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Don Rich
- Birthday
- April 10
- The Second Netanyahu Speech
February 27, 2015 03:42PM
- The Opportunity in Current
Middle Eastern Turmoil
February 14, 2015 03:18PM
- Whither and Where Ukraine?
February 07, 2015 04:24PM
- Airstrikes and ISIS
February 06, 2015 04:12PM
- Thinking Over the Meaning of
January 31, 2015 03:15PM
- "Interesting piece. Very
good, if was it always a
fantasy to
"have the
February 14, 2015 03:50PM
- "The Russians advance, if
they are hitting
February 14, 2015 03:17PM
- "That was funny, so
thanks for my status now of
February 06, 2015 04:25PM
- "hello.
It is of
course popular to see naming
as a victory, see abortion
February 06, 2015 03:58PM
- "The only freedom is in
your head."
January 31, 2015 03:11PM
Don Rich's Links
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The Second Netanyahu Speech
For MEB, may you live long and prosper.
No, this not about Mr. Spock passing away, if it's in the back of my mind and front too.
Mr. Spock of course was about jettisoning emotions and relying about reason.
People like him are often frustrated by the actual emotions that often… Read full post »
The Opportunity in Current Middle Eastern Turmoil
For MEB, forever.
Lately it has seemed like the Middle East has exploded or imploded. which you will.
In the long run view, such turbulence is understood to be normal in times of great change, especially in Yemen.
Yemen has a scary-looking at first glance, with a population closing in on… Read full post »
Whither and Where Ukraine?
for mebforever
The last ten years have been witness to oscillation between pro-Russian and anti-Russian forces, which at some times involved various uses of force.
Right now the anti-Russian forces in the Ukraine have the diplomatic high ground, since Russia's annexation of Crimea marks a serious bre… Read full post »
Airstrikes and ISIS
for mebforever
Airstrikes are central to modern war, managing the consequences an importand job of the insurgents or terrorists.
Some will say that airpower cannot win alone, citing the experience with the Nazis, and also in Korea and Vietnam.
In fact, since WWI so astonished people by being so blood… Read full post »
Thinking Over the Meaning of Terrorism
for meb forever
Using language in a way that makes what you want sound more favorable is common in political discourse.
Here that claim one can see as accurate in the abortion debates, as one side prefers that the key remains choice while the other side prefers that discussion to be… Read full post »
Libyan Policy Re-Considered
For MEB, forever.
Foreign policy often remains obscure to most average people, and more the realm of the 1 percent we see as talking heads, or people who would be plausible in that role.
That is because foreign policy deals with topics not likely to be known well by the… Read full post »
American Strategy with ISIS
For MEB, forever.
President Obama has been much criticized for his denial of having a strategy.
Announcing the strategy on the other hand could look like the SNL satire of the media, like the cast member who as satire asks "Sir, could you state precisely where and when our strik… Read full post »
The Curiosity of the Recognition Game and Gaza War
For MEB forever.
It has always struck the author's curiosity as to recognition of Israel in the Middle East.
They still talk, just through mediaries, like Egypt and Jordan.
Neither the Israelis nor the Hamas side will concede that Hamas or Israel have a right to coexist, at… Read full post »
Realism and Iraq
MEB forever
Realism predicts that states are driven by the fear of a lack of a world sovererign to settle disputes.
Realism posits in some, if not all, versions, that an individual President is unlikely to end up of doing different things that any President would come to, as to security… Read full post »
Obama's Move back to Iraq
For MEB forever
One of the major premises of his celection and re-election was to leave Iraq.
Thus, to go back in any fashion was bound to trigger partisanship, and yet, we really did have to do something to try to stabilize Iraq, especially when the opposition in Iraq are millitants who… Read full post »
What Price Ukraine, and What Not?
Forever MEB.
The sanctions we impose on foreign countries are a hard weapon to use with Will ususally thought to be important.
In the case of Ukraine, Russia was unlikely to just rollowever, and Western politicians in fact wil have to see what teh risks to doing more will entail for th… Read full post »
Reagan Proved Correct on Missile Defense?
For MEB, forever
When Reagan outlinedhis vision of missile defense, many ccrtics emerged.
That's fair enough, as the vision of Star Wars critics was in fact not ready.
That has now emerged as excessive too, as can be seen in Iron Dome.
It's scaled back to a single locati… Read full post »
Important dfferences with Srar Wars and Iron Dome
For MEB forever.
If Hamas can claim it a triumph of engineering under very difficult conditions, Israel can claim a similar triumph with the Iron Dome Missile Defense System.
They both have their limits, limits worth thinking about technically and politically.
As to the Qassam, Katyusha and … Read full post »
Ballistic Missile Defense, BMD, and Star Wars v Reality
FOr MEB forever.
Israel's much vaunted missile defense (BMD) system hasn't completely shut down Hamas, but if has worked pretty well in a real live fire excersise.
Now normally one would label Iron Dome a short range missile defense, unlike the SA 17 that may well have shot down the Malays… Read full post »
Why Hamas is Something of a Rational actor: Implications
For MEB, forever.
