
Pharmaceuticals have been studied in the environment since the 1990s. Most studies have been performed in surface waters, but wastewater (effluents), groundwater, drinking water, manure, soil and sediments were also studied. Pharmaceuticals have been observed in all these matrices in concentrations generally varying from µg/L to sub ng/L levels (Aus der Beek et al., 2016, Monteiro and Boxall, 2010). Various studies have related environmental loads and related concentrations to human consumption data. Basically such -so called- mass balance or immision-emission balancing studies work according the following principle:
Modelling pharmaceuticals in the environment
Since the application of human pharmaceuticals, the consumption, is relatively well documented. Environmental concentrations can be related to consumption. This prediction works the best for the most persistent pharmaceuticals, since variations in loss factors are marginal for these pharmaceuticals. When loss factors become larger, they generally also become more variable, trough seasonal variations in use as well as variation in loss during wastewater treatment and loss processes in the receiving rivers, respectively. This makes the loads and concentrations of more degradable pharmaceuticals are more difficult to predict (Ter Laak et al., 2010).
Loads in a particular riverine system (such as a tributary of the river Meuse in the example below) can be predicted with a very simplified model. Here the pharmaceutical consumption over a selected period is multiplied by the factor of the selected pharmaceuticals that are excreted unchanged by the human body (ranging from 0 to 1)and the fraction that is able to pass the wastewater treatment plant (WWTP) (ranging from 0 to 1):
When this is related to actual measured concentrations and loads calculated from these numbers, the correlation between predicted and measured loads can be plotted. Various studies have shown that environmental loads can be predicted within a factor of 3 for most commonly observed pharmaceuticals (Ter Laak et al., 2014; Ter Laak et al., 2010; Alder et al., 2010; Oosterhuis et al., 2013).
For veterinary pharmaceuticals this so called 'immision-emission balancing' is more difficult due to a number of reasons:
In a way the emissions and fate of veterinary pharmaceuticals is similar to emissions of pesticides used in agriculture, but then with a much poorer understanding on loads entering the system and the fate related to the various emission routes and emissions in combination with a complex matrix (urine, feces manure) (Guo et al., 2016). As a consequence, environmental fate studies of veterinary pharmaceuticals often describe specific cases, or cover laboratory studies to unravel specific aspects of the environmental fate of these pharmaceuticals (Kaczala and Blum, 2016; Kummerer, 2009).
Concluding remarks
Pharmaceuticals are commonly found in the environmental compartments such as surface water, soil, sediment and groundwater (Williams et al., 2016). Pharmaceuticals consist of a single or multiple active ingredients that have a specific biological activity. The therapeutic application and pharmacological mechanisms provide valuable information to evaluate the environmental hazard of these chemicals. Their physicochemical properties are of more relevance for the assessment of the environmental fate and exposure. The occurrence in the environment and the biological activity of this group of contaminants makes them relevant in environmental science.
References
Alder, A.C., Schaffner, C., Majewsky, M., Klasmeier, J., Fenner, K. (2010) Fate of [beta]-blocker human pharmaceuticals in surface water: Comparison of measured and simulated concentrations in the Glatt Valley Watershed, Switzerland. Water Research 44, 936-948
Aus der Beek, T., Weber, F., Bergmann, A., Hickmann, S., Ebert, I., Hein, A., Küster, A. (2016) Pharmaceuticals in the environment-Global occurrences and perspectives. Environ. Toxicol. Chem. 35, 823-835.
Guo, X.Y., Hao, L.J., Qiu, P.Z., Chen, R., Xu, J., Kong, X.J., Shan, Z.J., Wang, N. (2016) Pollution characteristics of 23 veterinary antibiotics in livestock manure and manure-amended soils in Jiangsu province, China. J. Environ. Sci. Health Part B Pestic. Food Contamin. Agric. Wastes 51 (6), 383-392
Kaczala, F., Blum, S.E. (2016) The occurrence of veterinary pharmaceuticals in the environment: A review. Curr. Anal. Chem., 12 (3), 169-182
Kümmerer, K. (2009) The presence of pharmaceuticals in the environment due to human use - present knowledge and future challenges. J. Environ. Manage., 90 (8), 2354-2366
Monteiro, S.C., Boxall, A.B.A. (2010) Occurrence and Fate of Human Pharmaceuticals in the Environment. Rev. Environ. Contam. Toxicol., 202, 53-154.
Oosterhuis, M., Sacher, F., ter Laak, T.L. (2013) Prediction of concentration levels of metformin and other high consumption pharmaceuticals in wastewater and regional surface water based on sales data. Sci. Total Environ. 442, 380-388
Ter Laak, T.L., Van der Aa, M., Houtman, C.J., Stoks, P.G., Van Wezel, A.P. (2010) Relating environmental concentrations of pharmaceuticals to consumption: A mass balance approach for the river Rhine. Environ. Intern. 36, 403-409
Ter Laak, T.L., Kooij, P.J.F., Tolkamp, H., Hofman, J. (2014) Different compositions of pharmaceuticals in Dutch and Belgian rivers explained by consumption patterns and treatment efficiency. Environ. Sci. Pollut. Res. 21, 12843-12855
Van der Aa, N.G.F.M., Kommer, G.J., van Montfoort, J.E. and Versteegh, J.F.M. (2011) Demographic projections of future pharmaceutical consumption in the Netherlands. Water Science and Technology, 825-832.
Williams, M., Backhaus, T., Bowe, C., Choi, K., Connors, K., Hickmann, S., Hunter, W., Kookana, R., Marfil-Vega, R., Verslycke, T. (2016) Pharmaceuticals in the environment: An introduction to the ET&C special issue. Environ. Toxicol. Chem. 35, 763-766.
2.3.3. Question 1
Name three reasons why pharmaceuticals are relevant environmental contaminants?
2.3.3. Question 2
Why is it easier to model human pharmaceuticals in the environment than veterinary pharmaceuticals?
2.3.3. Question 3
Ibuprofen (anti-inflammatory) is discharged with the wastewater to the river. The following information is known about the use and fate of this drug.
- consumption per 1000 inhabitants of 1 g/d
- excretion by human 10 %
- removal by STP 68%
How much iboprufen is potentially discharged to the river in g/d ?
2.3.4. Drugs of abuse
Author: Pim de Voogt
Reviewer: John Parsons, Félix Hernández
Leaning objectives:
You should be able to:
• distinguish between licit and illicit drugs
• know what sources cause illicit drugs to show up in the environment
• understand what wastewater-based epidemiology is
Keywords: Cocaine, ecstasy, speed, cannabis, wastewater analysis
Introduction
Since about little more than a decade, drugs of abuse (DOA) and their degradation products have been recognized as emerging environmental contaminants. They are among the growing number of chemicals that can be observed in the aquatic environment.
The residues of a major part of the chemicals used in households and daily life end up in our sewer systems. Among the many chemicals are cleaning agents and detergents, cosmetics, food additives and contaminants, pesticides, pharmaceuticals, and surely also illicit drugs. Once in the sewer, they are transported to wastewater treatment plants (WWTPs), where they may be removed by degradation or adsorption to sludge, or end up in the effluent of the plant when removal is incomplete.
The consumption of both pharmaceuticals and DOA has increased substantially over the last couple of decades as a result of several factors, including ageing of the population, medicalization of society and societal changes in life-style. As a result the loads in wastewater of drugs and their transformation products formed in the body after consumption have steadily increased. More recently, it has been observed that chemical waste from production sites of illicit drugs is being occasionally discharged into sewer systems, thereby dramatically increasing the loads of illicit drug synthesis chemicals and end products transported to WWTPs. As WWTPs are not designed to remove drugs, a substantial fraction of the loads may end up in receiving waters and thus pose a threat to both human and ecosystem health.
Drugs of Abuse (DOA)
Europe's most commonly used illicit drugs are THC (cannabis), cocaine, MDMA (ecstasy) and amphetamines. The structure of these drugs is given in Figure 1. Other important DOA include the opioids such as heroine and fentanyl, GHB, Khat and LSD.
Drugs of abuse are controlled by legislation, in The Netherlands by the Opium Act. The Opium Act encompasses two lists of substances. List one chemicals are called hard drugs while List II chemicals are known as soft drugs. Some narcotics are also being used for medicinal purposes, e.g., ketamine, diazepines, and one of the isomers of amphetamine. New psychoactive substances (NPS), also known as designer drugs or legal highs (because they are not yet controlled as they are not listed on the Opium Act lists), are synthesized every year and become available on the market in high numbers (see Figure 2).
Wastewater-based epidemiology
Central sewage systems collect and pool wastewater from household cleaning and personal care activities as well as excretion products resulting from human consumption and thus contain chemical information on the type and amount of substances used by the population connected to the sewer. Drugs that are consumed are metabolized in the body and subsequently excreted. Excretion products can include the intact compounds as well as the transformation products, that can be used as biomarkers. An example of the latter is benzoylecgonine, which is the major transformation product of consumed cocaine. The collective wastewater from the sewer system carrying the load of chemicals is directed to the WWTP, and this wastewater influent can be sampled at the point where it enters the WWTP. By appropriate sampling of the influent during discrete time-intervals, e.g. 24 h, a so-called composite sample can be obtained and the concentrations of the chemicals can be determined. The volume of influent entering the WWTP is recorded continuously. Multiplying the observed 24 h average concentration of a compound with the total 24 h volume yields the daily load of the chemical entering the WWTP. This load can be normalised to the number of people living in the sewer catchment, resulting in a load per inhabitant. The loads of drugs in wastewater influents are usually expressed as mg.day-1.1000 inh-1. Normalised drug load data allow comparison between sewer catchments, such as shown in Figure 3. Obtaining chemical information about the population through wastewater analysis is known as Wastewater-based epidemiology, WBE (Watch the video). While WBE was developed originally to obtain data on consumption of DOA, the methodology has been shown to have a much wider potential: in calculating the consumption of e.g., alcohol, nicotine, NPS, pharmaceuticals and doping, as well as for assessing community health indicators, such as incidence of diseases or stress biomarkers.
DOA and the environment
Barring direct discharges into surface waters or terrestrial environments, the major sources of DOA to the environment are WWTP effluents. Conventional treatment in municipal WWTPs has not been specifically designed to remove pharmaceuticals or DOA. Removal rates of DOA vary widely and depend on compound properties such as persistence and polarity as well as WWTP operational conditions and process configurations. Some DOA cross WWTPs almost unhindered, thus ending op in the receiving waters. Examples of the latter are MDMA and some diazepines (see Figure 4). Despite that several studies report the presence of DOA or their transformation products in surface waters, until now there is very little information about their aquatic ecotoxicity available in the scientific literature.
Recently, chemical waste from synthetic DOA manufacturing including their precursors and synthesis byproducts have been observed to be discharged directly into sewers. In addition, containers with chemical waste from DOA production sites have been dumped on soil or surface waters. Apart from solvents and acids or bases this waste often contains remainders of the synthesis products, which can then be dissipated in the aquatic environment or seep through the soil into groundwater.
Considering that DOA are highly active in the human body, it can be expected that some of them, in particular the more persistent ones, may exert some effects on aquatic biota when their levels increase in the aquatic environment.
References
Van der Aa, M., Bijlsma, L., Emke, E., et al. (2013). Risk assessment for drugs of abuse in the Dutch watercycle. Water Research 47(5), 1848-1857.
Bijlsma, L., Emke, E., Hernández, F., de Voogt, P. (2012). Investigation of drugs of abuse and relevant metabolites in Dutch sewage water by liquid chromatography coupled to high resolution mass spectrometry. Chemosphere 89(11), 1399-1406.
EMCDDA, European Drug Report 2018, Lisbon (www.emcdda.europa.eu/publications/edr/trends-developments/2018_en)
Thomas, K. V., Bijlsma, L., Castiglioni, S., et al. (2012). Comparing illicit drug use in 19 European cities through sewage analysis. Science of the Total Environment 432, 432-439.
2.3.4. Question 1
One way of mapping the consumption volumes of psychoactive substances in a city is by chemical analysis of drug residues and biomarkers in wastewater. To assess the consumption volume of cocaine by a population the concentrations in wastewater of the substance benzoylecgonine (BE) are determined. Why is BE used instead of cocaine itself?
2.3.4. Question 2
What possible sources can lead to the occurrence of an illicit drug in wastewater?
2.3.4. Question 3
Can illicit drugs in wastewater pose a threat to the environment and if so, why?
2.3.5. Hydrocarbons
Author: Pim N.H. Wassenaar
Reviewer: Emiel Rorije, Eric M.J. Verbruggen, Jonathan Martin
Learning objectives:
You should be able to
• explain the diversity/variation in hydrocarbon structures.
• explain the specific and non-specific toxicological effects of several hydrocarbons.
Keywords: Hydrocarbons, Paraffins, Naphthenics, Aromatics
Introduction:
Hydrocarbons are a class of chemicals that only consist of carbon and hydrogen atoms. But despite their simplicity in building blocks, this group of chemicals consists of a wide variety of structures, as there are differences in chain length, branching, bonding types and ring structures. The main sources of hydrocarbons are crude oil and coal, which are formed over millions of years by natural decomposition of the remains of plants, animals or wood, and are used to derive products we are using on a daily basis, including fuels and plastics Other natural sources include natural burning (forest fires) and volcanic sources
Hydrocarbon classification
The major classes of hydrocarbons are paraffins (i.e. alkanes), naphthenics (i.e. cycloalkanes) and aromatics (Figure 1), and within these classes, several subclasses can be identified. Paraffins are hydrocarbons that do not contain any ring structures. Paraffins can be subdivided in normal (n-) paraffins, which do not contain any branching (straight chain), and iso-paraffins (i-), which do contain a branched carbon-chain. When alkanes include at least one carbon-carbon double bond, they are considered olefins (or alkenes).
Naphthenic and aromatic hydrocarbons both contain ring-structures but differ in the presence of aromatic or non-aromatic rings. The naphthenics and aromatics can be further specified based on their ring count; often mono-, di- and poly-ring structures are distinguished from each other. Of all these classes, the polycyclic aromatic hydrocarbons (PAHs) are the best-studied category in terms of all kinds of environmental aspects.
Besides the classes considered in Figure 1., combinations of these classes also exist. Naphthenic or aromatic structures with an alkane side chain are mostly still considered as naphthenic or aromatic hydrocarbons, respectively. However, when a non-aromatic-ring is fused with an aromatic-ring, the hydrocarbon is classified as a naphthenic-aromatic structure. Depending on the ring-count several subclasses can be identified, including naphthenic-mono-aromatics and naphthenic-poly-aromatics.
Concerns for human health and the environment
Because of their lack of polar functional groups, hydrocarbons are generally hydrophobic and, as a consequence, many are able to cause acute toxic effects in aquatic animals by a non-specific mode of action known as narcosis (or baseline toxicity). Narcosis is a reversible state of inhibited activity of membrane structures within the cells of organisms. Narcosis type toxicity is considered the minimum toxicity that any substance will be able to have, just by reaching concentration levels in the phospholipid bilayer of the cell membranes that disturb membrane transportation process. Hence the name "baseline" or minimum toxicity. When these events take place above a certain threshold, systemic toxicity can be observed in the organism, such as lethality. This threshold concentration is also known as the critical body residue (CBR) (Bradbury et al., 1989; Parkerton et al., 2000; Veith & Broderius, 1990).
Nevertheless, hydrocarbons can also have a more specific mechanisms of action, resulting in greater toxicity than baseline toxicity. For example, the toxicity of several PAHs increases in combination with ultraviolet radiation due to photo-induced toxicity. Photo-induced toxicity may be caused by photoactivation, in which a PAH is degraded into an oxidized product with a higher toxicity, or rather by photosensitization, in which reactive oxygen species (ROS) are formed due to an excited state of the PAHs (Figure 2) (Roberts et al., 2017). PAHs are especially vulnerable to photodegradation as their absorption spectrum falls within the range of wavelengths reaching the earth's surface (> 290 nm), which is not the case for most monoaromatic and aliphatic hydrocarbons (EMBSI, 2015). The photo-induced effects are of particular concern for aquatic species with transparent bodies, like zooplankton and early life stages, as more UV-light can penetrate into their organs and tissues (Roberts et al., 2017).
Several hydrocarbons are also able to cause genotoxicity and cancer upon exposure, including benzene, 1,3-butadiene and some PAHs. The carcinogenicity of PAHs is caused by biotransformation into reactive metabolites, specifically into epoxides which are the first step in oxidation of aromatic ring structures into dihydrodiol ring systems (Figure 3). In general, the biotransformation step increases the water solubility of the hydrocarbons (Phase I metabolism) and promotes subsequent conjugation and excretion (Phase II metabolism). However, several epoxide metabolites - more specifically the most stable aromatic epoxides - can reach the cell nucleus and covalently react with DNA, forming DNA adducts, and induce mutations (Figure 3). Ultimately, if not repaired such mutations can accumulate and may result in the formation of tumors (Ewa & Danuta, 2016). Specifically, PAHs with a bay-like region are of concern as biotransformation results in relatively stable reactive epoxides that are not accessible to epoxide hydrolase enzymes (Figure 3) (Jerina et al. 1980). Similar to PAHs, 1,3-butadiene and benzene are also able to cause cancer via the effects of their respective reactive metabolites (Kirman et al., 2010; US-EPA 1998).
Besides their toxicity, some hydrocarbons such as the high molecular weight PAHs can be persistent in the environment and may accumulate in biota as a result of their hydrophobicity. It is therefore expected that internal concentrations are higher for such hydrocarbons and it is interesting that there is thus a relationship between narcosis and bioaccumulation potential. Consequently, these hydrocarbons might be of even greater concern.
Characterization of mixtures of hydrocarbons
As most research focused on specific hydrocarbons, including several PAHs, it is important to note that the biodegradation, bioaccumulation and toxicity potential of many hydrocarbons is still not fully known, such as for alkylated PAHs and naphthenics. As there is such a wide variety in hydrocarbon structures, it is impossible to assess the (potential) hazards of all hydrocarbons separately. Therefore, grouping approaches have been developed to speed up the risk assessment. Within a grouping approach, hydrocarbons can be clustered based on structural similarities. The underlying assumption is that all chemicals in a group are expected to have fairly similar physicochemical properties, and subsequently also fairly similar environmental fate and effect properties. As a result, such a group could potentially be assessed as if it is one single hydrocarbon.
The applicability of a hydrocarbon specific grouping approach, known as the Hydrocarbon Block Method (King et al., 1996), to assess the biodegradation and bioaccumulation potential of hydrocarbons is currently being investigated. Within this approach, all hydrocarbons are grouped based on their functional class (e.g. paraffin, naphthenic, aromatic) and the number of carbon atoms. The number of carbon atoms is thought to highly correlate with the boiling point of the hydrocarbons. An example matrix of the Hydrocarbon Block Method is presented in Figure 4. The composition of an oil substance could be expressed in such a matrix following GC-GC/MS analysis. Subsequently, the PBT-properties of the individual blocks could potentially be assessed by analyzing and extrapolating the PBT-properties of representative hydrocarbons for varying hydrocarbon blocks (see Figure 4).
References
Bradbury, S.P., Carlson, R.W., Henry, T R. (1989). Polar narcosis in aquatic organisms. In Aquatic Toxicology and Hazard Assessment: 12th Volume. ASTM International.
EMBSI (2015). Assessment of Photochemical Processes in Environmental Risk Assessment of PAHs
Ewa, B., Danuta, M.Š. (2017). Polycyclic aromatic hydrocarbons and PAH-related DNA adducts. Journal of applied genetics 58, 321-330.
Homburger, F., Hayes, J.A., Pelikanm E.W. (1983). A Guide to General Toxicology. Karger/Base, New York, NY.
Jerina, D.M., Sayer, J.M., Thakker, D.R., Yagi, H., Levin, W., Wood, A.W., Conney, A.H. (1980). Carcinogenicity of polycyclic aromatic hydrocarbons: the bay-region theory. In Carcinogenesis: Fundamental Mechanisms and Environmental Effects (pp. 1-12). Springer, Dordrecht.
King, D.J., Lyne, R.L., Girling, A., Peterson, D.R., Stephenson, R., Short, D. (1996). Environmental risk assessment of petroleum substances: the hydrocarbon block method. CONCAWE report no. 96/52.
Kirman, C.R., Albertini, R.A., & Gargas, M.L. (2010). 1, 3-Butadiene: III. Assessing carcinogenic modes of action. Critical reviews in toxicology 40(sup1), 74-92.
Parkerton, T.F., Stone, M.A., Letinski, D. J. (2000). Assessing the aquatic toxicity of complex hydrocarbon mixtures using solid phase microextraction. Toxicology letters 112, 273-282.
Roberts, A.P., Alloy, M.M., Oris, J.T. (2017). Review of the photo-induced toxicity of environmental contaminants. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 191, 160-167.