Rational actor doesn't mean good, just having a set of goals that link together. Hamas saying it will destroy Israel now, or in ten years, is rational within the framework of rational actor, although a stretch too, since it isn't very likely that Hamas will ever be strong enoug… Read full post »
Sevens and Serendipity in flight MH7 on a 777 on 7/7/14
MEB forever
It's curious how the number seven ties to the tragic plane crash of Malaysian flight designated flight MH 17 on a 777.
That is not to make light6 of what has happended, a massive loss of life.
Then the date, 7 July, 7, in a 777, and again on the seventh mon… Read full post »
Hamas and Nixon Goes to China viz Netanyahu
MEB forever
There are many problems facing America in foreign affairs, not however mainly of our making.
We didn't invent the 1667 conquest of Ukraine by the suceessor state to the original Russian Empire of Kievan Rus.
As to Netanyahu, there is an analogy to Nicon going to China,… Read full post »
The bias in Israel-Hamas Media Coverage
For MEB forever.
The author cannot be described as being pro-Israel or anti-Palestinian.
Rather that situation has tragic origins now is niniety years old.
Although that length of time doesn't induce hope, proplems are what they are too.
As to the current dispute, it is biased to repo… Read full post »
What is a reasonable hope for Iraq 2014?
For MEB, forever.
Iraq and the Middle East have had a very mixed bbag of experiences as independent states since the Great War ended in 1917.
It didn't reaaly end then, as France had to bomb areas of Damsxus in 1930, and the British even used mustard gas to beat down tri… Read full post »
Obams's Measured Appraoch in Iraq 12014
For mebforever.
My glasses are broken and so any errors as to typos have that cause.
IN the end, Iraq has to govern iitself, just like children have to leave their parents too, someday.
It is obvious that the Iraqi Security Foreces need something of an apron string, the… Read full post »
The Kurds Dreams of Independence
For MEB, forever.
When you hear certain towns fall, it's easy to see why ISIS chose the routes that they did, save for one: Kurds.
When you see their path and strongholds, it then is more obvious that the Kurds cannot be said to be so passive afterall, and that some are trying to… Read full post »
A Non-Partisan Debate on Iraq
For MEB, forever. And "Johnny Ramrod," for a 15 month tour.
It might have been predictable that Iraq would become a painful issue in the history of American foreign policy.
If you don't see that, think about what would have happened if Saddam had lived as long as Mubara… Read full post »
Iran and The Iraqi Crisis of 2014
For MEB, forever.
The best titles of works on the Middle East have a commonality as to title, if not optimistic ones; Quicksand, A Peace to End all Peace being my personal favorites.
Iran of course borders Iraq completely in the East of Iraq, for right around 1,000… Read full post »
Iraq in Modern History: Part I
For MEB.
Iraq is going back into the news cycle, because it looks like a civil war is a distinct possibly, a civil war that would endanger the contractors there, and the diplomats too, as in thousands of lives.
Deploying more American ships to the Gulf as has been announced is jus… Read full post »
Dienbienphu and Iraq
For MEB, forever.
It is never easy to make policy in international relations.
That is because there are just too many complexities to know with a very high degree of certainty what the policy options will actually do in practice.
That has led to some researchers using analogy as a method of … Read full post »
Don Rich's Favorites
ISIS beheading Iranian Mullahs? What's not to love?
1 Question God 'n Government
Howard and Me
Would Napoleon have been on Facebook?
PBS Tries Dumbing Down Content During Pledge Drive
Textures of a Walk ~ a photo essay
Fighting The Good Fight
Rookie Republican senator Cotton pens letter to Iran


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47-year-old Sheryl Lynn Lee lives in Beverly Hills, California. Other places in which Sheryl has lived are Malibu, California; Moorpark, California; Van Nuys, California and West Hollywood, California.
Companies in which Sheryl has a work history include Without A Trace, Twin Peaks, Bliss, Kiss The Sky, Jersey Girl, Love, Kingpin, Texas, Desperate Housewives, David, Manchild, One Tree Hill, Vampires, Fall Time, State Of Mind, Love Letters, Medicine Woman, Emerald City, Backbeat, Wild At Heart, Homage, Paradise, Television Actors, Csi: Ny, Stage Actors, House M.d., Twin Peaks: Fire Walk With Me, Mother Night, Dirty Sexy Money, Dr. Quinn, L.a. Doctors, Lies And Murder, Hitched, Follow The River, Aaahh Real Monsters, Red Shoe Diaries, Guinevere, Children On Their Birthdays, Crimes Of The Heart, This World, Then The Fireworks and Salome. One of Sheryl's favorite TV shows is Television Actors.
Some of Sheryl's relatives are Ralph Bryan Lee, Susan Leehoffman, Elizabeth L Kreger, William Wayne Lee, Paul Stephen Lee, Linda Anne Lee, Laura Diane Lee, Darian Nelson Shapiro, Summer Aubri Lee, Salvatore Paul Coppolecchia, Laura Lee Lambrecht, Hhs W Lee and Wayne W Lee.

subset: commoncrawl


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Hep C Acute
Here's a taste of what TheBody.com has to offer on this topic:
Ask the Experts
Urgent Question on treating acute Hep CUrgent Question: I may be in the acute phase of Hepatitis C, having possibly contracted it on Nov 8, 2004 (10 weeks ago). I am waiting on blood test results (PCR RNA, ALT/AST, Hep C AB). I understand that the cure rate is nearly 100% if I start...