US-EPA (1998). Carcinogenic Effects of Benzene: An Update. EPA/600/P-97/001F.
Veith, G.D., Broderius, S.J. (1990). Rules for distinguishing toxicants that cause type I and type II narcosis syndromes. Environmental Health Perspectives 87, 207.
2.3.5. Question 1
Mention at least three aspects that influence the variation in hydrocarbon structures?
2.3.5. Question 2
To which hydrocarbon class (or Hydrocarbon Block) do the following structures belong?
1.
2.
3.
2.3.5. Question 3
What kind of toxic effects can be observed for polycyclic aromatic hydrocarbons?
2.3.6. CFCs
(draft)
Authors: Steven Droge
Reviewer: John Parsons
Leaning objectives:
You should be able to:
• realize what the ozone layer depletion was all about
• understand why certain replacement chemicals are still problematic
Keywords: Ozone layer, refrigerator, volatile chemicals, spray cans, radicals
Introduction
CFCs (chlorofluorocarbons) were very common air pollutants in the 20th century because they were the basic components of refrigerants and air conditioning, propellants (in spray can applications), and solvents, since the 1930s. They are still very common air pollutants, because they are very persistent chemicals, and emissions do still continue. In the first years as refrigerants, they replaced the much more toxic components ammonia (NH3), chloromethane (CH3Cl), and sulfur dioxide (SO2). Particularly the CFCs leaking from old refrigerating systems in landfills and waste disposal sites caused high emissions into the environment. Typically, these volatile CFC chemicals are based on the smallest carbon molecules methane (CH4), ethane (C2H6), or propane (C3H8). All hydrogen atoms in these CFC molecules are replaced by a mixture of chlorine and fluorine atoms.
CFCs are less volatile than their hydrocarbon analogue, because the halogen atoms polarize the molecules, which causes stronger intermolecular attractions. Depending on the substitution with Cl or F, the boiling point can be tuned to the desired point for refrigerating cooling processes. The CFCs are also much less flammable than hydrocarbon analogues, making them much safer in all kinds of applications.
Naming of CFCs
CFCs were often known by the popular brand name Freon. Freon-12 (or R-12) for example stands for dichlorodifluoromethane (CCl2F2, boiling point -29.8 °C, while methane has -161 °C), as shown in Figure 1. The naming reflects the amount of fluor atoms as the most right number. The next value to the left is the number of hydrogen atoms plus 1, and the next value to the left is the number of carbon atoms less one (zeroes are not stated), and the remaining atoms are chlorine. Accordingly, Freon-113 could apply to 1,1,2-trichloro-1,2,2-trifluoroethane (C2Cl3F3, boiling point 47.7 °C, while ethane has -161 °C). The structure of any Freon-X number can also be derived from adding +90 to the value of X, so Freon-113 would give a value of 203. The first numerical is the number of C (2), the second numerical H (0), the third numerical F (3), and the remaining substitutions are by chlorine (C2X6 gives 3 chlorines).
The reason CFC depletes the ozone layer
The key issue with CFC emissions is the reaction under influence of light ("photodegradation") that ultimately reduces ozone concentrations ("ozone depletion") in the upper atmosphere ("stratosphere"). Ozone absorbs the high energy radiation of the solar UV-B spectrum (280-315nm), and the ozone layer therefore prevents this to reach the Earth's surface. The even more energetic solar UV-C spectrum (100-280nm) is actually causing the formation of ozone (O3) when reacting with oxygen (O2), as shown in Figure 2. Under the influence of intense light-energy in the upper atmosphere, CFC molecules can disintegrate into two highly reactive radicals (molecules with a free electron . ), for Freon-11:
It is the radical Cl. that catalyzes the conversion of ozone back into O2. The environmentally relevant role of the fluorine atoms in CFCs is that they make these chemicals very persistent after emission, because the C-F bond is one of the strongest covalent bonds known. With half-lives up to >100 years, high CFC levels can reach the upper atmosphere. James Lovelock was the first to detect the widespread presence of CFCs in air in the 1960s, while the damage caused by CFCs was discovered only in 1974. Another undesirable effect of CFC in the stratosphere is that they are a much more potent greenhouse gases than CO2.
CFC replacements.
In 1978 the United States banned the use of CFCs such as Freon in aerosol cans. After several years of observations of the ozone layer depletions globally (Figure 3), particularly above Antarctica, the Montreal Protocol was signed in 1987 to drastically reduce CFC emissions worldwide. CFCs were banned by the late 1990s in most EU countries, and e.g. in South Korea by 2010. Due to the persistency of CFCs it may take until 2050-2070 before the ozone layer will return to 1980 levels (which were bad already).
The key damaging feature of CFCs in terms of ozone depletion is their persistency, so that emissions reach and build up in the stratosphere (starting from 20km above the equator, but only at 7km above the poles). CFC replacement molecules were initially found simply by adding more hydrogens in the CFC structures and somewhat less Cl (HCFCs), but fractions still contributed to Cl. radicals. Later alternatives lack the chlorine atoms and have even shorter lifetimes in the lower atmosphere, and simply cannot form the Cl radicals. These "hydrofluorocarbons" (HFCs) are currently common in automobile air conditioners, such as Freon-134 (do the math to see that there is no Cl, boiling point -26.1 °C).
Still, HCFC as well as HFCs are still very potent greenhouse gasses, so the worldwide use of such chemicals remains problematic and gives rise to new legislations, regulations, and searches for alternatives. R-410A (which contains only fluorine) is becoming more widely used but is 1700 times more potent than CO2 as greenhouse gas, equal to Freon-22. Simple hydrocarbon mixtures such as propane/isobutane are already used extensively in mobile air conditioning systems, as they have the right thermodynamic properties for some uses and are relatively safe. Unfortunately, we did not have the technological skills, nor the awareness to apply this back in the 1930s.
2.3.7. Cosmetics/personal care products
(draft)
Author: Mélanie Douziech
Reviewers: John Parsons
Learning objectives:
You should be able to:
• Define what personal care products and cosmetics are
• Explain how chemicals from personal care products end up in the environment
• Cite and describe some of the most common chemicals found in personal care products
Keywords: wastewater, chemical function, surfactants, microbeads
Introduction
Personal care products (PCPs) cover a large range of products fulfilling hygiene, health, or beauty purposes (e.g. shampoo, toothpaste, nail polish). They are categorized into oral care, skin care, sun care, hair care, decorative cosmetics, body care and perfumes. Overall, most PCPs are classified as cosmetics and regulated accordingly. In the European Union (EU) the Cosmetic Regulation governs the production, safety of ingredients and the labelling and marketing of cosmetic products. The United States of America (USA), on the other hand, have a narrower definition of cosmetics so that products not fulfilling the definition are regulated as pharmaceuticals (e.g. sunscreen) (Food and Drug Administration, 2016).
PCPs come in a range of formats (e.g. liquids, bars, aerosols, powders) and typically contain a wide range of chemicals, each fulfilling a specific function within the product. For example, a shampoo can include cleansing agents (surfactants), chemicals to ensure product stability (e.g. preservatives, pH adjusters, viscosity controlling agents), diluent (e.g. water), perfuming chemicals (fragrances), and chemicals to influence the product's appearance (e.g. colourants, pearlescers, opacifiers). The chemicals present in PCPs ultimately enter the environment either through air during direct use, such as the propellants in aerosols, or through wastewater via down the drain disposal following product use (e.g. shower products, toothpaste). The release of PCP chemicals into the environment needs to be monitored and the safety of these chemicals understood in order to avoid potential problems. In developed countries, the use of wastewater treatment plants (WWTPs) is key to effectively removing the PCP chemicals and other pollutants from wastewater prior to their release to rivers and other watercourses. The removal mechanisms occurring in WWTPs include biodegradation, sorption onto solids, and volatilization to the air. The extent of removal is influenced by the physicochemical properties of the chemicals and the operational conditions of the WWTPs. In regions where wastewater treatment is lacking, the chemicals in PCPs enter the environment directly.
The wide scale daily use of PCPs and the associated large volumes of chemicals released explain why they are scrutinized by environmental protection agencies and regulatory bodies. The following sections will briefly review some of the classes of chemicals used in PCPs by describing their behavior in the environment and their potential effect on ecosystems.
Cleansing agents - surfactants
Surfactants are an important and widely used class of chemicals. They are the key components of many household cleaning agents as well as PCPs, such as shampoos, soaps, bodywash and toothpaste, because of their ability to remove dirt. These dirt-removing properties also make surfactants inherently toxic to aquatic organisms. The biodegradability of surfactants is a key legal requirement for their use in PCPs to minimize the likelihood of unsafe levels in the environment. Different types of surfactants exist and are often classified based on their surface charge. Anionic surfactants, which carry a negative surface charge, interact and help remove positively charged dirt particles from surfaces such as hair and skin. Sodium lauryl sulfate is a typical example of an anionic surfactant used in PCPs. Cationic surfactants, such as cetrimonium chloride, are positively charged and may be used as hair conditioning agents to make hair shinier or more manageable. Non-ionic surfactants (uncharged), such as cetyl alcohol, help formulate products or increase foaming. Amphoteric surfactants, such as sodium lauriaminodipropionate, carry both positive and negative charges and are commonly used to counterbalance the potentially irritating properties of anionic surfactants.
Fragrances
Fragrances are mixtures of often more than 20 perfumery chemicals used to provide the smell of PCPs. Typically, fragrances are present at very low levels in most PCPs (below 0.01%) so that their exact compositions are not disclosed. Disclosed, however, are any allergens present in the fragrance to help dermatologists and consumers avoid certain fragrance chemicals. Despite the wish to protect trade secrets, a recent trend increasingly sees companies disclose the full fragrance compositions of their products on their websites (e.g. L'Oréal, Unilever). Well-known examples of fragrances include hexyl cinnamal, linalool, and limonene. Potential concerns about the ecotoxicological impact of fragrances have arisen on the one hand because of a lack of disclosure of fragrance formulations and on the other hand because of the detection of certain persistent fragrances in the environment (e.g. nitromusks).
Preservatives
Preservatives are usually added to PCPs containing water for their ability to protect the product from contamination by bacteria, yeasts, and molds during storage or repeated use. Given their targeted action against living organisms, the use of preservative in chemical products including PCPs is under constant scrutiny. For example, in 2016 and 2017, the European Commission tightened the regulation around the use of methylisothiazolinone in cosmetics products due to human safety concerns. Other preservatives that have been restricted in use, because of both human safety and environmental safety concerns (e.g. endocrine disruption effects), include certain types of parabens and triclosan.
UV filters
UV filters are used in sunscreen products as well as in other PCPs such as foundation, lipstick, or moisturizing cream to protect users from UV radiation. UV filters can be organic or inorganic. Inorganic UV filters, like titanium oxide and zinc oxide, form a physical boundary protecting the skin from UV radiation. Organic UV filters, on the other hand, protect the skin by undergoing a chemical reaction with the incoming UV radiation. Organic UV filters commonly found in PCPs include butyl methoxydibenzoylmethane, ethylhexyl methoxycinnamate, and octocrylene. Organic UV filters are poorly biodegradable and have the potential to accumulate in organisms. Further, a number of organic UV filters have been shown to be toxic to coral organisms in laboratory tests. They are suspected to cause coral bleaching by, for example, promoting viral infections but research is still on-going to understand their potential ecotoxicological effects at realistic environmental concentrations.
Volatile chemicals
Certain chemicals used in PCPs are highly volatile and may end up in the air following product use. Examples include propellants, such as propane butane mixes or compressed air/nitrogen, used in aerosols to apply ingredients in hairsprays or deodorants and antiperspirants. Fragrances also volatilize when the product is applied to skin or hair to provide smell. Volatile silicones, chemicals used to assist the deposition of ingredients in liquids and creams, are another example of chemicals emitted to air upon PCP use.
The special case of plastic microbeads
Plastic microbeads, with a diameter smaller than 5mm, have been used in PCPs such as face scrubs or shower gels for their scrubbing and cleansing properties. The growing concern about plastic pollution in water has drawn attention to the use of microbeads in PCPs. As a result, a number of initiatives were launched both to highlight the use of plastic microbeads and to encourage replacement with natural alternatives. An example thereof is the "Beat the microbead" coalition (https://www.beatthemicrobead.org/) sponsored by the United Nations Environment Program, launched to help consumers identify and avoid PCPs containing microbeads. Such initiatives together with voluntary commitments by industry have led to a large decrease in the use of microbeads in wash-off cosmetic products: In the EU, for example, the use of microbeads in wash-off products was reduced by 97% from 2012 to 2017. Legislation to restrict the use of microbeads has also recently been put in place. In the USA microbeads in PCPs were banned in July 2017 and a number of EU countries (e.g. United Kingdom, Italy) have also banned their use in wash-off products.
Further reading
For more information on PCP chemicals and their function in products, please see (European commission 2009; Grocery Manufacturers Association 2017).
For more information on the different types of surfactants, please see Tolls et al. (2009) and Section 2.3.8.
Manova et al. (2013) list the different types of UV filters.
The report of Scudo et al. (2017) gives more information on the use of microplastics in Europe.
References
European Commission (2019). Cosing. 2009 03.2019]; Available from: http://ec.europa.eu/growth/tools-databases/cosing/.
Food and Drug Administration (2016). Are All "Personal Care Products" Regulated as Cosmetics? [cited 2019 03]; Available from: https://www.fda.gov/forindustry/fdabasicsforindustry/ucm238796.htm.
Grocery Manufacturers Association (2017). Smartlabel. [cited 2017 11]; Available from: http://www.smartlabel.org/.
Manova, E., von Goetz, N., Hauri, U., Bogdal, C., Hungerbuhler, K. (2013). Organic UV filters in Personal Care Products in Switzerland: A Survey of Occurrence and Concentrations. International Journal of Hygiene and Environmental Health 216, 508-514.
Scudo, A., Liebmann, B., Corden, C., Tyrer, D., Kreissig, J., Warwick, O. (2017). Intentionally Added Microplastics in Products. in: Limited A.F.W.E.a.I.U., ed. United Kingdom
Tolls, J., Berger, H., Klenk, A., Meyberg, M., Müller, R., Rettinger, K., Steber, J. (2009). Environmental safety aspects of Personal Care Products - a European perspective. Environmental Toxicology and Chemistry 28, 2485-2489.
2.3.7. Question 1
How do chemicals found in cosmetics end in the environment?
2.3.7. Question 2
Why are surfactants a concern for environmental toxicity?
2.3.7. Question 3
How did personal care products contribute to the microplastic pollution in the water?
2.3.8. Detergents and surfactants
Author: Steven Droge
Reviewer: Thomas P. Knepper
Leaning objectives:
You should be able to:
• explain why surfactants remove dirt
• discuss historical progress on surfactant biodegradability
• describe the different types of common surfactants.
• describe examples of how surfactants enter the environment.
Keywords: amphiphilic chemicals, micelle formation, biodegradability
Introduction
Surface active agents ("surf-act-ants") are a wide variety of chemicals produced in bulk volumes (>10.000 tonnes annually) as a key ingredient in cleaning products: detergents. Typical for surfactants is that they have a hydrophobic tail and a hydrophilic head group (Figure 1).
At relatively high concentrations in water (typically >10-100 mg/L), surfactants spontaneously form aggregated structures called micelles (Figure 1), often in spheres with the hydrophobic tails inward and the hydrophilic head groups towards the surrounding water molecules. These micelle super-structures allow surfactants to dissolve grease and dirt from e.g. textile or dishes into water, which can then be flushed away. Besides this common use of surfactants, their amphiphilic (i.e., both hydrophilic and lipophilic) properties allow for a versatile use in our modern world:
• During the large 2010 oil spill in the Mexican Gulf, enormous volumes (>6700 tonnes) of several types of surfactant formulations (e.g. "Corexit") were used to disperse the constant stream of oil leaking from the damaged deep water well into small dissolved droplets, in order to facilitate microbial degradation and prevent the formation of floating oil slabs that could ruin coastal habitats.
• The ability of a layer of surfactants to maintain hydrophobic particles in solution is a key process in many products, such as paints and lacquers.
• The ability to emulsify dirt particles is a key feature in process fluids during deep drilling in soil or sediment.
• Fabric softners, and hair conditioners, have cationic surfactants as key ingredients that stick with the positively charged head groups onto the negatively charged fibers of your towel or hair. After the final flushing, these cationic surfactants still stick on the fibers and because of the hydrophobic head groups sticking out make these materials feel soft and smooth. Often only during the next washing event (with anionic or nonionic surfactants) the cationic surfactants are flushed off the fibers.
• Many cationic surfactants have biocidal properties at relatively low concentrations and are therefore used in a few percent in many cosmetic products as preservatives, e.g. in cosmetics, or used to kill microbes in food processing, antibacterial hand wipes or during swimming pool cleaning. Examples are chloride salts of benzalkonium, benzethonium, cetylpyridinium.
• Surfactants lower the surface tension of water, and therefore are used (as "adjuvants") in pesticide products to facilitate the droplet formation during spraying and to improve contact of the droplets with the target leaves in case of herbicides. Examples are fluorinated surfactants, silicone based surfactants (Czajka et al. 2015), and polyethoxylated tallow amine (POEA) used for example in the glyphosate formulation Roundup.
The hydrophobic tail of surfactants is mostly composed of a chain of carbon atoms, although also fluorinated carbon (-CF2-) chains or siloxane (Si(CH3)3-O-[..]-Si(CH3)3) chains are also possible.
The first bulk volume produced surfactants for washing machines were branched anionic alkylbenzenesulfonates (ABS) and alkylphenolethoxylates (APEO), with the hydrocarbon source obtained from petroleum. Because of the variable petroleum source, these chemicals are often complex mixtures. However, hydrophobic branched alkylchains are poorly biodegraded, and the constant disposal of these surfactants into the waste water caused very high environmental concentrations, often leading to foaming rivers (Figure 2).
Surfactant producers 'voluntarily' switched to carbon sources such as palm oil or controlled polymerization of petrol-based ethylene, that could be used to generate surfactants with linear alkyl chains: linear alkylbenzenesulfonate (LAS) and alcohol ethoxylates (AEO). Some surfactants have the hydrophilic headgroup attached to two carbon chains, such as the anionic docusate (heavily used in the BP oil spill) and the cationic dialkyldimethylammonium chemicals. Common detergent surfactants are nowadays designed to pass ready biodegradability tests (>60% mineralisation to CO2 within a 10 d window following a lag phase, in a 28 d test). Early examples of fabric softners are double chain (dialkyl)dimethylammonium surfactants, but the environmental persistency of these compounds (DODMAC and DHTDMAC, see e.g. EU and RIVM-reports) has led to a large replacement by diesterquats (DEEDMAC), which degrade more rapidly through the weak ester linkages of the fatty acid chains (Giolando et al. 1995). A switch to sustainable production of the carbon sources is ongoing. Whereas petroleum based ethylene oil was mostly used, it is being replaced increasingly by the linear fatty acid carbon chains from either palm-oil (mostly C16/C18), coconu oil (mostly C12/C14), but also such raw materials needs to be as sustainably derived as possible.
The hydrophilic headgroups can vary extensively. Nonionic surfactants can have a simple polar functional group (amide), glucose based (polyglycoside), or contain a variable lengths of repetitive ethoxlyate and/or propoxylate units. Because the ethoxylation process is difficult to control, such surfactants are often complex mixtures. Anionic surfactants are often based on sulfate (SO4-) or sulfonate (SO4-), but also phosphonate and carboxylates are common. A key difference between anionic surfactants is that sulfate and sulfonates are fully anionic (pKa ~<0) over the entire environmental pH range (pH4-9), while carboxylates are weaker acids that are still partially neutral species (pKa ~5). Most cationic surfactants are based on permanently charged quaternary ammonium headgroups (R-(N+)(CH3)3), although several ionizable amine groups are applied in cationic surfactants too (e.g., diethanolamines).
The key ingredient property of most surfactants is the critical micelle concentration (CMC), which defines the dissolved concentration above which micellar aggregates start to form that can remove grease or fully emulsify particles. The CMC decreases proportionally with the hydrophobic tail length, and this means that with longer tails, you need less surfactant to start to form micelles. However, with increasing hydrophobic tails anionic surfactants more readily precipitate with dissolved inorganic cations such as calcium. Also, surfactant toxicity increases proportionally with hydrophobic tail lengths. If the alkyl chain is too long, the surfactant may bind strongly to all kinds of surfaces and not be available for micelle formation. The optimum hydrophobic chain length is thus often a balance between the desired properties of the surfactant and several critical processes that influence the efficiency and risk of surfactants.
References
Kümmerer, K. (2007). Sustainable from the very beginning: rational design of molecules by life cycle engineering as an important approach for green pharmacy and green chemistry. Green Chemistry 9, 899-907 . DOI: 10.1039/b618298b
Giolando, S.T., Rapaport, R.A. , Larson, R.J., Federle, T.W., Stalmans, M., Masscheleyn P. (1995). Environmental fate and effects of DEEDMAC: A new rapidly biodegradable cationic surfactant for use in fabric softeners. Chemosphere 30, 1067-1083. DOI: 10.1016/0045-6535(95)00005-S
Czajka, A., Hazell, G., Eastoe, J. (2015). Surfactants at the Design Limit. Langmuir 31, 8205−8217. DOI: 10.1021/acs.langmuir.5b00336
Scientific Committee on Toxicity, Toxicity and the Environment (CSTEE) (2001). Opinion on the results of the Risk Assessment of: Dimethyldioctadecylammonium chloride (DODMAC). EU-report C2/JCD/csteeop/DodmacHH22022002/D(02) https://ec.europa.eu/health/archive/ph_risk/committees/sct/documents/out143_en.pdf
Van Herwijnen, R. (2009). Environmental risk limits for DODMAC and DHTDMACRIVM Letter report 601782029 - https://www.rivm.nl/bibliotheek/rapporten/601782029.pdf
2.3.8. Question 1
Try to look up toxicity literature for the pesticide formulation Roundup and compare the apparent toxicities of the active ingredient glyphosate and the adjuvant surfactant to nontarget organisms. What do you notice?
2.3.8. Question 2
Surfactants do not just accumulate at the air-water interface, but also at water-solvent interfaces. The octanol-water partition coefficient is a critical property for risk assessment, but can you provide reasons why this property is problematic to be derived and applied for surfactants?
2.3.8. Question 3
Although detergents are designed to be readily biodegradable, some compounds appear to be pseudo-persistent as they are still found at detectable levels near the outflow of sewage treatment effluent pipes. What do you think is meant by this?
In preparation | textbooks/chem/Environmental_Chemistry/Environmental_Toxicology_(van_Gestel_et_al.)/02%3A_Environmental_Chemistry_Chemicals/2.03%3A_Pollutants_with_specific_use.txt |
3.1.1. Introduction
In order to understand and predict the effects of chemicals in the environment we need to understand the behaviour of chemicals in specific environments and in the environment as a whole. In order to deal with the diversity of natural systems, we consider them to consist of compartments. These are defined as parts of the physical environment that are defined by a spatial boundary that distinguishes them from the rest of the world, for example the atmosphere, soil, surface water and even biota. These examples suggest that three phases: gas, liquid, and solid, are important but compartments may consist of different phases. For example, the atmosphere consists of suspended liquids (e.g., fog) and solids (e.g., dust) as well as gases. Similarly, lakes contain suspended solids and soils contain gaseous and water-filled pore space. In detailed environmental models, each of these phases may also be considered to be a compartment.
The behaviour and fate of chemicals in the environment is determined by the properties of environmental compartments and the physicochemical characteristics of the chemicals. Together these properties determine how chemicals undergo chemical and biological reactions, such as hydrolysis, photolysis and biodegradation, and phase transfer processes such as air-water exchange and sorption.
In this chapter, we first introduce the most important compartments and their most important properties and processes that determine the behaviour of chemical contaminants: the atmosphere, the hydrosphere, sediment, soil, groundwater and biota. The emissions of chemicals into the environment from either point sources or diffuse sources is discussed and the important pathways and processes determining the fate of chemicals. The partitioning approach to phase-transfer processes is presented with sorption as a specific example. The impact of physicochemical properties on partitioning is also discussed.
Other important environmental processes are discussed in sections on metal speciation, processes affecting the bioavailability of metals and organic contaminants and the transformation and degradation of organic chemicals. These sections also include information on the basic methods to measure these processes.
Finally, approaches that are used to model and predict the environmental fate of chemicals, and thus the exposure of organisms to these chemicals are described in section 3.8.
3.1.2. Atmosphere
Authors: Astrid Manders-Groot
Reviewer: Kees van Gestel, John Parsons, Charles Chemel
Leaning objectives:
You should be able to:
• describe the structure of the atmosphere and mention its main components
• describe the processes that determine residence time and transport distances of chemicals in air
Keywords: atmosphere, transport distance, residence time
Composition and vertical structure of atmosphere
The atmosphere of the Earth consists of several layers that have limited interaction. The troposphere is the lowermost part of the atmosphere and contains the oxygen that we breathe and most of the water vapor. It contains on average 78% N2, 20% O2 and up to 4% water vapor. Greenhouse gases like CO2 and CH4 are present at 0.0038 % and 0.0002%, respectively. Air pollutants like ozone and NO2 have concentrations that are even a factor 1,000-10,000 lower, but are already harmful for the health of humans, animals and vegetation at these concentrations.
The troposphere is 6-8 km high near the poles, about 10 km at mid-latitudes and about 15 km at the equator. It has its own circulation and determines what we experience as weather, with temperature, wind, clouds and precipitation. The lowest part of the troposphere is the boundary layer, the part that is closest to the Earth. Its height is determined by the heating of the atmosphere by the Earth surface and the wind conditions and has a daily cycle, determined by the incoming sunlight. It is not a completely separate layer, but the exchange of air pollutants like O3, NOx, SO2, and xenobiotic chemicals between the boundary layer and the above layers is generally inefficient. Therefore it is also termed the mixing layer.
Above the troposphere there is the stratosphere, a layer that is less strongly influenced by the daily solar cycle. It is very dry and has its owns circulation, with some exchange with the troposphere. The stratosphere contains the ozone layer that protects life on Earth against UV radiation and extends to about 50 km altitude. The layers covering the next 50 km are the mesosphere and thermosphere, which are not directly relevant for the transport of the chemicals considered in this book.
Properties of pollutants in the air
Air pollutants include a wide range of chemicals, ranging from metals like lead and mercury to asbestos fibers, polycyclic hydrocarbons (PAH) and chloroform. These pollutants may be emitted into the atmosphere as a gas or as a particle or droplet with sizes of a few nanometer to tens of micrometers. The particles and droplets are termed aerosol, or, depending on the measurement method, particulate matter. The latter definition is used in air quality regulations. Note that a single aerosol can be composed of several chemical compounds. Once a pollutant is released in the atmosphere, it is transported by diffusion and advection by horizontal and vertical winds and may be ultimately deposited to the Earth's surface by rain (wet deposition), and by sticking to the surface (dry deposition). Large particles may fall down by gravitational settling, a process also called sedimentation. Air pollutants may interact with each other or with other chemicals, particles and water by physical or chemical processes. All these processes will be explained in more detail below. A summary of the relevant interactions is given in Figure 1.
It is important to realize that air pollutants can have an impact on meteorology itself, by acting as a greenhouse gas, scattering or absorbing incoming light when in aerosol form, or be involved in the formation of clouds. This aspect will not be discussed here.
Meteorology is relevant for all aspects, ranging from mixing and transport to temperature or light dependent reaction rates and absorption of water. Depending on the removal rates, species may be removed with timescales of seconds, like heavy sand particles, to decades or longer, like halogen (Cl, Br)-containing gases, and be transported over ranges of a few meters to crossing the globe several times. Concentrations of gases are often expressed in volume mixing ratios (parts per billion, ppb) whereas for particulate matter the correct unit is in (micro)gram per cubic meter as there is no molecular weight associated to it. For ultrafine particles, concentrations are expressed as numbers of particles per cubic meter, for asbestos, the number of fibers per cubic meter is used.
Physical and chemical processes determining the properties of air pollutants
The properties of air pollutants, like solubility in water, attachment efficiencies to the Earth's surface (water, vegetation, soil) and size of particles, are key elements determining the lifetime and transport distances. These properties may change due to the interaction with other chemicals and with meteorology.
The main physical processes are:
• Condensation or evaporation with decreasing/increasing temperature. A potentially toxic aerosol may thus be covered by semi-volatile chemicals like ammonium sulfate, whilea gas may condensate on an aerosol and be transported further as part of this aerosol. Some pollutants exist at the same time in aerosol and in gas phase with their partitioning depending on air temperature and relative humidity.
• Gases may cluster to form ultrafine particles of a few nanometers (nucleation) that will grow to larger sizes.
• Particles may grow by coagulation: rapidly moving small particles bump into large slow-moving particles and remain attached to it.
• Particles may take up water (hygroscopicity), leading to a larger diameter.
Chemical conversions include:
• Chemical reactions between gas-phase pollutants and ambient gas, which alters the characteristics of an air pollutant or can lead to the formation of pollutants (e.g. NO2 being directly emitted by combustion, leading to ozone formation).
• Chemical reactions between aerosols and gases, often involving water attached to aerosol.
• Cloud droplets or water attached to aerosols have their own role in the chemistry of the atmosphere, and gases may diffuse into the water .
• Some pollutants may act as a catalyst.
• Some air pollutants may be degraded by (UV) light (photodegradation).
Pollutants are characterized by their chemical composition but for aerosols also the size distribution of particles is relevant. Note that the conservation of atoms always applies, but particle size distribution and particle number can be changed by physical processes. This has to be kept in mind when concentrations are expressed in particles per volume instead of mass concentrations.
Transport of air pollutants
Several processes determine the mixing and transport of chemicals in the air:
• Diffusion due to the motion of molecules, or Brownian motion of particles (random walk).
• Turbulent diffusion: the mixing due to (small-scale) turbulent eddies which have a random nature, but the strength of this diffusion is related to friction in the flow.
• Advection: the process of transport with the large-scale flow (wind speed and direction).
• Mixing or entrainment of different air masses leads to further mixing of air pollutants over a larger volume. This process is for example relevant when the sun rises, and air in the boundary layer is heated, rises, and mixes with the air in the layer above the boundary layer.
Although the processes of diffusion and transport are well-known, it is not an easy task to solve the equations describing these processes. For stationary point and line sources under idealized conditions, analytical descriptions can be derived in terms of a plume with concentration profile with a Gaussian distribution, but for more realistic descriptions the equations must be solved numerically. For complex flow around a building, computational fluid dynamics is required for an accurate description, for long-range transport a chemistry-transport model must be used.
Wet deposition
Wet deposition comprises the removal processes that involve water:
1. Scavenging of particles by or dissolution of gases in cloud droplets. These cloud droplets may grow to larger size and rain out, thereby removing the dissolved air pollutants from the atmosphere (in-cloud scavenging).
2. Particles below the clouds may be scavenged by falling raindrops (below-cloud scavenging).
3. Occult deposition occurs when clouds are in contact with the surface (mountain areas) and cloud droplets containing air pollutants stick to the surface.
Wet deposition is a very efficient removal mechanism for both small (<0.1 µm diameter) and large aerosols (diameter >1 µm). Aerosols that are hygroscopic can grow in size by absorbing water, or shrink by evaporating water under dry conditions. This affects their deposition rate for wet or dry deposition.
Dry deposition
Dry deposition is partly determined by the gravitational forces on a particle. Heavy particles (≥ 5 µm) fall to the Earth's surface in a process called gravitational settling or sedimentation. In the lowest layer of the atmosphere, air pollutants can be brought close enough to the surface to stick to it or be taken up. In the turbulent boundary layer, air pollutants are brought close to the surface by the turbulent motion of the atmosphere, up to the very thin laminar layer (laminar resistance, only for gases) through which they diffuse to the surface. Aerosols or gases can stick to the Earth's surface or be taken up by vegetation, but they may also rebound. Several pathways take place in parallel or in series, similar to an electric circuit with several resistances in parallel and series. Therefore the resistance approach is often used to describe these processes.
Deposition above snow or ice is generally slow, since the atmosphere above it is often stably stratified with little turbulence (high aerodynamic resistance), the surface area to deposit on is relatively small (impactors) and aerosols may even rebound to an icy surface (collection efficiency of impactors) to which it is difficult to attach. On the other hand, forests often show high deposition velocities since they induce stronger turbulence in the lowermost atmosphere and have a large leaf surface that may take up gases by the stomata or provide sticking surfaces for aerosols. Deposition velocities thus depend on the type of surface, but also on the season, atmospheric stability (wind speed, cloud coverage) and ability of stomata to take up gases. When the atmosphere is very dry, for example, plants close their stomata and this pathway is temporarily shut down. For particles, the dry deposition velocity is lowest at sizes of 0.1-1 µm.
Re-emission
Once air pollutants are removed from the atmosphere, they can be part of the soil or water compartments which can act as a reservoir. This is in general only taken into account for a limited number of chemicals. Ammonia or persistent organic pollutants may be re-emitted from the soil by evaporation. Dusty material or pollutants attached to dust may be brought back into the atmosphere by the action of wind. This is relevant for bare areas like agricultural lands in wintertime, but also for passing vehicles that bring up the dust on a road by the flow they induce.
Atmospheric fate modelling
Due to the many relevant processes and interactions, the fate of chemical pollutants in the air has to be determined by using models that cover the most important processes. Which processes need to be covered depends on the case study: a good description of a plume of toxic material during an accident, where high concentrations, strong gradients and short timescales are important, requires a different approach than the chronic small release of a factory. Since it would require too heavy numerical simulations to include all aspects, one has to select the relevant processes to be included. Key input for all transport models are emission rates and meteorological input.
When one is interested in concentrations close to a specific source, next to emission rate the effective emission height is important, and processes that determine dispersion: wind speed, atmospheric stability. Chemical reaction rates and deposition velocities should be included when the time horizon is long or when the reactions are fast or deposition velocities are high.
When one is interested in actual concentrations resulting from releases of multiple sources and species over a large area of interest, like for an air quality forecast, the processes of advection, deposition and chemical conversions become more relevant, and input meteorology needs to be known over the area. Sharp gradients close to the individual sources are, however, no longer resolved. In particular rain can be a very efficient removal mechanism, removing most of the aerosol within one hour. Dry deposition is slower, but results in a lifetime of less than a week and transport distances of less than 1,000 km for most aerosols. For some gaseous compounds like halogens and N2O deposition does hardly play a role and they are chemically inert in the troposphere, leading to very long lifetimes.
To assess the overall long-term fate of a new chemical to be released to the market, the potential concentrations in air, water and soil have to be determined. Ideally, models for air, soil and water are used together in a consistent way, including their interaction For many air pollutants the atmospheric lifetime is short but determines where and in which form they are deposited onto ground and water surfaces, where they may accumulate. This means that even if a concentration in air is relatively low at a certain distance from a source, the deposition of an air pollutant over a year may still be significant. Figure 2 shows an example of annual mean modelled concentrations and annual total deposition of a hypothetical passive (non-reactive) soot-like tracer that is released at 1 kg/hour at a fictitious site in The Netherlands. Annual mean concentrations are small compared to ambient concentrations of particulate matter, but the footprint of the accumulated deposition is larger than that of the mean concentration, since the surface acts as a reservoir. This implies that re-emission to air can be relevant. It may take several years for soil or water before an equilibrium concentration is reached in these compartments from the deposition input, as different processes and time scales apply. Mountain ranges are visible in the accumulated wet deposition (Alps, Pyrenees), as they are areas with enhanced precipitations.
In addition to spatially explicit modelling, also box models exist that have the advantage that they can make long-term calculations for a continuous release of a species, including interaction between the compartments air, soil and water. They can be used to determine when an equilibrium concentration is reached within a compartment, but these models cannot resolve horizontal concentration gradients within a compartment.
Figure 2. Constant release of a passive tracer from a point source in The Netherlands. Upper panel shows the annual mean concentration, the lower panel shows the accumulated wet and dry deposition over one year. Note the nonlinear colour scale to cover the large range of values. Source: https://doi.org/10.3390/atmos8050084.
Learn more
EU air quality, policy and air quality legislation: http://ec.europa.eu/environment/air/index_en.htm
US hazardous air pollutants, including lists of toxics: https://www.epa.gov/haps
Plume dispersion approach: http://courses.washington.edu/cewa567/Plumes.PDF
Chemistry-transport models: www.narsto.org/sites/narsto-dev.ornl.gov/files/Ch71.3MB.pdF
Seinfeld, J., Pandis, S.N. Atmospheric Chemistry and Physics, from air pollution to climate change, Wiley, 2016, covering all aspects.
John, A. C., Küpper, M., Manders-Groot, A. M., Debray, B., Lacome, J. M., Kuhlbusch, T. A. (2017). Emissions and possible environmental implication of engineered nanomaterials (ENMs) in the atmosphere. Atmosphere, 8(5), 84.
3.1.2. Question 1
Which processes determine the concentration of a pollutant in the air very close to its point of emission?
3.1.2. Question 2
When fine particles (<1µm diameter) are released in the lowest 100 m of the atmosphere, how far will they be transported? And what processes do contribute to removal of the particles from the air?
3.1.2. Question 3
When gases are released in the lowest 100 m of the atmosphere, how far do they get?
3.1.3. Hydrosphere
Authors: John Parsons
Reviewers: Steven Droge, Sean Comber
Leaning objectives:
You should be able to:
• describe the most important chemical components and their sources
• describe the most important chemical processes in fresh and marine water.
• be familiar with the processes regulating the pH of surface water.
Keywords: Hydrogen bonding, carbonates, dissolved salts
The properties and importance of water
Water covers 71% of the earth's surface and this water, together with the smaller amounts present as gas in the atmosphere, as groundwater and as ice is referred to collectively as the hydrosphere. The bulk of this water is salt water in the oceans and seas with only a minor part of freshwater being present as lakes and rivers (Figure 1).
Water is essential for life and also plays a key role in many other chemical and physical processes, such as the weathering of minerals and soil formation and in regulating the Earth's climate. These important roles of water derive from its structure as a small but very polar molecule arising from the polarised hydrogen-oxygen bonds (Figure 2). As a consequence, water molecules are strongly attracted by hydrogen bonding, giving it relatively high melting and boiling points, heat capacity, surface tension, etc. The polarity of the water molecule also makes water an excellent solvent for a wide variety of ionic and polar chemicals but a poor solvent for large nonpolar molecules.
The freshwater environment
As mentioned above, freshwater is only very small proportion of total amount of water on the planet and most of this is present as ice. Since this water is in contact with the atmosphere and the soils and bedrock of the Earth's crust, it dissolves both atmospheric gases such as oxygen and carbon dioxide and salts and organic chemicals from the crust. If we compare the relative compositions of cations in the Earth's crust and the major dissolved species (Table 1) it is clear that these are very different. This difference reflects the importance of the solubility of these components. For ionic chemicals, this depends on both their charge and their size (expressed as z/r2, where z is the charge and r the radius of an ion). As well as reflecting the properties of the local crust, the composition of salts is also influenced by precipitation and evaporation and the deposition of sea salt in coastal regions.
Table 1. Comparison of the major cation composition of average upper continental crust and average river water. (*except aluminum and iron from Broecker and Peng (1982))
Upper continental crust (mg/kg) (Wedepohl 1995*)
River water (mg/kg)
(Berner & Berner 1987*)
Al
77.4
0.05
Fe
30.9
0.04
Ca
29.4
13.4
Na
25.7
5.2
K
28.6
1.3
Mg
13.5
3.4
The pH of surface water is determined by both the dissolution of carbonate minerals and carbon dioxide from the atmosphere. These components are part of the set of equilibrium reactions known as the carbonate system (Figure 3).
At equilibrium with the current atmospheric CO2 concentration and solid calcium carbonate, the pH of surface water is between 7 and 9 but this may reach more acidic values where soils are calcium carbonate (limestone) poor. This is illustrated by the pH values measured in a river in Northern England, where acidic, organic carbon-rich water at the source is gradually neutralised once the river encounters limestone rich bedrock (Figure 4).
As well as these natural processes, there are human influences on the pH of surface water including acidic precipitation resulting from fossil fuel combustion and acidic effluents from mining activities caused by oxidation and dissolution of mineral sulphides. Regions such as Southern Scandinavia with carbonate-poor soils are particularly vulnerable to acidification due to these influences and this is reflected in for example, reduced fish populations in these vulnerable regions (see Figure 5). More recently, reduced coal burning and the decline in heavy industry is resulting in the recovery of pH values in upland areas across Europe.
Dissolved oxygen is of course essential to aquatic life and concentrations are in general adequate in well mixed water bodies. Oxygen can become limiting in deep lakes where thermal stratification restricts the transport of oxygen to deeper layers, or in water bodies with high rates of organic matter decomposition. This may result in anoxic conditions with significant ecological impacts and on the behaviour of chemical contaminants.
The marine environment
Freshwater eventually moves into seas and oceans where the concentrations of dissolved species are much higher than in the freshwater environment. This is partly due to the effects of evaporation of water from the oceans but is also be due to specific marine sources of some dissolved components. Estuaries are the transition zones where freshwater and seawater mix. These are highly productive environments where increasing salinity has a major impact on the behaviour of many chemicals, for example on the speciation of metals and the aggregation of colloids as a result of cations shielding the negative surface change of colloidal particles (Figure 6). Increasing salinity also affects organic chemicals, with ionic chemicals forming ion pairs, and even reducing the solubility of neutral organics (the so-called salting-out effect). As well as these chemical effects due to increasing salinity, the lowering of flow rates in estuaries leads to the deposition of suspended particles.