Response from Barbara McGovern, M.D.
Tufts University School of Medicine...You are correct in saying that if you treat hepatitis C in the "acute stage" the chance of cure is much higher than when you have chronic infection. Not all people need treatment for Read more »
acute HIV/HepC co-infection dynamicsDear Doctor McGovern:Thanks to both you and Dr. Dieterich for finding time to help us. I hope you... Read more »
Acute Hepatitis C and HIVHi Dr. McGovern I know you said that if a person has Acute Hepatatis C ..then and only then will... Read more »
FAQs for the Public: Hepatitis C...C? What is hepatitis C? Statistics How common is acute hepatitis C in the United States? How common is chronic hepatitis C in the United States? How likely is it that acute hepatitis C will become chronic? Transmission /... Read more »
Hepatitis: Overview...hepatitis A, B, C, D, E and G viruses, or HAV, HBV, and so on. Over 90% of cases of hepatitis are caused by hepatitis A, B, or C. Viral hepatitis can be acute or chronic. Acute means the first few weeks to months after you get hepatitis. You may... Read more »
Rapid Liver Fibrosis Occurring More Frequently Among HIV-Infected Men Newly Coinfected With Hepatitis C...things out. Daniel Fierer: Right. And we plan to do that. But unfortunately, from fairly large experience of people with chronic hepatitis C, we don't expect to have useful results from that. Because at least in people with chronic hepatitis... Read more »
Hepatitis C and HIV... What Is Hepatitis C? How Is It Diagnosed? How Is Hep C Treated? Can Hep C Be Prevented? Hep C and HIV Together The Bottom Line Read more »
Connect With Others
Posted by Anonymous, 1 Reply...JOHN HOPKINS SITE SAID THAT CHRONIC HEP C CAN CAUSE DELAYED SEROCONVERSION SO IF THAT PERSON HAS BOTH BECAUSE OF HIV HE WOULD BE MORE LIKELY TO GIVE YOU HEP C SO IF YOUR Read more »
Haircut risk?
Posted by Anonymous, 3 Replies...recently. The barber nicked me when shaving my neck with the straight razor. I hardly felt it. What is the risk for aids or hepatitis c? I am freaking out here and worried sick. I haven't had any acute symptoms. Thanks. Read more »
Hep c delays seroconversion
Posted by Anonymous, 3 Replies...I just found info that Hep c will delay seroconversion for Hiv test John Hopkis web site says that Hiv test will show positive 10-14 days after infection Read more »
Posted by Anonymous, 2 Replies...vaccinated right away. DTD Here is that post I was wanting you to see. Did you use protection? I REALLY would not worry about hep C, it is extremely hard to catch. If you shared a needle with her sure, be worried. I have a boyfriend that... Read more »
Resource Centers
Guides for People With HIV


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Visit create.airbnb.com »
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SD Rancher Relief Fund nearing $1M
RAPID CITY (AP) — A fund aimed at helping South Dakota ranchers who lost livestock in an early autumn blizzard is nearing $1 million.
The Rapid City Journal reported the South Dakota Rancher Relief Fund began taking applications Friday from livestock producers who need assistance. The 45-day application period closes on Dec. 31.
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Nowatzki hired as Bismarck reporter for Forum News Service
BISMARCK -- Veteran news reporter Mike Nowatzki has been hired to staff the Bismarck bureau for Forum News Service.
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Page 1
Mukerjee and Ostrow BMC Biochemistry 2010, 11:16
Open Access
Interactions of unconjugated bilirubin with
vesicles, cyclodextrins and micelles: New modeling
and the role of high pKa values
© 2010 Mukerjee and Ostrow; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and repro-
duction in any medium, provided the original work is properly cited.
Research article
Pasupati Mukerjee†1 and J Donald Ostrow*†2
Background: Unconjugated bilirubin (UCB) is an unstable substance with very low aqueous solubility. Its aqueous pKa
values affect many of its interactions, particularly their pH-dependence. A companion paper shows that only our prior
solvent partition studies, leading to pKa values of 8.12 and 8.44, met all essential requirements for valid pKa
determinations. Other published values, generally lower, some below 5.0, were shown to be invalid. The present work
was designed to derive suitable models for interpreting published data on the pH-dependent binding of UCB with four
agents, mentioned below, chosen because they are not, themselves, sensitive to changes in the pH range 4-10, and the
data, mainly spectrometric, were of reasonable quality.
Results: These analyses indicated that the high pKa values, dianion dimerization constant and solubilities of UCB at
various pH values, derived from our partition studies, along with literature-derived pH- and time-dependent
supersaturation effects, were essential for constructing useful models that showed good qualitative, and sometimes
quantitative, fits with the data. In contrast, published pKa values below 5.0 were highly incompatible with the data for
all systems considered. The primary species of bound UCB in our models were: undissociated diacid for
phosphatidylcholine, dianion for dodecyl maltoside micelles and cyclodextrins, and both monoanions and dianion for
sodium taurocholate. The resulting binding versus pH profiles differed strikingly from each other.
Conclusions: The insights derived from these analyses should be helpful to explore and interpret UCB binding to more
complex, pH-sensitive, physiological moieties, such as proteins or membranes, in order to understand its functions.