Since the concentrations of pollutants are in general lower in the marine environment than in the freshwater environment, concentrations in estuaries decrease as freshwater is diluted with seawater. Measuring salinity at different locations in estuaries is a convenient way to determine the extent of this dilution. Components that are present in higher concentrations in seawater will of course show an increase with salinity unless. Plotting salinity against the concentrations of chemicals at different locations can yield information on whether they behave conservatively (i.e. only undergoing mixing) or are removed by processes such as degradation or partitioning into the atmosphere or sediments. Figure 7 shows examples of plots expected for conservative chemicals and those that are either removed in the estuary or have local sources there. Models describing the behaviour of chemicals in estuaries can be used with these data to derive the rates of removal or addition of the chemical in the system.
The open ocean is sufficiently mixed for the composition of major dissolved constituents to be fairly constant, except in local situations as a result of upwelling of deep nutrient-rich waters or the biological uptake of nutrients. In coastal regions the concentrations of chemicals and other components originating from terrestrial sources may also be locally higher. The major components in seawater are listed in Table 2 with their typical concentrations.
Table 2. Major ion composition of freshwater and seawater.
Seawater (mmol/L)
(Broecker and Peng, 1982)
River water (mmol/L)
(Berner and Berner, 1987)
Na+
470
0.23
Mg2+
53
0.14
K+
10
0.03
Ca2+
10
0.33
HCO3-
2
0.85
SO42-
28
0.09
Cl-
550
0.16
Si
0.1
0.16
These concentrations may be higher in waterbodies that are partly or wholly isolated from the oceans and are impacted by evaporative losses of water (e.g. Mediterranean, Baltic, Black Sea). In extreme case, concentrations of salts may exceed their solubility product, resulting in precipitation of salts in evaporate deposits.
As is the case in freshwater, carbonates play an important role in regulating the ocean pH. The fact that the oceans are supersaturated in calcium carbonate makes it possible for a variety of organisms to have calcium carbonate shells and other structures. The important processes and equilibria involved are illustrated in Figure 8. There is concern that one of the most important effects of increasing atmospheric carbon dioxide will be lowering of ocean pH to values that will result in destabilisation of these carbonate structures.
References
Andrews, J.E., Brimblecombe, P., Jicketts, T.D., Liss, P.S., Reid, B.J. (2004). An Introduction To Environmental Chemistry, Blackwell Publishers, ISBN 0-632-05905-2.
Baird, C., Cann, M. (2012). Environmental Chemistry, Fifth Edition, W.H. Freeman and Company, ISBN 978-1429277044.
Berner, E.K., Berner, R.A. (1987). Global water cycle: geochemistry and environment, Prentice-Hall.
Broecker, W.S., Peng, T.S. (1982). Tracers in the Sea, Lamont-Doherty Geol. Obs. Publ.
Henriksen, A., Lien, L., Rosseland, B.O., Traaen, T.S., Sevaldrud, I.S. (1989). Lake Acidification in Norway: Present and Predicted Fish Status. Ambio 18, 314-321
Wedepohl, K.H. (1995). The composition of the continental crust, Geochimica Cosmochimica Acta 59, 1217-1232.
3.1.3. Question 1
The concentrations of dissolved salts in rivers and lakes is determined by three processes. Describe these processes and the characteristic composition of waterbodies where one of these processes is dominant.
3.1.3. Question 2
In addition to the well-known effects on global climate, increasing atmospheric CO2 is also expected to have an impact on the pH of the oceans. Which processes are responsible for determining the pH of the oceans?
3.1.3. Question 3
How could increasing atmospheric CO2 affect these processes?
3.1.3. Question 4
What effects could increasing acidity of the oceans have on marine organisms?
In preparation
3.1.5. Soil
Author: Kees van Gestel
Reviewers: John Parsons, Jose Alvarez Rogel
Learning goals
You should be able to
• describe the main components of which soils consist
• describe how soil composition influences properties that may affect the fate of chemicals in soil
Keywords:
Particle size distribution, Porosity, Minerals, Organic matter, Cation Exchange Capacity, Water Holding Capacity
Introduction
Soil is the upper layer of the terrestrial environment that serves as a habitat for organisms and medium for plant growth. In addition, it also plays an important role in water storage and purification and helps to regulate the Earth's atmosphere (e.g. carbon storage, gas fluxes, …).
Soils are composed of three phases (Figure 1).
The solid phase is formed by mineral and organic components. Mineral components appear in different particle sizes from coarse particles (sand), intermediate (silt) and fine (clay) which combination determine soil texture. The particles can be arranged to form porous aggregates; soil pores being filled with air and/or water. The proportion of air in soils depends on soil moisture content. The composition of the soil solid phase may be quite variable.
The gaseous phase has a similar composition as the air, but due to the respiration of plant roots and the metabolic activity of soil microorganisms, O2 content generally is lower and CO2 content higher. Exchange of gases between soil pores and atmospheric air takes place by diffusion. Diffusion proceeds faster in dry soil and much slower when soil pores are filled with water.
The liquid phase of the soil, the soil solution or pore water, is an aqueous solution containing ions (mainly Na+, K+, Ca2+, Cl-, NO3-, SO42-, HCO3-) from dissolution of a variety of salts, and also contains dissolved organic carbon (DOC, also referred to as dissolved organic matter, DOM). The soil solution is part of the hydrological cycle, which involves input from among others rain and irrigation, and output by water uptake by plants, evaporation, and drainage to ground and surface water. The soil solution acts as a carrier for the transport of chemicals in soil, both to plant roots, soil microorganisms and soil animals and to ground and surface water.
Soil solids
The soil solid phase consists of mineral and organic soil particles. Based on their size, the mineral particles are divided into sand (63-2000 µm), silt (2- 63 µm), and clay (<2 µm). With increasing particle size, the specific surface area decreases, pore size increases and water retention capacity decreases. The sand fraction mainly consists of quartz (SiO2) and does not have any sorption properties because the quartz crystals are electrically neutral. Sandy soils have large pores, so a low capacity to retain water. In soils with a high silt fraction, smaller pores are better represented, giving these soils a higher water retention capacity. Also the silt fraction has no adsorptive properties. Clays are aluminium silicates, lattices composed of SiO4 tetrahedrons and Al(OH)6 octahedrons. Upon the formation of clay particles, isomorphic substitution occurred, a process in which Si4+ was replaced by Al3+, and Al3+ by Mg2+. Although having similar diameters, these elements have different valences. As a consequence, clay particles have a negative charge, making positive ions to accumulate on their surface. This includes ions important for plant growth, like NH4+, K+, Na+ and Mg2+, but also cationic metals (Figure 2). Many other minerals have pH-dependent charges (either positive or negative) which are also important in binding cations and anions.
In addition to mineral particles, soils also contain organic matter, which includes all dead plant and animals remains and their degradation products. Living biota is not included in the soil organic matter faction. Organic matter is often divided into: 1. humin, non-dissolved organic matter associated with clay and silt particles, 2. humic acids having a high degree of polymerization, and 3. fulvic acids containing more phenolic and carboxylic acid groups. Humic and fulvic acids are water soluble but their solubility depends on pH. For example, humic acids are soluble at alkaline pH but not at acidic pH. The dissociation of the phenolic and carboxylic groups gives the organic matter also a negative charge (Figure 3), the density of which increases with increasing soil pH. The soil organic matter acts as a reservoir of nitrogen and other elements, provides adsorption site for cations and organic chemicals, and supports the building of soil aggregates and the development of soil structure.
The binding of cations to the negatively charged sites on the soil particles is an exchange process. The degree of cation accumulation near soil particles depends on their charge density, the affinity of the cations to the charged surfaces (which is higher for bivalent than for monovalent cations), the concentration of ions in solution (the higher the concentration of a cation in solution, the higher attraction to soil particles), etc. Due to their binding to charged soil particles, cations are less available for leaching and for uptake by organisms. The Cation Exchange Capacity (CEC) is commonly used as a measure of the number of sites available for the sorption of cations. CEC is usually expressed as cmolc/kg dry soil. Soils with higher CEC have a higher capacity to bind cations, so cationic metals show a lower (bio)availability in high CEC soils (see the Section on metal speciation). CEC depends on the content and type of clay minerals, with montmorillonite having a higher CEC than e.g. kaolinite, organic matter content and pH of the soil. In addition to clay and organic matter, also aluminium and iron oxides and hydroxides may contribute to the binding of cations to the soil.
Soil water
The transport of water through soil pores is controlled by gravity, and by suction gradients which are the result of water retention by capillary and osmotic processes. Capillary binding of water is stronger in smaller soil pores, which explains why clayey soils have higher water retention capacities than sandy soils. The osmotic binding of water increases with increasing ionic strength, and is especially high close to charged soil particles like clay and organic matter where ions tend to accumulate.
The stronger water is retained by soil, the lower its availability is for plants and other organisms. The strength by which water is retained depends on moisture content, because 1. at decreasing moisture content the ionic strength of the soil solution and therefore osmotic binding increases, 2. when soil moisture content decreases the larger soil pores will be emptied first, leading to increasing capillary retention of the remaining water in smaller pores. Water retention curves describe the strength with which water is retained as a function of total water content and in dependence of the composition of the soil. Figure 4 shows pF curves for three different soil types.
A pF value of 2.2-2.5 corresponds with a binding strength of 200 to 300 hPa. This is called field capacity; water is readily available for plant uptake. At pF 4.2 (15,000 hPa), water is strongly bound in the soil and no longer available for plant uptake; this is called the wilting point. For soil organisms, not the total water content of a soil is of importance but rather the content of available water. Water retention curves may be important to describe the availability of water in soil. Toxicity tests with soil organisms are typically performed at 40-60% of the water holding capacity (WHC) of the soil, which corresponds with field capacity.
References/further reading
Schulten, H.-R., Schnitzer, M. (1997). Chemical model structure for soil organic matter and soils. Soil Science 162, 115-130.
Blume, H.-P., Brümmer, G.W., Fleige, H., Horn, R., Kandeler, E., Kögel-Knabner, I., Kretzschmar, R., Stahr, K., Wilke, B.-M. (2016). Scheffer/Schachtschabel Soil Science, Springer, ISBN 978-3-642-30941-0
3.1.5. Question 1
What are the main components of the solid phase of a soil?
3.1.5. Question 2
When considering two different soils, a sandy and a clayey soil, which one has the highest available volume of water when both soils have the same total water content? Explain your answer.
3.1.5. Question 3
What is the agricultural and environmental importance of the cation exchange capacity of soils?
3.1.5. Question 4
Explain the role of clay minerals and organic matter in the retention of cationic metals in soils.
3.1.5. Question 5
Which two factors explain the fact that the availability of cationic metals increases with decreasing soil pH?
3.1.6. Groundwater
(draft)
Author: Thilo Behrends
Reviewer: Steven Droge, John Parsons
Leaning objectives:
You should be able to:
• understand the significance of redox reactions for the fate of potentially toxic compounds in groundwaters and aquifers.
• apply the Nernst equation to assess the feasibility of redox reactions.
Keywords: Aquifer, Nernst equation, electron transfer, redox potential, half reactions
Introduction
Some definitions conceive all water beneath the earth's surface as groundwater while others restrict the definition to water in the saturated zone. In the saturated zone the pores are completely filled with water in contrast to the undersaturated zone in which some pores are filled with gas and capillary action are important for moving water. Geological formations, which host groundwater in the saturated zone, can be classified as 'aquifer', ' aquitard', or 'aquifuge' depending on their permeability. In contrast to aquitard and aquifuge, which have a low permeability, an aquifer permits water to move in significant rates under ordinary field conditions. Aquifers typically have a high porosity and the pores are well connected with each other. Examples of aquifers include sedimentary layers of sand or gravel, carbonate rocks, sandstones, volcanic rocks and fractured igneous rocks. The redox chemistry discussed in this chapter is focusing on aquifers in sedimentary formations.
Groundwaters are an important source for drinking water and the quality of groundwater is, therefore, of high importance for protecting human health. However, aquifers also represent a habitat for bacteria and aquatic invertebrates and are, therefore, also an object for ecotoxicological studies. Furthermore, groundwater can act as a transportation pathway connecting different environmental compartments e.g. soils with rivers or oceans. Groundwater thus plays a role in the distribution of contaminants in the environment.
Transport of contaminants in aquifers
The movement of a chemical in groundwater is controlled by three processes: advection, dispersion and reaction. Advection is the transport of a chemical in dissolved form together with the groundwater flow. When a chemical is released from a point source into groundwater with a constant flow direction, a plume is forming downstream of the source. The spreading of the chemical is due to dispersion. There are two reasons for this spreading: First, molecular diffusion causes transport of the chemical independently from advection; Second, differences in groundwater velocities at different scales causes mixing of the groundwater (mechanical dispersion) in the direction of groundwater flow but also perpendicular to it. Several process can retard the transport of chemicals or can cause its removal from the system (e.g. degradation). For the mobility of a chemical, the distribution between immobile solid phase and moving liquid phase is of key importance in groundwater (see chapter 3.4). There are several processes which can lead to the degradation of a compound in aquifers. Microbial activity can contribute to the degradation of chemicals but also abiotic reactions can be of importance. For some chemical, redox reactions are relevant which are discussed in the following section.
Redox reactions in aquifers
Many elements are redox-sensitive under environmental conditions. This means they occur naturally in different 'redox states'. For example oxidation or reduction of carbon plays a pivotal role in the energy metabolism of living organisms and carbon occurs in oxidation states from +IV in CO2 (because the two oxygen atoms both count as -II ((because oxygen is more electronegative than carbon)), and the total molecule should balance out) to -IV in CH4 (because each H-atom counts as +I ((because hydrogen is less electronegative than carbon))). Also potentially toxic elements, such as arsenic, are found in nature in different oxidation states. Important oxidation states of arsenic are +V, (e.g. AsO43-, arsenate), + III (e.g. AsO3-3, arsenite), 0 (elemental arsenic or arsenic associated with sulfide as in FeAsS, arsenopyrite). Arsenic can also have negative oxidation states when it forms arsenides such as FeAs2 (löllingite). Bioavailability, toxicity and mobility of redox sensitive elements are usually strongly dependent on their oxidation state. For example, arsenite tends to be more toxic and more mobile than arsenate. For this reason, assessing the redox state of potentially toxic elements is an important element of environmental risk assessment of groundwater.
Organic contaminants can also undergo redox transformations. At the earth surface, when oxygen is present, (photo-)oxidation is an important degradation pathway for organic contaminants. In subsurface environments, when oxygen concentrations are often very low (anoxic conditions), reduction can play an important role in degradation pathways. For example, the reductive dehalogenation of chlorinated hydrocarbons or the reduction of nitroaromatic compounds have been extensively investigated. The reduction of these compounds can be mediated by microorganisms but they can also occur abiotically on solid surfaces present in the subsurface. In any case, reduction of organic contaminants is only possible when the reaction is thermodynamically feasible. For this reason it is necessary to know the redox conditions in, for example, an aquifer.
Quantitative assessment of redox reactions
As the name indicates, redox reactions combine oxidation of one constituent in the system with the reduction of another and, hence, involve electron transfer. The oxidation of arsenite with elemental oxygen to arsenate has following stoichiometry:
It is important that the stoichiometries of redox reactions are not only charge- and mass-balanced but also electron-balanced. Here, arsenic releases two electrons when going from oxidation state +III to +V (arsenite becomes oxidized to arsenate) while one oxygen atom takes up the two electrons and goes from oxidation state 0 to -II (elemental oxygen becomes reduced). For this reaction an equilibrium constant can be obtained and based on the activities (or concentrations) of dissolved reactants and products it can be evaluated whether the reaction is in equilibrium or in which direction the reaction is thermodynamically favorable.
When a natural system contains several different redox-active constituents, a large number of possible redox reactions can be formulated and evaluated separately. In this situation it is more convenient to formulate and compare half reactions. For examples, the oxidation of arsenite with oxygen can be split up into the reactions of arsenic and oxygen.
Half reactions are typically formulated as reduction reactions (electrons are on the left hand side of the reaction). The Eho is the standard redox potential and represents the electrical potential, which would be measured in a standardized electrochemical cell which contains on one side, H3AsO4, H3AsO3 and H+, all with activities of 1 mol l-1, and a solution containing 1 mol l-1 H+ in equilibrium with H2 gas with a pressure of 1 bar, on the other side.
In natural environments the pH is usually not 0 and the activities of arsenite and arsenate are not 1 mol l-1. The redox potential, Eh under these conditions can be calculated using the Nernst equation:
where:
R is the ideal gas constant ( 8.314 J mol-1 K-1),
T the temperature in K,
z is the number of electrons which are transferred in the reaction,
F the Faraday constant (96485 mol C-1).
In the ratio ox/red, 'ox' represents the activities or pressures of the constituents on the right hand side of the half reaction, whereby the stoichiometric factor becomes the corresponding exponent, while 'red' represents the right hand side of the half reaction.
The redox potentials of different half reactions can be compared:
• The half reaction with the higher redox potential provides the electron acceptor in the thermodynamically favorable redox reaction,
• The half reaction with the lower potential provides the electron donor.
In other words, it is thermodynamically favorable that the half reaction with the high potential proceeds from left to right and the half reaction with the low potential from right to left.
Redox conditions in aquifers
The redox conditions in an aquifer depends on the inherited inventory of oxidants and reductants during the formation of the geological formation and the processes which have been occurring throughout its history. Oxidants and reductants can have entered the aquifer by diffusion or with the infiltrating water and slowly progressing redox reaction can have modified the assemblage of oxidants and reductants. In the absence of (microbial) catalysis redox reactions often have very slow kinetics. Furthermore, due to photosynthesis, redox reactions are not in equilibrium at the earth's surface and the upper part of the underlying subsurface. As a consequence, the redox conditions in an aquifer can usually not be represented in one unique redox potential. This implies that values obtained for groundwaters with electrochemical measurements, e.g. potentiometric measurements using redox electrodes, might be not representative for the redox conditions in the aquifer. Furthermore, relevant half reactions in the aquifer often involve solids (heterogeneous reactions) with low solubility, implying that the concentrations in solution (for example of Fe3+) are too low to be detected. Hence, evaluating the redox conditions in subsurface environments is often challenging.
Oxygen concentrations in groundwaters are often virtually zero, as oxygen in infiltrating rain water or entering the subsurface by molecular diffusion is often consumed before it can reach the aquifer. Hence, 'reducing conditions' typically prevail in aquifers. The redox potential measured in a system may reflect the dominant electron acceptors besides oxygen that are present in the system (Figure 1).
In sediments or sedimentary rocks, redox reactions after deposition are predominately driven by the oxidation of organic matter, which entered the sediment during its deposition. However, the aquifer might also have received dissolved organic matter via infiltrating water. The oxidation of organic matter is predominately microbially mediated and predominately coupled to the reduction of elemental oxygen (if present). However, when elemental oxygen is depleted, which is usually the case, other electron acceptors are used by microorganisms. Relevant electron acceptors (see Figure 1) in anoxic environments include:
• Nitrate (in dissolved form),
• Mn(IV) (as solid surface),
• Mn(III) (as solid surface),
• Fe(III) (as solid surface),
• Sulphate (in dissolved form).
Nitrate and sulphate can be present in dissolved form while Mn(IV), Mn(III), Fe(III) occur as solids with low solubility. The (hydr)oxide solids of these metals, such as goethite (FeOOH) or manganite (MnOOH) are mostly accessible for microbial reduction while Mn(III) or Fe(III) in silicates can only be partially reduced or are not bioavailable for reduction. When also these electron acceptors run short, methanogenesis can be initiated.
Microorganisms, which reduce sulphate, Mn or Fe(III), can use the products of fermentative organisms. These fermentative organisms produce short-chain fatty acids, such as acetate or lactate, but often also release hydrogen gas. That is, hydrogen concentrations in groundwater reflect a steady state of hydrogen production and consumption, and are typically limited by the rates of production. As a consequence, hydrogen concentrations in groundwater are often at the physiological limit of the consuming organism. The concentrations are just sufficient to allow the organism to conserve energy from oxidizing the hydrogen. This limit increases according to the sequence of electron acceptors (Figure 1): nitrate reduction < Mn reduction < Fe reduction < sulphate reduction < methanogenesis when the corresponding compounds are present in relevant amounts or concentrations. For this reason, concentrations of dissolved hydrogen can be a useful indicator to identify the dominant, anaerobic respiration pathway in an aquifer. For example, one can determine whether sulphate reduction is enabled or methanogenesis has set in. The hydrogen concentrations in the groundwater can also directly be used to assess whether the microbial reduction of metals, metalloids, chlorinated hydrocarbons, nitro aromatic compounds or other organic contaminants is feasible.
The reduction of Fe(III)(hydr)oxides are sulphate leads to the formation of Fe(II) and sulphide, which, in turn, typically results in the precipitation of ferrous solids such as FeCO3 (siderite), FeS (mackinawite) or FeS2 (pyrite). These Fe(II) containing minerals often play an important role in the abiotic reduction of organic or inorganic contaminants in aquifers. When the composition of the groundwater and the mineral assemblage is known, the Nernst equation can be used to calculate the redox potential of relevant half reactions in the aquifer. These redox potential can be then used for evaluating whether reduction of potentially toxic compounds is possible or not. For example, the half reaction for the reduction of an amorphous ferric iron hydroxide coupled to the precipitation of siderite is given by:
At given pH and carbonic acid concentration, the corresponding redox potential can be calculated using the Nernst equation. This redox potential can be compared to that obtained from the Nernst equation for the reductive dichlorination of tetrachloroethylene (Cl2C=CCl2)
With this approach the feasibility of redox reactions involving potentially organic and inorganic compounds can be evaluated in aquifers. That does, however, not imply that the corresponding reactions also occur within the relevant time scale. For this the kinetics of the reaction have to be known and have been studied for many reactions of potential relevance in aquifer systems. However, the kinetics of redox reactions are not subject of this section.
References
Sparks, D. (2002). Environmental Soil Chemistry, Second Edition, Academic Press, Chapters 5 and 8, ISBN 978-0126564464.
Essington, M.E. (2004). Soil and Water Chemistry: An Integrative Approach, Chapters 7 and 9, CRC Press, ISBN 978-0849312588
3.1.6. Question 1
Which processes control the movement of chemicals in groundwater? Define each of these processes.
3.1.6. Question 2
Redox reactions of contaminants in groundwater are controlled by the redox potential. What are the most important chemicals and processes determining the redox potential?
3.1.6. Question 3
How can the redox potential in aquifer be determined? What are the disadvantages of these methods?
3.1.7. Biota
(draft)
Author: Steven Droge,
Reviewer: Nico van der Brink, John Parsons
Leaning objectives:
You should be able to:
• explain the effect of cell and body composition on toxicokinetics of compounds
• describe the role of biota in the environmental fate of chemicals
Keywords: cellular composition, body composition, exposure routes, absorption, distribution
Introduction
Just like soil, water, and air, the organic tissue of living organisms can also be regarded as a compartment of the ecosystem where chemical pollutants can accumulate or can be broken down. The internal concentration in living organisms provide important information on chemical exposure and ultimately determines the environmental risk of pollution, but it is important to understand the key features of tissue that influence chemical partitioning into organisms. Chemical accumulation in the tissue of living organisms is a series of chemical and biological processes, briefly based on:
- chemical uptake (mostly permeation from bulk media over certain membranes into cells);
- internal distribution (e.g. via blood flows through organs);
- metabolism (e.g. biotransformation processes in for instance the liver).
- excretion (e.g. through urine and feces, but also via gills, sweat, milk, or hairs)
These four processes are the basis of toxicokinetic modeling, and are often summarized as Absorption, Distribution, Metabolism, and Excretion, or "ADME". These ADME processes can strongly vary for different polluting compounds due to the properties of the chemical structure. These ADME processes can also strongly vary for different organisms, because of:
- the physiological characteristics (e.g. having gills, lungs, or roots, availability of specific chemical uptake mechanisms, presence of specific metabolic enzymes, size-related properties like metabolic rate),
- the position in the polluted environment (flying birds or midge larvae living in sediment),
- the interaction with the polluted environment (living in soil or water, food choice, etc.)
- the behaviour in the polluted environment (being sessile or able to move (temporarily) away from a polluted spot).
More details of these toxicokinetic processes are presented in section 4.1 on Toxicokinetics and bioaccumulation. The current module aims to provide a summary of the key features of different tissue components that explain the internal distribution of chemicals (distribution), the different types of contact between pollutants and organisms (exposure-absorption), and temporal changes in physiology that may affect internal exposure (e.g. excretion, which includes examples such as release of POPs via lactation, and increasing POP concentrations during starvation). Before we discuss how chemicals are taken up into biota, it is important to first define the key chemical properties and the molecular composition of tissue that influence the way chemicals are absorbed from the surrounding environment and distributed throughout an organism.
Absorption-distribution: Tissue building blocks
All organisms are composed of cells, which are composed of a cell membrane, surrounding the largely watery solution filled with inner organelle membranes, protein structures, and DNA/RNA. Prokaryote organisms such as bacteria, but also algae, fungi and plants have reinforced membranes with cell walls to prevent water leaking by high osmotic pressures, and to protect the cell membrane. Metabolic energy is stored in large molecules such as fatty esters and sugars. Remarkably, for all existing living organism species, these tissue components are mostly structures made out of relatively simple and repetitive molecular building blocks, with minor variations on side chains. See examples in Figure 1. The composition of organs, as a collection of specific cells, in terms of the percentage of lipids, proteins and carbohydrates is important for the overall toxicokinetics of chemicals in the whole organism.
Cell walls are mostly made from highly polar polysaccharides, e.g.:
• cellulose, a polymer of sugary molecules, and chitin in fungi, which is highly polar and therefore permeable for water.
• peptidoglycan semi-crystal structures surrounding bacteria (90% of the dry weight of Gram-positive bacteria but only 10% of Gram-negative strains), a mixed polymer between N-acetylglucosamine (alike chitine) and short interconnecting 4 or 5 amino acid chains.
• lignin, a polar (~30% oxygen) but more hydrophobic supra-structure of polymerized phenolic molecules lining the main plant vessels that transport water.
The specific algae group of diatoms have a cell wall composed of biogenic silica (hydrated silicon dioxide), typically as two valves that overlap each other surrounding the unicellular species. Diatoms generate about 20 percent of the oxygen annually produced on the planet, and contribute nearly half of the organic material found in the oceans. With their specific cell wall structure, diatoms take in over 6.7 billion metric tons of silicon each year from the waters in which they live, which creates huge deposits when they die off.
Cell membranes are made up mostly of a phospholipid bilayer, with each phospholipid molecule basically having a polar and ionized headgroup connected to two long alkyl chains (Figure 1 example with POPC type phospholipid). The outer sides of a phospholipid bilayer are hydrophilic (water-loving), the inside is hydrophobic (water-fearing). Ions (inorganic salts, nutrients, metals, strong acids and ionized biomolecules) do not readily permeate through such a membrane passively, and require specific transport proteins that can transport as well as regulate ions in and out of the cell interior. Cholesterol molecules stabilize the fluidity of the membrane bilayers in cells of most organisms, but for example not in most Gram negative bacteria. Dissolved neutral chemicals may passively diffuse through phospholipid bilayers into and out of cells.
Proteins are chains of a variety of amino acids, 21 of which are known to be genetically coded, and of which humans can only produce 12. The other nine must be consumed, and are therefore called essential amino acids (coded H, I, L, K, M, F, T, W, V). Proteins form complex 3 dimensional structures that allow for enzymatic reactions to occur effectively and repeatedly. There are two amino acids with side chains that carry a positive charge at neutral pH: Arginine (pKa 12) and Lysine (pKa 10.6), and two amino acids with side chains that carry a negative charge at neutral pH: Aspartic acid (pKa 3.7) and Glutamic acid (pKa 4.1). Some amino acids carry typical hydrophobic side chains: amongst others Leucine and Phenylalanine. Cysteine has a thiol (SH) moiety that can form strong connective disulfide interactions with spatially nearby cysteine side groups in the 3D structure. The key blood transport protein albumin, for example, contains about 98 anionic amino acids, and 83 cationic amino acids, and about 35 cysteine residues.
DNA and other genetically encoding chains are composed of 4 different nucleotides that form a double helix of two opposing strands, held together by hydrogen bonds connecting the complementary bases: A and T (or A and U in RNA), and G and C. DNA can be densely packed around histone proteins, and is either or part of the cellular cytoplasm (in prokaryotic species) or separated within a membrane (in eukaryotic species). DNA is not a critical accumulation phase for chemicals, but of course it is a cellular structure where pollutants can strongly impact all kinds of cellular processes when they react with DNA components or affect the structural organisation otherwise.
Storage fat provides for many animals and fruits of plants an important energy reserve, but also insulates warm-blooded animals in cold climates, lubricates joints to move smoothly, and protects organs from shocks (e.g. eyes and kidneys). Seeds and nuts may contain up to 65% (walnuts) of fatty components, which of course provides energy for initial growth, but from which also oil can be pressed. Storage fat in most animals is present in the form of triglycerides, and as such neutral and very hydrophobic phases within tissue. Polyunsaturated fatty acid esters like omega-6 and omega-3 fatty acids are abundant in fish (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) and in seeds and plants (mostly alpha-linolenic acid (ALA), but algae also contain EPA and DHA). The high intake of algae by fish in aqueous food webs based on algae, results in the high EPA/DHA levels in many fish species, as they mostly cannot make it themselves (https://www.pufachain.eu). Humans can make some EPA and DHA from ALA.
The average composition of living organisms based on the key tissue components lipid, protein, and carbohydrate can range widely, as illustrated in Table 1.
Table 1. Tissue structure composition of the average dry weight of different organisms.
Organism
lipid
% of d.w.
protein
% of d.w.
carbohydrate
% of d.w.
Grass
0.5-4
15-25
60-84
Phytoplankton
20
50
30
Zooplankton
15-35
60-70
10
Oyster
12
55
33
Midge larvae
10
70
20
Army cutworms (moth larvae)
72% of body
Pike filet
3.7
96.3
0
Lake trout
14.4
85
0
Eel (farmed for 1.5y)
65
~34
1
Deer game meat
10
90
0
Table 2. Estimates on the tissue structure composition of a woman (BW= 60 kg, H = 163 cm, BMI = 22.6 kg/m2), (taken from Goss et al., 2018). Bones are not included.
Organ
Total organ volume (mL)
moisture
content
phospholipid
% of d.w.
storage lipid
% of d.w.
protein
% of d.w.
Adipose
22076
26.5%
0.3%
93.6%
6.1%
Brain
1311
80.8%
35.4%
22.6%
42.1%
Gut
1223
81.8%
9.9%
22.0%
68.1%
Heart
343
77.3%
19.1%
17.7%
63.2%
Kidneys
427
82.4%
16.5%
5.9%
77.7%
Liver
1843
79.4%
19.4%
8.0%
72.6%
Lung
1034
94.5%
13.1%
13.7%
73.2%
Muscle
19114
83.7%
2.6%
2.5%
95.0%
Skin
3516
71.1%
2.6%
22.9%
74.5%
Spleen
231
83.5%
5.6%
2.4%
92.0%
Gonads
12
83.3%
18.8%
0.0%
81.3%
Blood
4800
83.0%
2.5%
2.4%
95.1%
total
55929
60.2%
1.7%
71.0%
27.3%
Different organs in a single species can also largely differ in their composition, as well as their contribution to the overall body, as shown for a human in Table 2. Most organs have a moisture content >75%, but overall the moisture content is considerably lower, due to the low moisture content of bones and adipose tissue. Adipose tissue is by far the largest repository of lipids, but made up mostly of storage lipid, while the brain is also particularly rich in both lipids, but particularly enriched in phospholipids of cell membranes. Muscles and blood contain relatively high protein content.
Absorption-distribution: Chemical properties
The influence of chemical structure on the accumulation of chemicals in the biotic compartments is largely dependent on their bioavailability, as discussed in more detail in section 4.1 on Toxicokinetics and bioaccumulation, as well as on the basic binding properties as the results of the chemical's hydrophobicity and volatility (section 3.4 on Partitioning and partitioning constants) and ionization state (section 2.2.6 on Ionogenic organic chemicals). In brief, the more non-polar the composition of a chemical, the more hydrophobic it is, the higher its affinity to partition from dissolved phases (both externally as well as internally) into poorly hydrated tissue phases such as storage fat and cell membranes. For this reason, the main issue with classical organic pollutants such as dioxins, DDT, and PCBs, is often their high hydrophobicity which results in strong accumulation in tissue. Such chemicals often take very long times to excrete from the tissue if they are not made less hydrophobic via biotransformation processes. This leads to foodweb accumulation and specific acute or chronic toxic effects at a certain organism level (section 4.1.6 on Food chain transfer). Proteins and sugary carbohydrates are mostly comprised of extended series of polar units and thus strongly hydrated, and bind hydrophobic chemicals to a much lower extent. Proteins may have three dimensional pockets that could fit either hydrophilic of hydrophobic chemicals, and as such act as transport proteins in blood throughout the body (transporting fatty acids for example), based on the specific binding affinity. Many protein based receptors are also based on a specific binding affinity, and in many cases this involves (combinations of) polar and electrostatic interactions that also have an optimum three-dimensional fitting space. Volatile chemicals are more abundantly present in the gas phase rather than being dissolved, and are more readily in contact with biota via gas-exchange on the extensive surfaces of lungs of animals and leaves of plants.
In order to be taken up into cells, or into organs, chemicals have to permeate through membranes. For most organic pollutants, the passive diffusion through phospholipid bilayers has an optimum at a certain hydrophobicity. The high accumulation in the membrane ensures desorption into the adjacent cellular solution. It is assumed that ionized chemicals have a passive permeation rates that are either negligible or at least orders of magnitude lower than that of corresponding neutral chemicals. For this reason, all kinds of molecular intra-extracellular gradients can be readily maintained, for example for protons (H+) or sodium-potassium (Na+/K+). The movement of very polar and ionic chemicals can be tightly regulated by transport proteins protruding the membrane bilayer. Specific molecules can be actively excreted from cells (e.g. certain drugs) or reabsorbed (e.g. in the kidneys back into the blood stream). This again is based on three dimensional fitting in the transport pocket and stepwise movement through the protein structure, and costs energy. For acids and bases with a very small fraction of neutral species at physiological pH, the passive permeation over membranes may still be dominated by the neutral species.
Exposure: Contact between biota and various environmental compartments
There are multiple routes by which chemicals can enter the tissue of biota, for example via respiratory organs, through digestion of contaminated food, or dermal contact. Most animals need to take in a more or less constant flux of oxygen and water, and periodically food, to release nutrients and energy from the food. Of course they also need to release CO2 (and other chemicals) as waste. Pollutant chemicals are taken up alongside these basic processes, and it depends on the chemical properties and the efficiency of the uptake route how much the organism will take in from these different exposure routes.
Plants need plenty of water and during daytime photosynthesis need CO2, but also require oxygen during the night. High algal densities can deplete the oxygen levels in shallow aquatic systems during the night, and replenish oxygen levels during daytime. Oxygen is plentiful in air (200,000 parts per million in the air), but it is considerably less accessible in water (15 parts per million in cool, flowing water), and often depleted below the first few mm of sediment. To obtain sufficient oxygen, water and food, aquatic organisms have to pass large volumes of waters through their gills. Sediment-dwelling organisms either have hemoglobin to bind oxygen, or constantly pump fresh overlying water through burrows created in sediment, often lined with mucus. Living organisms are thus constantly in contact with water dissolved pollutants, and air breathing organism are readily exposed to air pollutants. To simplify the domain of living organisms as part of this module about the biotic compartment and how they get into contact with chemicals relevant to Environmental Toxicology, they can for example be divided in:
• , which often take in large amounts of water from their surroundings via their roots, driven by the evaporation of water at the leaves and resulting internal flows.
• water breathing organisms, which pass large amounts of water through gills or gill like structures (tubules or other thin skin structures close to where water is passing) in order to take in enough oxygen and reduce the built up of CO2. Filter feeders like oysters and mussels, that populate enormous surfaces as reefs or banks, can turnover a huge volume of water on a daily basis, and thus allow dissolved chemicals get in close contact to the outer membranes.
• air breathing organisms, which can effectively exchange large quantities of volatile and gaseous chemicals with the air (oxygen but also organic compounds), but typically take in less/non-volatile chemicals via food and require active excretion and metabolism for the emission of less/non-volatile chemicals.
Plants
Nearly all plants have roots below ground, a sturdy structure of stem and branches above ground, and leaves. Along with soil pore water, soluble chemicals are readily transported from roots of the plant in the internal circulation stream through xylem cells, which are lined with water impenetrable lignin (see Figure 2). Moderately hydrophobic chemicals (Kow of 1-1000) are rapidly transported from roots to shoots to leaves, while hydrophobic chemicals may be strongly retained on the membranes and cell walls and mostly accumulate in root sections, limiting transport to above-ground plant tissues. Roots may also actively release considerable quantities of chemicals to influence the immediate surrounding media of the roots (rhizosphere), e.g. to stimulate microbial processes or pH in order to release nutrients. These plant root 'exudates' can be ions, small acids, amino acids, sterols, etc. Chemicals that enter plants via leaves, such as pesticides or semi-volatile organic pollutants, can be redistributed to other plant parts via the phloem streams.
The transport through xylem up to higher plant tissues occurs via capillary forces and is enhanced by three passive phenomena:
• the high sugar content in the phloem causing osmotic pressure to attract water from other parts.
• the evaporation of water creates surface tension on thousands of cells that pulls water from the soil through the xylem system.
• the osmotic pressure of root cells compared to soil pore water. Root pressure is highest in the morning before the stomata open and allow transpiration to begin.
As a result of the capillary forces needed to pull water up against gravity, and a certain maximum diameter of the vessels to do so, there is a maximum possible plant height of 122-130 m (Koch et al., 2004) which compares to Redwood trees (Sequoias) reaching a maximum height of 113 m.
Most leaves are covered with a waxy layer, to prevent damage and water evaporation. This wax layer may be 0.3-4.6 µm thick (Moeckel et al., 2008). Large forests provide enormous hydrophobic surfaces to which semi-volatile organic chemicals (SVOC) can bind out of air, which influences the global distribution of chemicals such as PCBs. Partitioning of SVOCs on the vegetation of extended grasslands contaminates the base of the food chain, as well as agricultural and cattle sectors used by humans. The grass/corn-cattle-milk/beef food chain accounts for the largest portion of background exposure of the European and North American population to many persistent SVOCs. The absorption rate of chemicals on the leaves often also depends on the air boundary layer surrounding leaves, which limits diffusion into the leave surfaces. Of course, all kinds of other factors such as wind speed, canopy formation and cuticle thickness also control exchange between leaves and gas phase (see also section 3.1.2 on the Atmosphere). Tiny openings or pores on the lower side of the leaves, called stomata, allow furthermore for gas exchange. In warm conditions, stomata can close to prevent water evaporation, but gas exchange is needed in many plant types to allow for CO2 to be metabolized and the release of O2 that is produced. The waxy layer on leaves can trap gaseous organic chemicals. Many plants, like coniferous trees, produce resins to provide effective defense against insects and diseases, and these resins release large amounts and structurally highly diverse organic volatiles such as terpenes and isoprenes (Michelozzi, 1999). These plant-produced volatiles can even contribute to ozone formation. Plants thus accumulate chemicals from their environment, but also release chemicals into the environment. It thus also matters for the exposure of grazing organisms to certain types of pollutants whether they eat roots, shoots, leaves, seeds or fruits of plants living in contaminated environments.
Organisms using dissolved oxygen
The 'gill' movements of water breathers create a constant flux of chemicals dissolved in bulk water along outer cell membranes (or mucus layers surrounding cell membranes) of gills. A 1 kg rainbow trout fish ventilates about 160 mL/min, so 230 L/day (Consoer et al., 2014). The total gill surface area in a fish depends on species behaviour and weight (active large fish require a lot of oxygen), and equals to about 1-6 cm2 per g fish (Palzenberger & Pohla, 1992). For a 1 kg fish of ~20 cm length, the ~1000 cm2 gill area compares to a ~500 cm2 outer body surface. This results in an effective partitioning of chemicals between water and cell membranes. Within the gills, the cells are in close contact with the blood system of the organism, and the build-up of chemical concentrations in the outer cells provides an effective exchange with the blood stream (or other internal fluids, Figure 2) flushing along that redistribute chemicals to the inner organs. The reverse equally occurs: chemicals dissolved in blood stream coming from organs will also rapidly exchange with bulk external water if concentrations are lower.
Of course, many pollutants can also enter water breathing organisms via food, but the gills-water exchange is very effective in controlling the distribution of chemicals. The salinity of water plays a strong role in the need of water breathing organisms to "drink" water, and hence take in contaminants via this route.