The true pKa values of unconjugated bilirubin (UCB) are
an important determinant of the proportion of the three ion-
ization species of UCB in present in solution, and of the
overall aqueous solubility of UCB, at any given pH value
[1]. In a companion paper [2] we re-examined many pub-
lished studies that assessed pKa values for UCB in simple
solutions, determined by a wide variety of methods. We
critically assessed the reliability of the methods used, in
relationship to minimal criteria for validity, as well as other
considerations. We summarized the deficiencies in the
many reports which suggested that pKa values of UCB
were below 7.0 and even below 5.0 (see Table eight in Boi-
adjiev et al. [3]). The only experiments which fulfilled all
the validity criteria were our solvent partition data [4,5],
which indicated that the two pKa values were much higher,
8.12 and 8.44.
pKa values of UCB are clearly of great importance in deter-
mining the effects of pH on the interactions of UCB with
other molecules [1]. In the present paper, we critically re-
examine and reinterpret several reports dealing with the
effects of varied pH on the interactions of UCB with phos-
pholipid vesicles, cyclodextrins, and dodecylmaltoside and
bile salt micelles. Some general approaches have been
developed to deal with the binding equilibria involved. We
show that these studies are incompatible with proposed low
aqueous pKa values for UCB. In several cases, the reinter-
pretations, using high pKa's and incorporating the effects of
binding ratios, self-association and pH-dependent supersat-
uration, lead to some interesting new models and findings.
* Correspondence: email@example.com
2 GI/Hepatology Division, Dept. Medicine, Box 356424, Univ. Washington
School of Medicine, 1959 NE Pacific St., Seattle, WA 98195-6424, USA
† Contributed equally
Full list of author information is available at the end of the article
Page 2
Mukerjee and Ostrow BMC Biochemistry 2010, 11:16
Page 2 of 12
Criteria for Acceptability and Pitfalls in Interpretation
Studies of UCB binding vs. pH should meet the same crite-
ria of validity proposed in the companion paper that deals
with UCB alone in simple systems [2]: 1) The UCB is pure;
2) There should be no significant degradation of UCB; 3)
Measurements are made after a rapidly achieved equilib-
rium; 4) Unbound UCB concentrations are below to mini-
mally above aqueous saturation; 5) The pH range studied
should encompass all suggested pKa values of UCB, and
sufficient data points should be available to permit mathe-
matical modeling.
In addition, for studies of the effects of pH on UCB bind-
ing: 6) The large molecule or components of the aggregate
must also be purified; 7) Changes with pH in the ionization
and/or conformation of constituent regions of the large mol-
ecule or aggregate must not affect its intrinsic affinity for
each UCB species. This last criterion renders it difficult to
perform detailed mathematical modeling of pH effects on
interactions of UCB with proteins, natural membranes and
other biologically-relevant systems, which are often impure
and include molecules whose conformation and binding
properties for UCB are affected by pH in incompletely
known ways.
Further discussion is needed regarding criterion 4, above,
including the thermodynamic solubility of UCB crystals
[4], and the possibility of stable supersaturation [6,7]. Brod-
ersen & Theilgaard [7], showed that, after extensive sedi-
mentation at 100,000 xg, the UCB concentrations were 0.1
μM at pH 7.40, 0.5 μM at pH 7.83, 17 μM at pH 8.05, and
34 μM at pH 8.2. Solubility calculated from our partition
data [4] was 0.062 μM at pH 7.40, in reasonable agreement
with the data of Brodersen's & Theilgaard [7], but our pari-
tion-derived solubilities at the higher pH values were con-
siderably lower than theirs: 0.084 μM at pH pH 7.83, 0.112
μM at pH 8.05, and 0.148 μM at pH 8.2. Thus, for reasons
discussed previously [4,6,7], stable supersaturation with
UCB, up to fairly high UCB concentrations, may be
expected at pH values of 8.0 and above, particularly with
the short time intervals between solution preparation and
data gathering in most spectroscopic studies. At modestly
high concentrations of UCB, such supersaturation may
actually be promoted by amphipathic additives, for exam-
ple bile salts [8,9]. In the reinterpretations attempted below,
we have made comparisons of results expected in the pres-
ence and absence of supersaturation and assuming high vs.
low pKa values for UCB.
Selection of Publications for Further Analysis
To find papers for possible review, we electronically
searched PubMed (1967-date), and ISI and Chemical
Abstracts databases back to 1950, using the keywords "bili-
rubin, bile pigments, binding, hydrogen-ion concentration,
pH, pKa, ionization", as well as the reference lists in papers
thus discovered. To locate papers published earlier than
1968, we manually searched T.K. With's two comprehen-
sive compendia of studies related to bilirubin [10,11]. After
eliminating papers that dealt only with bile pigments other
than biladienes, or with bilirubin ester conjugates, papers
were then eliminated that failed to meet the majority of the
criteria of validity summarized above. In line with validity
criterion 7, above, we then focused on studies of interac-
tions of UCB with host systems not expected to be sensitive
to pH in the range of 5.5 to 10. These 12 papers and one
abstract are summarized in Additional files 1, 2 and 3
(Tables S1, S2 & S3), which specify experimental deficien-
cies for each study. Finally, we confined our detailed analy-
ses to the four best studies; binding of UCB to
phosphatidylcholine (PC) [12], dodecylmaltoside micelles
[13], β-cyclodextrins [14], and sodium taurocholate [15].