BOX 1. Osmoregulation (MSc level)
Most aquatic vertebrate animals are osmoregulators: their cells contain a concentration of solutes that is different than the water around them. Fish living in freshwater typically have a cellular osmotic level (300 millOsmoles per Liter, mOsm/L) that is higher than the bulk fresh water (~20-40 mOsm/L), so a lot of water flows passively via the gills (not via the skin) into the tissue of fish. They are thus constantly taking in water (water molecules only) via the gills, which needs to be controlled, e.g. by strongly diluting the urine. They do also take in some water in their gastro-intestinal tract (GIT). Marine fish have similar cellular osmotic levels as freshwater fish, but the salty water (1000 mOsm/L) causes water to move out of the gill tissue through the linings of the fish's gills by osmosis, which needs to be replenished by active intake of salty water, and separate excretion of the salts. Most invertebrate organisms in oceans have an internal overall concentration of dissolved compounds comparable to the water they live in, so that they don't suffer from strong osmotic pressures on their soft tissue (osmoconformers).
A single adult oyster can cleanse about 200 liters of water per day (https://www.cbf.org/about-the-bay/mo...act-sheet.html). Plans to re-populate the harbour of New York with 1 billion oysters on artificial substrates can have enormous impacts on chemical redistribution. A single 2 cm zebra mussel (Dreissena polymorpha) that inhabits the shallow Lake IJsselmeer (6.05x1012 L) in can filter about 1 L per day, and the high densities of these and related species in this fresh water lake can turn over the lake volume once or twice per month (Reeders et al., 1989).
Even many soil organism are constantly in contact with wet soil surfaces, and contact between soil pore water and the outer surfaces (gills/soft skin areas) dominates the routes of chemical exchange for many chemicals. Earthworms, for example, do not have lungs and they exchange oxygen through their skin. Earthworms eat bacteria and fungi that grow on dead and decomposing organic matter, and thus act as major organic matter decomposers and recycling of nutrients. Earthworms dramatically alter soil structure, water movement, nutrient dynamics, and plant growth. It is estimated that earthworms turn over the top 15 cm of soil in ten to twenty years (LINK), and so they also are able to mix surface bound pollution into a substantial soil layer. In terms of biomass, earthworms dominate the world of soil invertebrates, including arthropods. In order to better understand how much contamination earthworms take in via food or via their skin, several studies have used earthworms in exposure tests with part of the organisms having their mouth parts sealed with surgical glue (Vijver et al., 2003). Uptake rates of the metals Cd, Cu and Pb in sealed and unsealed earthworms exposed to two contaminated field soils were similar (Vijver et al., 2003), indicating main uptake through the skin of the worms. The uptake rates as well as the maximum accumulation level for several organic contaminants from artificially contaminated soil were also comparable between sealed and non-sealed worms (Jager et al., 2003). The dermal route is thus a highly important uptake route for organic chemicals too. Dermal uptake by soil organisms is generally from the pool of chemicals in the soil pore water, hence the distribution of chemicals between solid particles and organic materials in the soil and the soil pore water is extremely important in driving the dermal uptake of chemicals by earthworms (See section 3.4 on Partitioning and partitioning constants, section 3.5 on Metal speciation and 3.6 on Availability and bioavailability).
Air breathing organisms
Air breathing organisms typically take in less/non-volatile chemicals via food and require active excretion and metabolism for the elimination of these chemicals via e.g. feces and urine. Dermal uptake is generally assumed to be negligible, while the intake of chemicals via air particles can be relatively high, for example of pollutants present in house dust, or contaminants on aerosols. The excretion via air particles is clearly not a dominant route. The chemicals in the food matrix inside the gastro-intestinal tract often first need to be fully dissolved in gut fluids, before they can pass the mucus layers and membranes lining the gastrointestinal tract and enter blood streams for redistribution. However, 'endocytosis' may also result in the uptake of (small) particulate chemicals in cells by first completely surrounding the particle by the membrane, after which the encapsulating membrane buds buds off inside the cell and forms a vesicle. Notwithstanding this endocytosis, chemical fractions of pollutants strongly sorbed to non-digestible parts may not always automatically be taken up from food. Grazing animals typically require microbial conversion in their gut to digest plant material like cellulose and lignin into chemical components that can be taken up as energy source.
Many aquatic foodwebs are structured such that they begin with aquatic plants being eaten by water breathing organisms, with air breathing marine animals or birds on top of the food chain. These air breathing top predators take in pollutants largely through their diet, but lack the effective blood-membrane-water exchange through gills. The blood-membrane-air partitioning in lungs is far less effective in removing chemicals via passive partitioning. For this reason, many top predators of foodwebs have the highest concentrations of pollutants. The chemical distribution in foodwebs will be discussed in more detail in section 4.1.6.
References
Palzenberger & Pohla 1992, Reviews in Fish Biology and Fisheries, 2, 187-216.
Jager, T.; Fleuren, R.H.L.J.; Hogendoorn, E.A.; de Korte, G. 2003. Elucidating the routes of exposure for organic chemicals in the earthworm, Eisenia andrei (Oligochaeta). Environ. Sci. Technol. 37 (15), 3399-3404
Koch et al. 2004, Nature (428) 851-854.
Moeckel et al. 2008, Environ. Sci. Technol. 42, 100-105.
Michelozzi 1999, Defensive roles of terpenoid mixtures in conifers, Acta Botanica Gallica 146 (1), 73-84.
Consoer et al. 2014, Aquatic Toxicology 156, 65-73.
Reeders et al. 1989, Freshwater Biology 22 (1), 133-141.
Vijver et al. 2003, Soil Biology and Biochemistry 35, 125-132.
Goss et al. 2018, Chemosphere 199, 174-181.
3.1.7. Question 1
Explain whether a lean fish of 1 kg likely contains a lower concentration of hydrophobic pollutant as a 1 kg fatty fish caught in the same lake.
3.1.7. Question 2
Explain how bivalves (clams, muscles, oysters) can reduce contamination with a hydrophobic chemical from a lake
3.1.7. Question 3
In many countries, the levels of water pollution have been strongly reduced compared to 30 years ago, but a lot of sediments are still contaminated with historic pollutants that strongly bind to the sediment particles. Explain how historic contamination in a sediment can be brought into suspension by benthic organisms.
3.1.7. Question 4
Discuss whether the internal concentrations of very hydrophobic chemicals in fatty fish gets higher or lower during an extended period of starvation, e.g. during migration towards breeding grounds. Note that very hydrophobic chemicals may have elimination half lives of months or even longer.
3.1.7. Question 5
Discuss whether the internal concentrations of very hydrophobic chemicals in female organisms gets higher or lower during a reproduction cycle, e.g. by generating offspring and or lactation. Note that very hydrophobic chemicals may have elimination half lives of months or even longer. | textbooks/chem/Environmental_Chemistry/Environmental_Toxicology_(van_Gestel_et_al.)/03%3A_Environmental_Chemistry_-_From_Fate_to_Exposure/3.01%3A_Environmental_compartments.txt |
Under review
3.03: Pathways and processes determining chemical fate
3.3. Pathways and processes determining chemical fate
Authors: Dik van de Meent, Michael Matthies
Reviewer: John Parsons
Leaning objectives:
You should be able to
• explain how emission of chemicals into the environment leads to exposure of ecosystems, populations, and organisms including man.
• understand how quantitative knowledge of process kinetics is of use in mathematical modeling of environmental fate
• understand the concept of characterization of fate processes in terms of rate constants and half-lives
• give examples of relevant fate processes and briefly describe them
Keywords: fate processes, degradation, transport, partitioning
Characterizing 'fate'
Chemicals can escape during all steps of their life cycle, e.g. manufacturing, processing, use, or disposal. Release of chemicals into the environment necessarily leads to exposure of ecosystems, populations, and organisms including man. Exposure assessment science seeks to analyze, characterize, understand and (quantitatively) describe the pathways and processes that link releases to exposure. Chemicals in the environment undergo various transport, transfer and degradation processes, which can be described and quantified in terms of loss rates, i.e. the rates at which chemicals are lost from the environmental compartment into which they are emitted or transferred from adjacent compartments. Exposure assessment science aims to capture the 'environmental fate' of chemicals in process descriptions that can be used in mass balance modeling, using mathematical expressions borrowed from thermodynamic laws and chemical reaction kinetics (Trapp and Matthies, 1998).
The 'fate' of a chemical in the environment can be viewed of as the net result of a suite of transport, transfer and degradation processes (see Section 3.4 on partitioning and partitioning constants, Section 3.6 on availability and bioavailability, Section 3.7 on degradation) that start to act on the chemical directly after its emission (see Section 3.2 on sources of emission) and during the subsequent environmental distribution. Environmental fate modeling (see Section 3.8 on multimedia mass balance modelling) builds on this knowledge by implementing the various degradation, transfer and transport processes derived in exposure assessment science in mathematical models that simulate 'fate of chemicals in the environment'.
First-order kinetics
In chemical reaction kinetics, the amount of chemical in a 'system' (for instance, a volume of surface water) is described by mass balance equations of the kind:
(eq. 1)
where is the rate of change (kg.s-1) of the mass (kg) of chemical in the system over time t (s), i is the input rate (kg.s-1) and k (s-1) is the reaction rate constant. Mathematically, this equation is a first-order differential equation in m, meaning that the loss rate of mass from the systemis proportional to the first power of m. Equation 1 is widely applied in description and characterization of environmental fate processes: environmental fate processes generally obey first-order kinetics, and can generally be characterized by a first-order reaction rate constant k1st:
(eq. 2)
Such loss rated equations can also be formulated in integral format, which is obtained by integration of equation (2) over time t with initial mass m0 = m(0):
(eq. 3)
As shown in Figure 1, first-order loss processes are expected to result in exponential decrease of mass from which concentration can be calculated by dividing m with the compartment volume. Using the value for t in equation 3, it follows directly that the value of is inversely proportional to the first-order loss rate constant :
(eq. 4)
which shows that half-life time constant, i.e. independent of the concentration of the chemical considered. This is the case for all environmental loss processes that obey first-order kinetics. First-order loss processes can therefore be sufficiently characterized by the time required for disappearance of 50% of the amount originally present.
The disappearance time DT50 is often used in environmental regulation but is only identically with the half-life if the loss process is of first order. Note that the silent assumption of constancy of half-life implies that the process considered is assumed to obey first-order kinetics.
Abiotic chemical reactions
Occurrence of true first-order reaction kinetics in chemistry is rare (see Section 3.7 on degradation). It occurs only when substances degrade spontaneously, without interaction with other chemicals. A good example is radio-active decay of elements, with a reaction rate proportional to the (first power of) the concentration (mass) of the decaying element, as in equation 3.
Most chemical reactions between two substances are of second order:
(eq. 5)
or, when a chemical reacts with itself:
(eq. 6)
because the reaction rate is proportional to the concentrations (masses) of both of the two reactants. It follows directly from equation 2. As the concentrations (masses) of both reactants decrease as a result of the reaction taking place, the reaction rate decreases during the reaction, more rapidly so at high initial concentrations. When second-order kinetics applies, half-life is not constant, but increases with ongoing reaction, when concentrations decrease. In principle, this is the case for most chemical reactions, in which the chemical considered is transformed into something else by reaction with a transforming chemical agent.
In the environment, the availability of second reactant (transforming agent) is usually in excess, so that its concentration remains nearly unaffected by the ongoing transformation reaction. This is the case, for oxidation (reaction with oxygen) and hydrolysis (reaction with water). In these cases, the rate of reaction decreases with the decreasing concentration of the first chemical only:
(eq. 7)
and reaction kinetics become practically first-order: so-called pseudo first-order kinetics. Pseudo first-order kinetics of chemical transformation processes is very common in the environment.
Biotic chemical reaction
Chemical reactions in the biosphere are often catalyzed by enzymes. This type of reaction is saturable and the kinetics can be described by the Michael-Menten kinetic model for single substrate reactions. At low concentrations, there is no effect of saturation of the enzyme and the reaction can be assumed to follow (pseudo) first order kinetics. At concentrations high enough to saturate the enzyme, the rate of reaction is independent of the concentrations (masses) of the reactants, thus constant in time during the reaction, and the reaction obeys zero-order kinetics. This is true for catalysis, where the reaction rate depends only on the availability of catalyst (usually the reactive surface area):
= constant. (eq. 8)
One could say that the rate is proportional to the zero-th power of the mass of reactant present. In case of zero-order kinetics, the half-life times are longer for greater initial concentrations of chemical.
An example of zero-order reaction kinetics is the transformation of alcohol (ethanol) in the liver. It has been worked out theoretically and experimentally that human livers remove alcohol from the blood at a constant rate, regardless the amount of alcohol consumed.
Microbial degradation
Microbial degradation (often referred to as biodegradation) is a special case of biotic transformation kinetics. Although this is an enzymatically catalysed process, the microbial transformation process can be viewed of as the result of the encounter of molecules of chemical with microbial cells, which should result in apparent second-order kinetics (first order with respect to the number of microbial cells present, and first order with respect to the mass of chemical present):
(eq. 9)
where mbio stands for the concentration (mass) of active bacteria present in natural surface water, and kdeg represents a pseudo-first-order degradation rate constant.
Advective and dispersive transport
Chemicals can be moved from one local point to another by wind and water currents. Advection means transport along the current axis whereas dispersion is the process of turbulent mixing in all directions. Advective processes are driven by external forces such as wind and water velocity, or gravity such as rain fall and leaching in soil. In most exposure models these processes are described in a simplified manner, e.g. the dispersive air plume model. An example of a first-order advective loss process is the outflow of a chemical from a lake:
(eq. 10)
where Q stands for the flow rate of lake water [m³/s] and V is the lake volume [m³]. Q/V is known as the renewal rate constant kadv of the transport medium, here water. More sophisticated hydrological, atmospheric, or soil leaching models consider detailed spatial and temporal resolution, which require much more data and higher mathematical computing effort (see sections 3.1.2 and 3.8.2).
Transfer and partitioning
Due to Fick's first law the rate of transfer through an interface between two media (e.g. water and air, or water and sediment) is proportional to the concentration difference of the chemical in the two media (see section 3.4 on partitioning, and Schwarzenbach et al., 2017 for further reading). As long as the concentration in one media is higher than in the other, the more molecules are likely to pass through the interface. Examples are volatilization of chemicals from water (to air) and gas absorption from air (to water or soil), adsorption from water (to sediments, suspended solids and biota) and desorption from sediments and other solid surfaces.
When two environmental media are in direct contact, (first-order) transfer can take place in two directions, in the case of water and air by volatilization and gas absorption: Each at a rate proportional to the concentration of chemical in the medium of origin and each with a (first-order) rate constant characteristic of the physical properties of the chemical and of the nature of the interface (area, roughness). This is known as physical intermedia partitioning (see section 3.4 on partitioning), usually represented by a chemical reaction formula:
(eq. 11)
where [M] stands for a (mass) concentration (unit mass per unit volume) and the double arrow represents forward and reverse transport. Intermedia partitioning proceeds spontaneously until the two media have come to thermodynamic equilibrium. In the state of equilibrium, forward and backward rates (here: volatilization from water to air and gas absorption from air to water) have become equal. At equilibrium, the total (Gibbs free) energy of the system has reached a minimum: the system has come to rest, so that
(eq. 12)
and the ratio of concentrations of the chemical in the two media has reached its (thermodynamic) equilibrium value, called equilibrium constant or partition coefficient (see section 3.4 on partitioning).
Challenge
Challenge to environmental chemists is to describe and characterize the various processes of chemical and microbial degradation and transformation, of intra-media transport and intermedia transfer rate constants and of equilibrium constants, in terms of (i) physical and chemical properties of the chemicals considered and (ii) of the properties of the environmental media.
References
Schwarzenbach, R.P., Gschwend, P.M., Imboden, D.M. (2017). Environmental Organic Chemistry, Third Edition, Wiley, ISBN 978-1-118-76723-8.
Trapp, S., Matthies, M. (1998). Chemodynamics and Environmental Modeling. An Introduction. Springer, Heidelberg, ISBN 3-540-63096-1.
3.3. Question 1
Name and explain in your own words the essential environmentally relevant property of (pseudo) first-order kinetics.
3.3. Question 2
Give examples of transformation or transport processes that obey zero-order, first-order, second-order and pseudo first-order kinetics.
3.3. Question 3
Why is it useful to formulate environmental fate processes in terms of process rates and rate constants and equilibrium constants? | textbooks/chem/Environmental_Chemistry/Environmental_Toxicology_(van_Gestel_et_al.)/03%3A_Environmental_Chemistry_-_From_Fate_to_Exposure/3.02%3A_Sources_of_chemicals.txt |
3.4. Partitioning and partitioning constants
3.4.1. Relevant chemical properties
Authors: Joop Hermens, Kees van Gestel
Reviewer: Steven Droge, Monika Nendza
Learning Objectives
You should be able to:
• define the concept of hydrophobicity and to explain which chemical properties affect hydrophobicity.
• define which properties of a chemical affect its tendency to evaporate from water.
• calculate fractions ionized for acids and bases.
Keywords: Hydrophobicity, octanol-water partition coefficients, volatility, Henry's Law constant, ionized chemicals
Introduction
Different processes affect the fate of a chemical in the environment. In addition to the transfer and exchange between compartments (air-water-sediment/soil-biota), also degradation determines the concentration in each of these compartments (Figure 1).
Some of these processes are discussed in other sections (see sections on Sorption and Environmental degradation of chemicals). Some chemicals will easily evaporate from water to air, while others remain mainly in the aqueous phase or sorb to sediment and accumulate into biota.
These differences are related to only a few basic properties:
• Hydrophobicity (tendency of a substance to escape the aqueous phase)
• Volatility (tendency of a substance to vaporize)
• Degree of ionization
Hydrophobicity
Hydrophobicity means fear (phobic) of water (hydro). A hydrophobic chemical prefers to "escape from the aqueous phase" or in other words "it does not like to dissolve in water". Water molecules are tightly bound to each other via hydrogen bonds. For a chemical to dissolve in water, a cavity should be formed in the aqueous phase (Figure 2) and this will cost energy.
Hydrophobicity mainly depends on two molecular properties:
• Molecular size
• Polarity / ability to interact with water molecules, for example via hydrogen bonding
It will take more energy for a chemical with a larger size to create the cavity making the chemical more hydrophobic, while interactions of the chemical with water will favour its dissolution making it less hydrophobic. Figure 3 shows chemicals with increasing hydrophobicity with increasing size and a decreasing hydrophobicity by the presence of polar groups (amino or hydroxy).
Most hydrophobic chemicals are non-polar organic micro pollutants. Well-known examples are the chlorinated hydrocarbons, such as polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). Water solubility of these chemicals in general is rather low (in the order of a few ng/L up to a few mg/L).
The hydrophobic nature mainly determines the distribution of these chemicals in water and sediment or soil and their uptake across cell membranes. Additional Cl- or Br-atoms in a chemical, as well as additional (CH)x units, increase the molecular size, and thus a chemical's hydrophobicity. The increased molecular volume requires a larger cavity to dissolve the chemical in water, while they only interact with water molecules via VanderWaals interactions.
Polar groups, such as the -OH and -NH units on the aromatic chemicals in Figure 3, can form hydrogen-bonds with water, and therefore substantially reduce the hydrophobicity of organic chemicals. The hydrogen bonding of hydroxy-substituents works in two ways: The oxygen of -OH bridges to the H-atoms of water molecules, while the hydrogen of -OH can form bridges to the O-atoms of water molecules. Nearly all molecular units consisting of some kind of (carbon-oxygen) combination reduce the hydrophobicity of organic contaminants, because even though they increase the molecular volume they interact via hydrogen bonds (H-bonds) with surrounding water molecules. Additional polar groups in a chemical typically decrease a chemical's hydrophobicity.
Octanol-water partition coefficient:
A simple measure of the hydrophobicity of chemicals, originating from pharmacology, is the octanol-water partition coefficient, abbreviated as Kow (and sometimes also called Pow or Poct): this is the ratio of concentrations of a chemical in n-octanol and in water, after establishment of an equilibrium between the two phases (Figure 4). The -OH group in n-octanol does allow for some hydrogen bonding between octanol-molecules in solution, and between octanol and dissolved molecules. However, the relatively long alkyl chain only interacts through VanderWaals interactions, and therefore the interaction strength between octanol-molecules is much smaller than that between water-molecules, and it is energetically much less costly to create a cavity to dissolve any molecule.
Experimentally determined Kow values were used in pharmacological research to predict the uptake and biological activity of pharmaceuticals. Octanol was selected because it appears to closely mimic the nonionic molecular properties of most tissue components, particularly phospholipids in membranes. Since the beginning of the 1970s, Kow values have also been used in environmental toxicology to predict the hazard and environmental fate of organic micro pollutants. Octanol may partially also mimic the nonionic molecular properties of most organic matter phases that sorb neutral organic chemicals in the biotic and abiotic environment.