Results and Discussion
Ionization and Binding Equilibria of Unbound UCB Species
The equilibria governing the binding of unconjugated bili-
rubin (UCB) involve its three unbound species, the proto-
nated diacid (H2B), the monoanions (HB-) and the dianion
(B=), one or more of which binds independently to a larger
host system [1], such as phospholipid vesicles, cyclodex-
trins, or micelles. For each UCB species, this binding can
then be described in terms of a distribution ratio, K = mols
bound/mols unbound (free).
The ionization equilibria of the unbound species apply
whether or not a binder is present. Thus, with a binder pres-
ent, at any pH, the relative fraction (f) of each unbound
monomeric species of UCB (fH2B, fHB-, and fB=) will be
equal to f for that species, at the same pH, in an aqueous
phase containing only monomeric UCB species [1]. There-
fore, as described in the appendix in Hahm et al. [4], these
unbound fractions are determined solely by the pH and the
assumed pKa values of the monomeric species. The total
concentration of unbound, monomeric UCB species (Bm)
is given by Bm = [H2B] + [HB-] + [B=] = [H2B] × (1 + K1/
[H+] + K1.K2/[H+]2). Note that these illustrative calculations
and simulations (Figures 1, 2 and 3 and Table 1) deal only
with pH effects on monomeric UCB species, so that fH2B +
fHB- + fB= = 1.00. Additional effects of the formation of B=
dimers [4] and supersaturation [7,9,16] have been consid-
ered below in the actual fitting of experimental data on
UCB binding to phospholipid vesicles, cyclodextrins and
micelles (Figures 4, 5 and 6 and Table 2).
Figure 1 shows the fractions of UCB species over the pH
range of 4 to 10, using the high pKa values of 8.12 and 8.44
[4]. Table 1 shows the fractions derived by applying the low
pKa values of 4.2 and 4.9, advocated in recent papers [3,17-
20]. For such low pKa values, B= is the dominant fraction at
pH values above 7.0 (fB= ≥ 0.992, close to unity), with
fH2B < 0.001 at pH values above 6.0 and fHB- < 0.001 at
Page 3
Mukerjee and Ostrow BMC Biochemistry 2010, 11:16
Page 3 of 12
pH values above 8.0. In stark contrast, for pKa values
above 8.0 [4], the f values of H2B, HB- and B= are moder-
ately high over wide ranges of pH and are of comparable
magnitude at most pH values between 6 and 10 (Figure 1).
At pH ≤ 6.0, only fH2B is above 0.99 and, over the pH
range of 6.5 to 10.0, significant roles can be played by any
of the three UCB species, each of which will show signifi-
cant dependence on pH (Figure 1). The inescapable qualita-
tive conclusion from these considerations is that numerous
studies which show UCB interactions increasing with pH in
range of 6.5-10.0 are far more compatible with the high
pKa values of UCB (> 8.0) than the low ones (< 5.0).
General Effects of Binding Affinities and pKa Values on
Binding Curves
The values of pKa affect some characteristic features of the
uptake and binding curves of UCB as a function of pH. As
the pH increases, the equilibrium among the unbound spe-
cies of UCB will shift to the more ionized forms (Figure 1,
Table 1). The binding of UCB will, therefore, increase with
pH if the preferentially bound species is B= (as proposed for
sodium taurocholate micelles [1,21]), but will decrease with
pH if H2B is preferentially bound (as proposed for PC vesi-
cles [4,12]).
Figure 1 Fractions (f) of unbound monomeric UCB at various pH
values. Calculated assuming high pKa values for UCB of 8.12 and 8.44
[4]. Abbreviations: H2B, UCB diacid; HB-, UCB monoanions; B=, UCB dian-
ion. fH2B + fHB- + fB= = 1.
6789 10
Fraction (f)
Figure 2 Fraction of total UCB bound (F) at various pH values, as-
suming preferential binding of H2B: Effect of distribution ratio (K
= bound/free UCB). Calculated assuming high pKa values for UCB of
8.12 and 8.44 [4]. H2B, fully protonated UCB diacid; shaded star, mid-
point pH of titration curve.
5.0 5.56.06.57.07.5
8.08.59.0 9.5
Fraction of Total UCB
Bound (F)
K= Bound/Free H2B
Figure 3 Fraction of total UCB bound (F) at various pH values, as-
suming preferential binding of B=: effect of distribution ratio (K' =
bound/free UCB). Calculated assuming high pKa values for UCB of
8.12 and 8.44 [4]. B=, UCB dianion; shaded star, midpoint pH of titration
curve; open star, midpoint for TC binding (K' for B= = 730, [21]).
5.0 5.56.0 6.57.0 7.5
8.08.5 9.09.510.0
Fraction of Total UCB
Bound (F)
K'= Bound/Free B=
Figure 4 Modeling UCB binding to phosphatidylcholine vesicles
at various pH values. Experimental data derived from Tipping et al.,
1979 [12]. Curve A, calculated assuming pKa values for UCB of 8.12 and
8.44 and the dimerization constant for the B= dianion of 0.26 μM-1 [4].
Curve B, calculated assuming constant maximum solubility of H2B of
51 nM [4]. Curve C, calculated assuming pKa values of UCB of 4.2 and
4.9 [3,17-20]. Shaded star, mid-point pH of binding curve A.
Mols UCB Bound per Mol PC
Tipping's Data
Calc. High pKa
Calc. H2B at
Saturation (B)
Calc. Low pKa
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Mukerjee and Ostrow BMC Biochemistry 2010, 11:16
Page 4 of 12
The mid-point pH values of plots of binding vs. pH may
shift significantly from the pKa of the unbound UCB ligand
according to its affinity for the large molecule or aggregate,
especially if the binding affinity for one of the three UCB
species, H2B, HB- and B=, is strongly dominant. This effect
is easily modeled by assuming that only one of the three
species is bound and that the unbound UCB concentrations
are low enough for self-association of the unbound UCB
species to be negligible. The proportions of the unbound
species, H2B, HB- and B=, are determined by the pKa values
and the pH and calculated from the relevant terms in the
model equation [4], as described above. If the relevant UCB
species is not the only one bound, but is the predominant
bound species, these analyses still apply qualitatively and
the plots will closely resemble those derived by assuming
that only a single UCB species binds.
Figures 2 and 3 show some simple representations of such
modeling, using the high pKa values of 8.12 and 8.44 [4];
the fraction F = [UCB]bound/[UCB]total is plotted against pH.
For Figure 2, it is assumed that only H2B binds, e.g. to a
phospholipid vesicle [12]. The fraction F is calculated for
values of the partition ratio K = [H2B]bound/[H2B]free,
increasing from 0.2 to 104. As expected (Figure 2), with
increasing pH, F decreases sigmoidally due to the decreas-
ing fraction (fH2B) of the unbound UCB diacid (Figure 1).
For each curve, its mid-point pH, where the binding of H2B
is 50% of the maximum, is represented in Figure 2 by a
shaded star. These mid-point pH values increase from 8.255
for K = 1, to 8.780 for K = 10, 9.251 for K = 102 and 9.778
for K = 103; for very low K values <<1 (e.g. for K = 0.2),
the limiting mid-point pH is 7.994. This increase in the
mid-point pH of the binding curves of H2B with increasing
K is a general result that applies to binding to any large
molecule and to any pKa values that are chosen. An exam-
ple is binding of UCB to PC (Figure 4, curve A)
When the UCB dianion, B=, is the only interacting species,
the uptake or binding is expected to increase with pH (Fig-
ure 3), due to the increasing fraction (fB=) of the unbound
UCB dianion (Figure 1). Figure 3 plots F vs. pH at values of
K' (= [B=]bound/[B=]free) of 0.2, 1, 10, 102, 103 and 104,
assuming the pKa values are 8.12 and 8.44. The mid-point
pH values of these sigmoidal curves (shaded stars), where
binding of B= is 50% of the maximum, decrease from 8.348
for K' = 1, to 7.863 for K' = 10, 7.315 for K' = 102, 6.794 for
K' = 103 and 6.287 for K' = 104. The limiting value of the
mid-point pH, when K' values are <<1 (e.g. K = 0.2), is
8.574. This trend of decreasing mid-point pH values with
increasing K' is also a general result for binding of B= to any
large molecule for any pKa values chosen.
Many curves of UCB interactions as a function of pH show
binding increasing with increasing pH (Figure 5; Additional
files 1, 2 and 3, Tables S1, S2 & S3), indicating a primary
role for the binding of B= and dictating that the mid-point
pH values must be lower than the true pKa values of
unbound UCB (Figure 3). Therefore, the large number of
these titration curves with mid-point pH values between 6
and 8 are compatible with high pKa values of 8.12 and 8.44
[4], and quite incompatible with pKa values below 5.0. If
pKa values were low, e.g. 4.2 and 4.9 [3,17-20], curves
Figure 5 UCB binding to β-cyclodextrin at various pH values. Ex-
perimental data (black squares) from Kano et al., 1995 [14]. Curve A, cal-
culated ellipticity assuming pKa values for UCB of 8.12 and 8.44, with B=
dimers [4]. Curve B, calculated ellipticity assuming pKa values of UCB of
4.2 and 4.9 [3,17-20]. Curve C, calculated ellipticity assuming constant
maximum solubility of H2B of 51 nM [4].
CD Ellipticity
Kano's Data
Calc. High pKa with
B= dimers (A)
Calc. Low pKa (B)
Calc. H2B at
saturation (C)
Figure 6 Experimental vs. calculated CD ellipticities (103θ) of UCB
in 50 mM sodium taurocholate solutions. Data for plotting taken
from Table 2. *103θ values, obtained with 4.6 μM UCB, have been mul-
tiplied by the factor, 34/4.6 to render them comparable with data ob-
tained at 34 μM UCB. Experimental values are from D'Alagni M, et al.
[15]. Calculated values are derived as described in the text and summa-
rized in Table 2.
-15-10 -505 10
103θ, Expeimental
103θ, Calculated
[UCB] = 34 µM
[UCB] = 4.6 µM*
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Mukerjee and Ostrow BMC Biochemistry 2010, 11:16
Page 5 of 12
showing increasing binding of UCB with increasing pH
and, therefore, binding of B=, would yield mid-point pH
values lower than about 5.0.