Not unexpectedly, water solubility is negatively correlated with octanol-water partition coefficients.
In practice, three methods can be used to determine or estimate the Kow:
Equilibration methods
In the shake-flask method (Leo et al., 1971) and the 'slow-stirring' method (de Bruijn et al., 1989), the distribution of a chemical between octanol and water is determined experimentally. For highly lipophilic chemicals (log Kow > 5-6), the extremely low water solubility, however, hampers a reliable analytical determination of concentrations in the water phase. For such chemicals, these experimental methods are not suitable. During the last two decades, the use of generator columns has allowed for quantification of higher Kow values. Generator columns are columns packed with a sorbing material (e.g. Chromosorb®) onto which an appropriate hydrophobic solvent (e.g. octanol) is coated that contains the compound of interest. In this way, a large interface surface area between the lipophilic and water phases is created, which allows for a rapid establishment of equilibrium. When a large volume of (octanol-saturated) water (typically up to 10 litres) is passed slowly through the column, an equilibrium distribution of the compound is established between the octanol and the water. The water leaving the column is passed over a solid sorbent cartridge to concentrate the compound and allow for a quantification of the aqueous concentration. In this way, it is possible to more reliably determine log Kow values up to 6-7.
Chromatography
Kow values may also be derived from the retention time in a chromatographic system (Eadsforth, 1986). The use of reversed-phase High Performance Liquid Chromatography (HPLC), thin-layer chromatography or gas chromatography results in a capacity factor (relative retention time; retention of the compound relative to a non-retained chemical species), which may be used to predict the chemical distribution over octanol and water. HPLC systems have shown most successful, because they consist of stationary and mobile phases that are liquid. As a consequence, the nature of the phases can be most closely arranged to resemble the octanol-water system. Of course, this requires calibration of the capacity factors by applying the chromatographic method to a number of chemicals with well-known Kow values. Chromatographic methods may reliably be applied for estimations of log Kow values in the range of 2-8. For more lipophilic chemicals, also these methods will fail to reliably predict Kow values (Schwarzenbach et al., 2003).
Calculation
Kow values may also be calculated or predicted from parameters describing the chemical structure of a chemical. Several software programs are commercially available for this purpose, such as KOWWIN program of the US-EPA. These programs make use of the so-called fragment method (Leo, 1993; Rekker and Kort, 1979). This method takes into account the contribution to Kow of different chemical groups or atoms in a molecule, and in addition corrects for special features such as steric hindrance or other intramolecular interactions (equation 1):
log Kow = Ʃ fn + Ʃ Fp (eq.1)
in which fn quantifies the contributions of each fragment n in a particular chemical (see e.g. Table 1) and Fp accounts for any special intramolecular interaction p between the fragments.
This fragment approach has been improved during the last decades and is available in the EPISUITE program from the US Environmental Protection Agency. Other programs for the calculation of Kow values are: ChemProp, and ChemAxon from Chemspider.
Table 1. Fragment constants (Kow) for a few fragments. (from the EPISUITE program)
Fragment
Fragment constant (f)a
-CH3 aliphatic carbon
0.5473
Aromatic Carbon
0.2940
-OH hydroxy, aromatic attach
-0.4802
-N aliphatic N, one aromatic attach
-0.9170
Note: the above calculations are given for non-ionized chemicals. The hydrophobicity of ionic chemicals is also highly affected by the degree of ionization (see below).
Kow values can also be retrieved from databases like echemportal or ECHA and others.
Volatility
Volatility of a chemical from the aqueous phase to air (see Figure 5) is expressed via the Henry's law constant (KH).
Henry's law constant (KH, in Pa⋅m3/mol) is the chemical distribution between the gas phase and water, as
(eq.2)
where in an equilibrated water-gas system:
Caq is the aqueous concentration of the chemical (units in mol/m3), and Pi is the partial pressure of the chemical in air (units in Pascal, Pa), which is the pressure exerted by the chemical in the total gas phase volume (occupied by the mixture of gases the gas-phase above the water solution of the chemical). Note that Pi is a measure of the concentration in the gas phase, but not yet in the same units as the dissolved concentration (discussed below)!
For compounds that are slightly soluble in water, KH can be estimated from:
(eq.3)
where:
KH: Henry's law constant (Pa⋅m3/mol), Vp is the (saturated) vapor pressure (Pa), which is the pressure of the chemical above the pure condensed (liquid) form of the chemical, and Sw is the maximum solubility in water (mol/m3).
The advantage of equation 3 is that both Vp and Sw can be experimentally derived or estimated. The rationale behind equation 3 is that two opposite forces will affect the evaporation of a chemical from water to air:
(i) the vapor pressure (Vp) of the pure chemical - high vapor pressure means more volatile, and
(ii) solubility in water (Sw) - high solubility means less volatile.
Benzene and ethanol (see Table 2) are good illustrations. Both chemicals have similar vapor pressure, but the Henry's law constant for benzene is much higher because of its much lower solubility in water compared to ethanol; benzene is much more volatile from an aqueous phase.
Table 2. Air-water partition coefficients (Kair-water) calculated for five chemicals (ranked by aqueous solubility) by equation 3.
Chemical
Vapor pressure
(Pa)
Solubility (mol/m3)
KH
(Pa.m3/mol)
Kair-water
(L/L, or m3/m3)
Ethanol
7.50⋅103
1.20⋅104
6.25⋅10-1
2.53⋅10-4
Phenol
5.50⋅101
8.83⋅102
6.23⋅10-2
2.52⋅10-5
Benzene
1.27⋅104
2.28⋅101
5.57⋅102
2.25⋅10-1
Pyrene
6.00⋅10-4
6.53⋅10-4
9.18⋅101
3.71⋅10-4
DDT
2.00⋅10-5
2.82⋅10-6
7.08
2.86⋅10-3
Note: all chemicals at equilibrium have a higher concentration (in e.g. mol/L) in the aqueous phase than in the gas phase. Of these five, benzene is the chemical most prone to leave water, with an equilibrated air concentration about 4 times lower (22.5%) than the dissolved concentration.
Equations 2 and 3 are based on the pressure in the gas phase. Environmental fate is often based on partition coefficients, in this case the air-water partition coefficient (Kair-water). These partition coefficients are more or less 'dimensionless', because the concentrations are based on equal volumes (such as L/L), while KH has the unit Pa⋅m3/mol or something equivalent to the applied units (equation 4).
(eq.4)
where:
Cair is the concentration in air (in e.g. mol/m3) and Caq is the aqueous concentration (in e.g. mol/m3).
Kair-water can be calculated from KH according to equation 5:
(eq.5)
where R is the gas constant (8.314 m3⋅Pa⋅K−1⋅mol−1), and T is the temperature in Kelvin (Kelvin = oCelsius + 273).
This use of "RT" converts this gas phase concentration to a volume based metric, and applies the ideal gas law which relates pressure (P, in Pa) to temperature (T, in K), volume (V, in m3), and amount of gas molecules (n, in mol), according to the gas constant (R: 8.314 m3⋅Pa⋅K-1⋅mol-1):
P⋅V = n⋅R⋅T (note that the units of both terms will cancel out) (eq.6)
At 25 oCelcius (298 K), the product RT equals 2477 m3⋅Pa⋅K-1⋅mol-1.
Examples of calculated values for Kair-water are presented in Table 2.
The influence of the chemical structure on volatility of a chemical from a solvent fully depends on the cost of creating a cavity in the solvent (interactions between solvent molecules) and the interactions between the chemical and the solvent molecules. For partitioning processes, the gas phase is mostly regarded as an inert compartment without chemical interactions (i.e. gas phase molecules hardly ever touch each other).
The molecules of a strongly dipolar solvent such as water that contain atoms that can interact as hydrogen acceptor (the O in an OH group) and hydrogen donor (the H in an OH group) strongly interact with each other, and it costs much energy to create a cavity. This cost increases strongly with molecular size, for nearly all molecules more than the energy regained by interactions with the surrounding solvent molecules. As a result, for most classes of organic chemicals, affinity with water decreases and volatility out of water into air slightly increases with molecular volume. For chemicals that are not able to re-interact via hydrogen bonding, e.g. alkanes, the overall volatility is much higher than for chemicals that do have specific interactions with water molecules besides van der Waals.
Degree of ionization
Acids and bases can be present in the neutral (HA and B) or ionized form (A- and BH+, respectively). For acids, the neutral form (HA) is in equilibrium with the anionic form (A-) and for bases the neutral form (B) is in equilibrium with the cationic form (BH+). The degree of ionization depends on the pH and the acid dissociation constant (pKa). Table 3 shows the equations to calculate the fraction ionized for acids and bases and examples of two acids (phenols) are presented in Table 4.
Table 3. Calculation of the fraction ionized for acids and bases.
Acids
Bases
pKa = - log Ka, where Ka is dissociation constant of the acidic form (HA or BH+).
The degree of ionization is thus determined by the pH and the pKa value and more examples for several organic chemicals are presented elsewhere (see Chapter Ionogenic organic compounds).
Table 4. The degree of ionization of two phenolic structures (acids).
Pentachlorophenol
Phenol
pKa = 4.60
pKa = 9.98
% ionized versus pH
% ionized versus pH
at pH of 7.0: 99.6 % ionized
at pH of 7.0: 0.1 % ionized
Examples for several organic chemicals are presented elsewhere (see section on Ionogenic organic compounds).
The fate of ionic chemicals is very different from that of non-ionic chemicals. The sediment-water sorption coefficient of the anionic species is substantially (>100x) lower than that of the neutral species. If the percentage of ionization is less than ~99 % (at a pH 2 units above the pKa), the sorption of the anion may be neglected (Kd is still dominated by the >1% neutral species) (Schwarzenbach et al., 2003). The reason for the low sorption affinity of the anionic acid form is twofold: anions are much better water soluble, but also most sediment particles (clay, organic matter, silicates) are negatively charged, and electrostatically repulse the similarly charged chemical. In that case the sorption coefficient Kd can be calculated from the sorption coefficient of the non-ionic form and the fraction of the non-ionized form (α):
(eq. 7)
In environments where the pH is such that the neutral acid fraction <1% (when pH >2 units above the pKa), the sorption of the anionic species to soil/sediment may significantly contribute to the overall "distribution coefficient" of both acid species.
For basic environmental chemicals of concern, among which many illicit drugs (e.g. amphetamine, cocaine) and non-illicit drugs (e.g. most anti-depressants, beta-blockers), the protonated forms are positively charged. These organic cations are also much more soluble in water than the neutral form, but at the same time they are electrostatically attracted to the negatively charged sediment surfaces. As a result, the sorption affinity of organic cations to sediment should not be considered negligible relative to the neutral species. The sorption processes, however, may strongly differ for the neutral base species and the cationic base species. Several studies have shown that the sorption affinity of cationic base species to DOM or sediment is even stronger than that of the neutral species.
References
De Bruijn, J., Busser, F., Seinen, W., Hermens, J. (1989). Determination of octanol/water partition coefficients for hydrophobic organic chemicals with the "slow-stirring" method. Environmental Toxicology and Chemistry 8, 499-512.
Eadsforth, C.V. (1986). Application of reverse-phase HPLC for the determination of partition coefficients. Pesticide Science 17, 311-325.
Leo, A., Hansch, C., Elkins, D. (1971). Partition coefficients and their uses. Chemical Reviews 71, 525-616.
Leo, A.J. (1993). Calculating log P(oct) from structures. Chemical Reviews 93, 1281-1306.
Rekker, R.F., de Kort H.M. (1979). The hydrophobic fragmental constant; an extension to a 1000 data point set. Eur.J. Med. Chem. - Chim. Ther. 14:479-488.
Schwarzenbach RP, Gschwend PM, Imboden DM (Eds.) 2003. Environmental organic chemistry. Wiley, New York, NY, USA.
Further reading:
Mackay, D., Boethling, R.S. (Eds.) 2000. Handbook of property estimation methods for chemicals: environmental health and sciences. CRC Press.
van Leeuwen, C.J., Vermeire, T.G. (Eds.) 2007. Risk assessment of chemicals: An introduction. Springer, Dordrecht, The Netherlands
3.4.1. Question 1
Explain the term hydrophobicity and mention two major properties that affect hydrophobicity of chemicals.
3.4.1. Question 2
What is the definition of Kow. Rank the following chemicals from the Table below from low to high Kow. 1. Pentachlorobenzene, 2. Monochlorobenzene, 3. Monochloroaniline, 4. DDT.
1.Pentachlorobenzene
2.Monochlorobenzene
3.Monochloroaniline
4.DDT
3.4.1. Question 3
Rank the chemicals in the Table according to their volatility as pure compounds and their solubility in water and volatility from water.
3.4.1. Question 4
Which two basic properties determine the volatility of a chemical from water to air.
3.4.1. Question 5
Calculate the percentage ionized for 2,3,4-trichlorophenol (pKa = 4.6) at pH 3, 5, 7 and 9.
3.4.2. Sorption
Authors: Joop Hermens
Reviewers: Kees van Gestel, Steven Droge, Philipp Mayer
Leaning objectives:
You should be able to:
• understand why information on sorption is important for risk assessment
• give examples that illustrate the importance of sorption for risk assessment
• understand the concept of sorption isotherms
• be familiar with different sorption isotherms (linear, Freundlich, Langmuir).
Keywords: Sorption isotherm, absorption and adsorption, organic matter, Freundlich model, Langmuir model, organic carbon content
Introduction
Sorption processes have a major influence on the fate of chemicals in the environment (Box 1). In general, sorption is defined as the binding of a dissolved or gaseous chemical (the sorbate) to a solid phase (the sorbent) and this may involve different processes, including:
(i) binding of dissolved chemicals from water to sediments and soils
and (ii) binding of gaseous phase chemicals from air to soils, plants, and trees.
Information about sorption is relevant because of a number of reasons:
• sorption controls the actual fate and thereby the risk of (many) organic and inorganic contaminants in the environment,
• sorbed chemicals cannot evaporate, are not available for (photo)chemical or microbial breakdown, are not as easily transported as dissolved/vapor phase chemicals, and are not available for uptake by organisms,
• sorption also plays an important role in toxicity tests, affecting exposure concentrations.
Box 1.
The Biesbosch is a wetland area in the Netherlands, an area in between the Rivers Rhine and Meuse and estuaries that are connected to the North Sea. The water flow is relatively low and as a consequence there is a strong sedimentation of particles from the water to the sediment. Chemicals present in the water strongly sorb to these particles, which in the past were polluted with hydrophobic organic contaminants such as dioxins and PCBs. The concentrations of these organic compounds in sediment are still relatively high because they are highly persistent. The reason for this persistence of these compounds is that these sorbed compounds are not easily available for degradation by bacteria. Also, the concentrations in organisms that live close to or in the sediment are high. These concentrations are so high that fishing on eel, for example, is not allowed in the area. This example shows the importance of sorption processes on fate, but also on effects in the environment.
Measurement of sorption is a simple procedure. A chemical X is spiked (added) to the aqueous phase in the presence of a certain amount of the solid phase (sediment or soil). The chemical sorbs to the solid phase and when the system is in equilibrium, the concentrations in the sediment (Cs) and in the aqueous phase (Ca) are measured. The solid phase is collected via centrifugation or filtration.
The sorption coefficient Kp (equation 1 and box 2) gives information about the degree of sorption of a chemical to sediment and is defined as:
(1)
Box 2:
The concentration of a chemical X in sediment (Cs) is 30 mg/kg and the concentration in the aqueous phase (Ca) is 0.1 mg/L.
The sorption coefficient Kp = Cs / Ca = 30 mg/kg / 0.1 mg/L = 300 L/kg
Note the units of a sorption coefficient: L/kg
In the environmental risk assessment of chemicals, it is very useful to understand the fraction of the total amount of chemical (Atotal) in a system that is sorbed (fsorbed) or dissolved (fdissolved) (e.g. due to an accidental spill in a river):
fdissolved = Adissolved / Atotal , and thus fsorbed = 1 - fdissolved
This is related to the sorption coefficients of X and the volume of the solvent and the volume of the sorbent material. The equation derived for calculating fdissolved is based on the mass balance of chemical A, which relates the concentration of X (C) to the amount of X (A) in each volume (V):
C = A / V, and thus A = C ⋅ V
which for a system of water and sediment (air not included for simplification) relates to:
Atotal = Adissolved + Asorbed = Cwater ⋅ Vwater + Csediment ⋅ Vsediment = Cwater ⋅ Vwater + (Kp ⋅Cwater)⋅Vsediment
fdissolved = Adissolved / Atotal = Cwater ⋅ Vwater / (Cwater ⋅Vwater + Kp ⋅Cwater⋅Vsediment)
This way of separating out Csediment from the equation using Kp can result, after rearranging (by dividing both parts of the ratio by Cwater ⋅ Vwater) to the following simplified equation:
fdissolved = 1 / (1 + Kp⋅(Vsediment / Vwater))
in this equation, 'sediment' can be replaced by any sorbent, as long as the appropriate sorption coefficient is used.
Let's try to calculate with chemical X from above, in a wet sediment, where 1L wet sediment contains ~80% water and 20% dry solids. The dissolved fraction of X with Kp = 300 kg/L, is only 0.013 in this example. Thus, with 1.3% of X actually dissolved, this indicates that 98.7% of X is sorbed to the sediment.
Sorption processes
There are two major sorption processes (see Figure 2):
• Absorption - partitioning ("dissolution") of a chemical into a 3-D sorbent matrix. The concentration in the sorbing phase is homogeneous.
• Adsorption - binding of a chemical to a 2-D sorbent surface. Because the number of sorption sites on a surface is limited, sorption levels off at high concentrations in the aqueous phase.
A sorption isotherm gives the relation between the concentration in a sorbent (sediment) and the concentration in the aqueous phase and the isotherm is important in identifying a sorption process.
Absorption of a chemical is similar to its partitioning between two phases and comparable to its partitioning between two solvents. Distribution of a chemical between octanol and water is a well-known example of a partitioning process (see Section 3.4.1 on Relevant chemical properties for more detailed information on octanol-water partitioning). The isotherm for an absorption process is linear (Figure 3A) and the slope of the y-x plot is the sorption coefficient Kp.
In an adsorption process, where the sorbing phase is a surface with a limited number of sorption sites, the sorption isotherm is non-linear and may reach a maximum concentration that is adsorbed when all sites are occupied. A mechanistic model for adsorption is the Langmuir model. This model describes adsorption of molecules to homogeneous surfaces with equal adsorption energies, represented by the adsorption site energy term (b) and a limited number of sorption sites (Cmax) that can become saturated (Figure 3B). The Langmuir adsorption coefficient (Kad) is equal to the product (b ⋅ Cmax) at relatively low aqueous concentrations, where the product (b ⋅ Caq) << 1 (note that the denominator term will then be ~1). Indeed, the isotherm curve on a double log scale plot shows a slope of 1 at such low concentrations, indicating linearity.
Another mathematical approach to describe non-linear sorption is the Freundlich isotherm (Figure 3C), where KF is the Freundlich sorption constant and n is the Freundlich exponent describing the sorption process non-linearity. Using logarithmic values for aqueous and sorbed concentrations, the Freundlich isotherm can be rewritten as:
Log Cs = n ⋅ log Caq + log KF (eq. 2)
This conveniently yields a linear relationship (just as y = a⋅x + b) between log Cs and log Caq, with a slope equal to n and the abscissa (crossing point with the Y-axis) equal to log KF. This allows for easy fitting of linear trend lines through experimental data sets. When n = 1, the isotherm is linear, and equals the one for absorption. In case of saturation of the sorption sites on the solid phase, 1/n will be smaller than 1. The Freundlich isotherm can, however, also yield a 1/n value > 1; this may occur for example if the chemical that is sorbed itself forms a layer that serves as a new sorbing phase and examples are described for surfactants.
Sorption phases
Soils and sediments may show large variations in composition and particle size distribution. The major components of soils and sediments are:
Sand
63 - 2 mm
Silt
2 - 63 µm
Clay
<2 µm
Organic matter
includes e.g. detritus, humic acids, especially associated with the clay and silt fractions
CaCO3
Figure 4 gives a schematic picture of a sediment or soil particle. In addition to the presence of clay minerals and (soil or sediment) organic matter (SOM), sediment and soil may contain soot particles (a combustion residue).
Organic matter is formed upon decomposition of plant material and dead animal or microbial tissues. Upon decomposition of plant material, the first organic groups to be released are phenolic acids, some of which have a high affinity for complexation of metals. One example is salicylic acid (o-hydroxybenzoic acid), which occurs in high concentrations in leaves of willows, poplar and other deciduous trees. Further decomposition of plant material may result in the formation of humic acids, fulvic acids and humin. Humic and fulvic acids contain a series of functional groups, such as carboxyl- (COOH), carbonyl- (=C=O), phenolic hydroxyl- (-OH), methoxy- (-OCH3), amino- (-NH2), imino (=NH) and sulfhydryl (-SH) groups (see for more details the section on Soil).