The mid-point pH values of sigmoidal titration curves, like
those in Figures 2 & 3, are sometimes misinterpreted as
average pKa values. This is clearly wrong, since all of the
curves in Figures 2 &3 assume the pKa values of unbound
monomeric UCB to be 8.12 and 8.44.
UCB Binding to Phospholipid Vesicles (Additional file 1,
Table S1)
Unilamellar vesicles of phosphatidylcholine (PC) are bilay-
ers with a hydrophobic core of the fatty acid polymethylene
chains, and hydrophilic exterior and interior surfaces com-
posed of phosphryl-choline sidechains whose terminal -
N+(CH3)3 groups are on the surfaces [22]. In the pH range 4
to 10, PC's are zwitterions, with both the amine and phos-
phoric acid groups fully ionized [23,24]; thus, changes in
UCB binding to PC may be attributed to altered proportions
Table 1: Fractions (f) of unbound UCB species at different pH values, assuming low pKa values of 4.2 and 4.9*.
0.26390.5265 0.2096
* Calculated assuming pKa values of UCB of 4.2 and 4.9 [3,17-20].
† Bilirubin species: H2B, diacid; HB-, monoanions; B=, dianion.
Table 2: Circular dichroism (ellipticity, 103θ) of UCB in 50 mM sodium taurocholate solutions*
[UCB] Exptl.
103θ†fsHB-‡fsB= ‡ 103θ†
34.0 -11.800.1661 0.5790 -13.42
4.6-12.600.14740.6780 -11.06
4.6 0.69 0.03450.95501.50
34.01.900.03300.9570 1.66
4.65.100.00370.9946 4.75
34.0 5.500.0000120.99865.13
* Data from D'Alagni M, et al., 1994 [15]. For experimental problems, see Additional file 3, Table S3.
† 103θ values obtained with 4.6 μM UCB have been multiplied by a factor, 34/4.6, to render them comparable with 103θ values obtained at
34 μM UCB.
‡ fsHB- and fsB=, fractions of total UCB bound to TC as monoanion and dianion respectively, calculated utilizing the partition ratios of each
UCB species in water [4] and 50 mM TC [21], plus fitted 103θ values for bound HB- and B=, derived from modeling the experimental changes
in 103θ with pH, as described in the text.
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Mukerjee and Ostrow BMC Biochemistry 2010, 11:16
Page 6 of 12
of the three ionization states of unbound UCB in this pH
range. Depending upon the molar ratios and experimental
conditions under which the UCB and PC are mixed, either
H2B or B= may be the UCB species that is preferentially
bound [25].
pH effects on the interactions of UCB with PC vesicles
have been reported in a number of studies [12,16,22,25-27].
Many of them involve time-dependent processes in non-
equilibrium situations. Some used PC systems containing
fluorescent probes, which are difficult to interpret due to
uncertainties regarding the localization of the probes and
the nature of their interactions with UCB.
The report by Tipping et al. [12], which assessed binding of
UCB to egg PC vesicles from the effects of binding on the
spectrum of UCB, most fulfills the above criteria of an
acceptable study (Additional file 1, Table S1), although nei-
ther the UCB (10 μM) nor the PC (100 μM) were purified.
We have, therefore, amplified our previous analysis [4] of
this study.
The data points in Figure 4 (black squares) show values of ñ
(moles of UCB bound per mole of total PC) vs. pH over the
pH range of 6.54 to 9.54, obtained from Figure 10 in the
paper of Tipping et al. [12]. ñ decreases from a maximum
of 0.049 as pH increases, indicating (see Figure 2) that the
binding of H2B is the predominant interaction. The concen-
tration of bound UCB (Bb = ñ × 100 μM) is then used to
calculate the concentration of unbound UCB (Bf = 10 μM -
Bb). Bf ranges from 5.1 μM at pH 6.54 to 9.5 μM at pH
At each pH, the fraction of unbound H2B (= [H2B]f/Bt) in
the presence of 100 μM PC [12], is assumed to be equal to
the fraction of H2B ([H2B]/Bw) in the aqueous phase in the
absence of PC, calculated using Equation four in Hahm et
al. [4]) and applying the high pKa values of 8.12 and 8.44
and the dimerization constant for the B= dianion of 0.26
Assuming that unbound UCB is in a solution state and that
H2B is the only species that binds to the PC yields a simple
model of distribution of H2B between the free and vesicle-
bound state, ñ = K" [H2B]f. Applying this equation to the 9
data points of Tipping et al. [12], yields a mean K" =
0.00932 ± 0.00053 μM-1) and curve A in Figure 4). The fit
of curve A with the experimental ñ values (black squares) is
quite good (r = 0.953), and suggests that our assumptions
are valid and that the H2B partition model is reasonable.
The deviation of the data points from Curve A at the highest
two pH values possibly reflects weak binding of the B=
dianion [16,22,27], which constitutes over 80% of the UCB
species above pH 9.0.