Hydrophobic organic chemicals mainly sorb to organic matter. Because organic matter has the characteristics of a solvent, the sorption is clearly an absorption process and the sorption isotherm is linear. Because binding is mainly to organic matter, the sorption coefficient (Kp) depends on the fraction of organic matter (fom) or the fraction of organic carbon (foc) present in the soil or sediment. Please note that as a rule of thumb, organic matter contains 58% organic carbon (foc = 0.58⋅fom). Figure 5A shows the increase in sorption coefficient with increasing fraction organic carbon in soils and sediments. In order to arrive at a more intrinsic parameter, sorption coefficients are often normalized to the fraction organic matter (Kom) or organic carbon (Koc). These Koc or Kom values are less dependent of the sediment or soil type (Figure 5B).
(3)
(4)
Hydrophobic chemicals can have a very high affinity to soot particles relative to the affinity to SOM. If a sediment contains soot, Kp values are often higher than predicted based on the fraction organic carbon in the organic matter (Jonker and Koelmans, 2002).
Suggested reading
van Leeuwen, C.J., Vermeire, T.G. (Eds.) (2007). Risk Assessment of Chemicals: An Introduction. Springer, Dordrecht, The Netherlands. Chapter 3 and 9.
Schwarzenbach, R.P., Gschwend, P.M., Imboden, D.M. (2003). Environmental Organic Chemistry. Wiley, New York, NY, USA. chapters 9, 11. Detailed information about sorption processes and sorption mechanisms.
3.4.2. Question 1
What is a sorption isotherm and what is the difference between absorption and adsorption?
3.4.2. Question 2
Why is the sorption isotherm to clay non-linear?
3.4.2. Question 3
What are the main sorption phases in sediment or soil?
3.4.3. QSPRs
Author: Joop Hermens
Reviewer: Steven Droge, Monika Nendza
Learning objectives:
You should be able to:
• indicate which properties of chemicals are applied in a QSPR
• list different techniques to derive a QSPR
• indicate which interactions may occur between molecules.
Keywords: Quantitative structure-property relationships (QSPR), quantitative structure-activity relationships (QSAR), octanol-water partition coefficients, hydrogen bonding, multivariate techniques.
Introduction
Risk assessment needs input data for fate and effect parameters. These data are not available for many of the existing chemicals and predictions via estimation models will provide a good alternative to actual testing. Examples of estimation models are Quantitative Structure-Property Relationships (QSPRs) and Quantitative Structure-Activity Relationships (QSARs). The term "activity" is often used in relation to models for toxicity, while "property" usually refers to physical-chemical properties or fate parameters.
In a QSAR or QSPR, a certain environmental parameter is related to a physical-chemical or structural property, or a combination of properties.
The elements in a QSPR or QSAR are shown in Figure 1 and include:
• the parameter for which the estimation model has been developed: the Y-variable (upper right),
• the properties of the chemical or chemical parameter: the X-variable (upper left),
• the model itself (center), and
• the prediction of a fate or effect parameter from the chemical properties (bottom).
The Y-variable
Estimation models have been developed for many endpoints such as sorption to sediment, humic acids, lipids and proteins, chemical degradation, biodegradation, bioconcentration and ecotoxic effects.
The X-variable
An overview of the chemical parameters (the X-variable) used in estimation models is given in Table 1. Chemical properties are divided in three categories: (i) parameters related to hydrophobicity, (ii) parameters related to charge and charge distribution in a molecule and (iii) parameters related to the size or volume of a molecule. Hydrophobicity is discussed in more detail in the section on Relevant chemical properties.
Other QSPR approaches use large number of parameters derived from chemical graphs. The CODESSA Pro software, for example, generates molecular (494) and fragment (944) descriptors, classified as (i) constitutional, (ii) topological, (iii) geometrical, (iv) charge related, and (v) quantum chemical (Katritzky et al. 2009). Some models are based on structural fragment in a molecule. The polyparameter linear free energy relationships (pp-LFER) use parameters that represent interactions between molecules (see under pp-LFER).
Table 1. Examples of parameters related to hydrophobicity and electronic and steric parameters (the X variable).
Hydrophobic parameters
Aqueous solubility
Octanol-water partition coefficient (Kow)
Hydrophobic fragment constant π
Electronic parameters
Atomic charges (q)
Dipole moment
Hydrogen bond acidity (H bond-donating)
Hydrogen bond basicity (H bond-accepting)
Hammett constant σ
Steric parameters
Total Surface Area (TSA)
Total Molecular Volume (TMV)
Taft constant for steric effects (Es)
The model
Most models are based on correlations between Y and X. Such a relationship is derived for a "training set" that consists of a limited number of carefully selected chemicals. The validity of such a model should be tested by applying it to a "validation set", i.e. a set of compounds for which experimental data can be compared with the predictions. Different techniques can be used to develop an empirical model, such as:
• graphical presentations,
• linear or non-linear equations between Y and X,
• linear or non-linear equations based on different properties (Y versus X1, X2, etc.),
• multivariate techniques such as Principal Component Analysis (PCA) and Partial Least Square Analysis (PLS).
Linear equations take the form:
Y(i) = a1X1(i) + a2X2(i) + a3X3(i) + ... + b (1)
where Y(i) is the value of the dependent parameter of chemical i (for example sorption coefficients); X1-X3(i) are values for the independent parameters (the chemical properties) of chemical i; a1-a3 are regression coefficients (usually 95% confidence limits are given); b is the intercept of the linear equation. The quality of the equation is presented via the correlation coefficient (r) and the standard error of estimate (s). The closer r is to 1.0, the better the fit of the relationship is. More information about the statistical quality of models can be found under "limitation of QSPR".
The classical approach in QSAR and QSPR studies is the Hansch approach that was develop in the 1960s. The Hansch equation (Hansch et al., 1963) describes the influence of substituents on the biological activity in a series of parent compounds with a certain substituent (equation 2). Substituents are for example a certain atom or chemical group (Cl, F, B,. OH, NH2) attached to a parent aromatic ring structure.
log 1/C = c π + c' σ + c'' Es + c''' (2)
in which:
C is the molar concentration of a chemical with a particular effect,
π is a substituent constant for hydrophobic effects,
σ is a substituent constant for electronic effects, and
Es is a substituent constant for steric effects.
c are constants that are obtained by fitting experimental data
For example, the hydrophobic substituent constant is based on Kow and is defined as is defined as:
π (X) = log Kow (RX) - log Kow (RH) (3)
where RX and RH are the substituted and unsubstituted parent compound, respectively.
The Hammett and Taft constants are derived in a similar way.
Multivariate techniques may be very useful to develop structure-activity relationships, in particular in cases where a large number of chemical parameters is involved. Principal Component Analysis (PCA) can be applied to reduce the number of variables into a few principal components. The next step is to find a relationship between Y and X via, for example, Partial Least Square (PLS) analysis. The advantage of PCA and PLS is that it can deal with a large number of chemical descriptors and that is can also cope with collinear (correlated) properties. More information on these multivariate techniques and examples in the field of environmental science are given by Eriksson et al. (1995).
Poly-parameter Linear Free Energy Relationship (pp-LFER)
The pp-LFER approach has a strong mechanistic basis because it includes the different types of interactions between molecules (Goss and Schwarzenbach, 2001). For example, the sorption coefficient of a chemical from an aqueous phase to soil or to phospholipids (the sorbent) depends on the interaction of a chemical with water and the interaction with the sorbent phase. One of the driving forces behind sorption is the hydrophobicity. Hydrophobicity means fear (phobia) for water (hydro). A hydrophobic chemical prefers to "escape from the aqueous phase" or in other words "it does not like to dissolve in water". Water molecules are tightly bound to each other via hydrogen bonds. For a chemical to dissolve, a cavity should be formed in the aqueous phase (Figure 2) and this will cost energy. More hydrophobic compounds will often have a stronger sorption (see more information in the section on Relevant chemical properties).
Hydrophobicity mainly depends on two molecular properties:
• Molecular size
• Polarity / ability to interact with water molecules, for example via hydrogen bonding
In the interaction with the sorbent (soil, membrane lipids, storage lipids, humic acids), major interactions are van der Waals interactions and hydrogen bonding (Table 2). Van der Waals interactions are attractive and occur between all kind of molecules and the strength depends on the contact area. Therefore, the strength of van der Waals interactions are related to the size of a molecule. A hydrogen bond is an electrostatic attraction between a hydrogen (H) and another electronegative atom bearing a lone pair of electrons. The hydrogen atom is usually covalently bound to a more electronegative atom (N, O, F). Table 2 lists the interactions with examples of chemical structures.
A pp-LFER is a linear equation developed to model partition or sorption coefficients (K) using parameters that represent the interactions (Abraham, 1993). The model equation is based on five descriptors:
(2)
with:
E
excess molar refraction
S
dipolarity/polarizability parameter
A
solute H-bond acidity (H-bond donor)
B
solute H-bond basicity (H-bond acceptor)
V
molar volume
The partition or sorption coefficient K may be expressed as the sum of five interaction terms, with the uppercase parameters describing compound specific properties. E depends on the valence electronic structure, S represents polarity and polarizability, A is the hydrogen bond (HB) donor strength (HB acidity), B the HB acceptor strength (HB basicity), V is the so-called characteristic volume related to the molecule size, and c is a constant. The lower-case parameters express the corresponding properties of the respective two-phase system, and can thus be taken as the relative importance of the compound properties for the particular partitioning or sorption process. In this introductory section, we only focus on the volume factor (V) and the two hydrogen bond parameters (A and B).
Numerous pp-LFERs have been developed for all kinds of environmental processes and an overview is given by Endo and Goss (2014).
Table 2. Types of interactions between molecules and the phase to which they sorb with examples of chemicals (Goss and Schwarzenbach, 2003).
Compounda)
Interactions
Examples
Apolar
only van der Waals
alkanes, chlorobenzenes, PCBs
Monopolar
van der Waals +
H-acceptor (e-donor)
alkenes, alkynes,
alkylaromatic compounds
ethers, ketones, esters, aldehydes
Monopolar
van der Waals +
H-donor (e-acceptor)
CHCl3, CH2Cl2
Bipolar
van der Waals
+ H-donor
+ H-acceptor
R-NH2, R2-NH,
R-COOH, R-OH
a) Apolar: no polar group present; mono/dipolar: one or two polar groups present in a molecule
Examples of QSPR for bioconcentration to fish
Kow based model
Predictive models for bioconcentration have a long history. The octanol-water partition coefficient (KOW) is a good measure for hydrophobicity and bioconcentration factors (BCF's) are often correlated to Kow (see more information in section on Bioaccumulation). The success of these KOW based models was explained by the resemblance of partitioning in octanol and bulk lipid in the organisms, at least for neutral hydrophobic compounds. A well-known example of a linear QSAR model for the log BCF (Y variable) based on the log KOW (X variable) (Veith et al., 1979):
log BCF = 0.85 log KOW - 0.70 (5)
Figure 3 gives a classical example of such a correlation for BCF to guppy of a series of chlorinated benzenes and polychlorinated biphenyls. When lipophilic chemicals are metabolised, the relation shown in Figure 3 is no longer valid and BCF will be lower than predicted based on KOW. Another deviation of this BCF-Kow relation can be found for highly lipophilic chemicals with log Kow>7. For such chemicals, BCF often decrease again with increasing Kow (see Figure 3). The apparent BCF curve with Kow as the X variable tends to follow a nonlinear curve with an optimum at log Kow 7-8. This phenomenon may be explained from molecular size: molecules of chemicals like decachlorobiphenyl may be so large that they have difficulties in passing membranes. A more likely explanation, however, is that for highly lipophilic chemicals aqueous concentrations may be overestimated. It is not easy to separate chemicals bound to particles from the aqueous phase (see box 1 in the section on Sorption) and this may lead to measured concentrations that are higher than the bioavailable (freely dissolved) concentration (Jonker and van der Heijden 2007; Kraaij et al. 2003). For example, at a dissolved organic carbon (DOC) concentration of 1 mg-DOC/L, a chemical with a log Koc of 7 will be 90% bound to particles, and this bound fraction is not part of the dissolved concentration that equilibrates with the (fish) tissue. This shows that these models are also interesting because they may show trends in the data that may lead to a better understanding of processes.
Examples of QSPR for sorption to lipids
Kow based models are successful because octanol probably has similar properties than fish lipids. There are several types of lipids and membrane lipids have different properties and structure than for example storage lipids (see Figure 4, and more details in the section on Biota). More refined BCF models include separation of storage and membrane lipids and also proteins as separate sorptive phases (Armitage et al. 2013). pp-LFER is a very suitable approach to model these sorption or partitioning processes and results for two large data sets are presented in Table 3. The coefficients e, s, b and v are rather similar. The only parameter that is different in these two models is coefficient a, which represents the contribution of hydrogen bond (HB) donating properties (A) of chemicals in the data set. This effect makes sense because the phosphate group in the phospholipid structure has strong HB accepting properties. This example shows the strength of the pp-LFER approach because it closely represents the mechanism of interactions.
Table 3. LFERs for storage lipid-water partition coefficients (KSL-W) and membrane lipid-water partition coefficients (KML-W (liposome)). Listed are the parameters (and standard error), the number of compounds with which the LFER was calibrated (n), the correlation coefficient (r2), and the standard error of estimate (SE). log K = c + eE + sS + aA + bB + vV.
Para-
meter
c
e
s
a
b
v
n
r2
SE
KSL-W
-0.07
(0.07)
0.70
(0.06)
-1.08
(0.08)
-1.72
(0.13)
-4.14
(0.09)
4.11
(0.06)
247
0.997
0.29
From (Geisler et al. 2012)
KML-W
(liposome)
0.26 (0.08)
0.85
(0.05)
-0.75
(0.08)
0.29
(0.09)
-3.84 (0.10)
3.35 (0.09)
131
0.979
0.28
From (Endo et al. 2011)
KSL-W: storage lipid partition coefficients are mean values for different types of oil. Raw data and pp-LFER (for 37 oC) reported in (Geisler et al. 2012).
KML-W (liposome): data from liposomes made up of phosphatidylcholine (PC) or PC mixed with other membrane lipids. Raw data (20-40 oC) and pp-LFER reported in (Endo et al. 2011).
Examples of QSPR for sorption to soil
Numerous QSPRs are available for soil sorption (see section on Sorption). Also the organic carbon normalized sorption coefficient (Koc) is linearly related to the octanol-water partition coefficient (see Figure 5).
The model in Figure 5 is only valid for neutral, non-polar hydrophobic organic chemicals such as chlorinated aromatic compounds, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCBs) and chlorinated insecticides or, in general, compounds that only contain carbon, hydrogen and halogen atoms. It does not apply to polar and ionized organic compounds nor to metals. For polar chemicals, also other interactions may influence sorption and a pp-LFER approach would also be useful.
The sorption of ionic chemicals is more complex. For the sorption of cationic organic compounds, clay minerals can be an equally important sorption phase as organic matter because of their negative surface charge and large surface area. The sorption of organic cations is mainly an adsorption process that reaches a maximum at the cation exchange capacity (CEC) of a particle (see section on Soil). Also models for the prediction of sorption of cationic compounds are more complicated and first attempts have been made recently (Droge and Goss, 2013). Major sorption mechanism for anionic chemicals is sorption into organic matter. The sorption coefficient of anionic chemicals is substantially lower than for the neutral form of the chemical, roughly a factor 10-100 for KOC (Tülp et al. 2009). In case of weakly dissociating chemicals such as carboxylic acids, the sorption coefficient can often be estimated from the sorption coefficient of the non-ionic form and the fraction of the chemical that is present in the non-ionized form (see section on Relevant chemical properties).
Reliability and limitations of QSPR
Predictive models have limitations and it is important to know these limitations. There is not one single model that can predict a parameter for all chemicals. Each model will have a domain of applicability and it is important to apply a model only to a chemical within that domain. Therefore, guidance has to be defined on how to select a specific model. It is also important to realize that in many computer programs (such as fate modeling programs), estimates and predictions are implicitly incorporated in these progams.
Another aspect is the reliability of the prediction. The model itself can show a good fit (high r2) for the training set (the chemicals used to develop the model), but the actual reliability should be tested with a separate set of chemicals (the validation set) and a number of statistical procedures can be applied to test the accuracy and predictive power of the model. The OECD has developed a set of rules that should be applied in the validation of QSPR and QSAR models.
References
Abraham, M.H. (1993). Scales of solute hydrogen-bonding - their construction and application to physicochemical and biochemical processes. Chemical Society Reviews 22, 73-83.
Armitage, J.M., Arnot, J.A., Wania, F., Mackay, D. (2013). Development and evaluation of a mechanistic bioconcentration model for ionogenic organic chemicals in fish. Environmental Toxicology and Chemistry 32, 115-128.
Bruggeman, W.A., Opperhuizen, A., Wijbenga, A., Hutzinger, O. (1984). Bioaccumulation of super-lipophilic chemicals in fish. Toxicological and Environmental Chemistry 7, 173-189.
Droge, S.T.J., Goss, K.U. (2013). Development and evaluation of a new sorption model for organic cations in soil: Contributions from organic matter and clay minerals. Environmental Science and Technology 47, 14233-14241.
Endo, S., Escher, B.I., Goss, K.U. (2011). Capacities of membrane lipids to accumulate neutral organic chemicals. Environmental Science and Technology 45, 5912-5921.
Endo, S., Goss, K.U. (2014). Applications of polyparameter linear free energy relationships in environmental chemistry. Environmental Science and Technology 48, 12477-12491.
Eriksson, L., Hermens, J.L.M., Johansson, E., Verhaar, H.J.M., Wold, S. (1995). Multivariate analysis of aquatic toxicity data with pls. Aquatic Sciences 57:217-241.
Geisler, A., Endo, S., Goss, K.U. (2012). Partitioning of organic chemicals to storage lipids: Elucidating the dependence on fatty acid composition and temperature. Environmental Science and Technology 46, 9519-9524.
Goss, K.-U., Schwarzenbach, R.P. (2001). Linear free energy relationships used to evaluate equilibrium partittioning of organic compounds. Environmental Science and Technology 35, 1-9.
Goss, K.U., Schwarzenbach, R.P. (2003). Rules of thumb for assessing equilibrium partitioning of organic compounds: Successes and pitfalls. Journal of Chemical Education 80, 450-455.
Hansch, C., Streich, M., Geiger, F., Muir, R.M., Maloney, P.P., Fujita, T. (1963). Correlation of biological activity of plant growth regulators and chloromycetin derivatives with hammett constants and partition coefficients. Journal of the American Chemical Society 85, 2817-&.
Jonker, M.T.O., van der Heijden, S.A. (2007). Bioconcentration factor hydrophobicity cutoff: An artificial phenomenon reconstructed. Environmental Science and Technology 41, 7363-7369.
Katritzky, A.R., Slavov, S., Radzvilovits, M., Stoyanova-Slavova, I., Karelson, M. (2009). Computational chemistry approaches for understanding how structure determines properties. Zeitschrift Fur Naturforschung Section B-a Journal of Chemical Sciences 64:773-777.
Könemann, H., Van Leeuwen, K. (1980). Toxicokinetics in fish: Accumulation and elimination of six chlorobenzenes by guppies. Chemosphere 9, 3-19.
Kraaij, R., Mayer, P., Busser, F.J.M., Bolscher, M.V., Seinen, W., Tolls, J. (2003). Measured pore-water concentrations make equilibrium partitioning work - a data analysis. Environmental Science and Technology 37, 268-274.
Sabljic, A., Güsten, H., Verhaar, H.J.M., Hermens, J.L.M. (1995). Qsar modelling of soil sorption. Improvements and systematics of log koc vs. Log kow correlations. Chemosphere 31, 4489-4514.
Tülp, H.C., Fenner, K., Schwarzenbach, R.P., Goss, K.U. (2009). pH-dependent sorption of acidic organic chemicals to soil organic matter. Environmental Science and Technology 43, 9189-9195.
Veith, G.D., Defoe, D.L., Bergstedt, B.V. (1979). Measuring and estimating the bioconcentration factor of chemicals in fish. Journal of the Fisheries Research Board of Canada 36, 1040-1048.
3.4.3. Question 1
What is a QSPR and why is it useful?
3.4.3. Question 2
Which techniques are applied to derive a QSPR.
3.4.3. Question 3
Which chemical parameters are applied in a QSPR? | textbooks/chem/Environmental_Chemistry/Environmental_Toxicology_(van_Gestel_et_al.)/03%3A_Environmental_Chemistry_-_From_Fate_to_Exposure/3.04%3A_Partitioning_and_Partitioning_Constants.txt |
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