Tipping et al. [12] reported that "The spectral differences
developed within the time required to mix the solutions and
start recording and were the same whether multilamellar or
unilamellar vesicles were used". This rapid analysis and
establishment of equilibrium are necessary conditions for
our detailed interpretation of the data. With the total con-
centrations of UCB = 10 μM and PC = 100 μM (molar ratio
of UCB/PC, 0.1) used in the pH-variation study, the
unbound UCB concentrations (Bf) at equilibrium were
modestly above the aqueous solubility of UCB at all pH
values ≤ 9.0 [4]. If supersaturation were absent, the concen-
tration of H2B would be at its saturation value over the
entire pH range, and ñ should maintain its value of 0.049 at
pH 6.54 (the horizontal dashed line B, in Figure 4). The
experimental data indicate, therefore, that stable supersatu-
ration was indeed likely present in these short term experi-
If, by contrast, the low pKa values of 4.2 and 4.9 [3,17-20],
are used to fit the ñ value at pH 6.54, a much higher value
of K", 94 μM-1, is needed to yield the calculated [H2B].
Using this constant and [H2B], determined from Bf and the
low pKa values, yields curve C in Figure 4. As pH increases
from 6.5 to 7, ñ decreases steeply, and is below 0.01 at a pH
of 6.9 or higher. The low pKa values are, thus, not compati-
ble with ñ vs. pH data determined for the binding of UCB
by neutral PC vesicles [12]. Since, in this study, K ≈ 1 [12],
the mid-point pH value of 8.1 (shaded star, Figure 4, curve
A) would be close to the mid-point value of the curve for K
= 1 in Figure 2, and thus compatible with our proposed high
pKa values in the absence of a UCB binder [4].
Comments are warranted on an often-cited study, by Erik-
sen, et al., of the binding of UCB to egg PC vesicles [16].
The concentrations were 12.8 μM UCB and 130 μM PC.
Figure two in that paper plots the changes with pH of the
quenching by bound UCB of the fluorescence of diphenyl-
hexatriene incorporated into the lipid bilayer, measured 3
hours after adding the UCB in an aqueous alkaline solution
(pH 11). The sigmoidal curve exhibits decreasing fluores-
cence (increasing quenching by bound UCB) as pH
increases, with a mid-point near pH 7.8. This evidence for
increased affinity of UCB binding to PC as pH increases
would indicate a primary role of binding of the dianion, B=.
Since the dianion fraction for low pKa values is already
0.992 at pH 7.0 (Table 1), any significant increase in bind-
ing at higher pH values is incompatible with low pH values
(see above). The increasing affinity of UCB for PC with
increasing pH is in stark contrast to Tipping's data [12] and
to our model based on uptake of H2B (above). However,
examination of data in Table one of the Eriksen paper,
reveals that results obtained "immediately" after adding the
UCB at pH 7.4 and 8.3 are just the reverse of those obtained
three hours later (as in their Figure two).
Eriksen's Figure one [16] indicates complex, pH-dependent
time effects. Using the percent decrease in fluorescence, P,
as a rough measure of the extent of binding of UCB, the
data show a high value of P = 79% at pH 7.43 "immedi-
ately" after addition of UCB. Within one minute, there is
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Mukerjee and Ostrow BMC Biochemistry 2010, 11:16
Page 7 of 12
rapid reversal of binding and P is decreased to 28%, fol-
lowed by a far less rapid decrease in P to 23% after 3 hours.
Results at pH 6.79 are similar. In contrast, at the highest pH
in their Figure one, 8.34, the "immediate" value of P = 57%
remains about the same for three hours. At the intermediate
pH of 7.90, the "immediate" value of P = 73% increases a
little over the first minute, remains about the same for four
more minutes, and then decreases gently over the next two
hours or so to a plateau of P = 22%. These complex time
effects yield very different sequences of P values decreas-
ing with pH: 7.43>7.90>8.34 for "immediate" measure-
ments; the irregular sequence 7.90>8.34>7.43 between 1
and 20 minutes; 8.43>7.90>7.43 at 60 minutes; and
8.34>7.90 ≈ 7.43 at 3 hours. Some discrepancies among
data in Eriksen's Table one, Figure one and Figure two [16],
suggest irreproducible time effects.
These observations can be rationalized roughly on the basis
of the progressive formation of aggregates of UCB, after
the initial rapid binding of UCB. Cestaro et al. [22] have
made a similar proposal, and Eriksen et al. [16] themselves
warn that, as the pH falls, supersaturation of the aqueous
medium with UCB must be avoided to prevent progressive
formation of H2B aggregates. Their gradient ultracentrifu-
gation data of 3 hour incubations (Figures three-d&e of
Eriksen et al.) [16] showed large aggregates of UCB, with
or without phospholipids, at pH 7.0 and smaller aggregates
at pH 8.2. The dependence on pH, of the rates of formation
of colloids and precipitates from supersaturated solutions of
UCB itself, is clearly relevant. As discussed earlier, after
extensive sedimentation at 100,000 xg, the measured super-
natant concentrations of UCB at pH values of 7.40, 7.83,
8.05 and 8.2 were 0.1 μM, 0.5 μM, 17 μM and 34 μM
respectively [7]. These are much higher than the calculated
solubilities of UCB at the same pH values [4], which were
low (0.062 μM, 0.084 μM, 0.112 μM and 0.148 μM) and
increased only a little as pH increased. This indicates that
the egg PC-UCB systems of Eriksen et al. [16] were highly
supersaturated when first prepared, and that aggregates of
UCB formed to relieve the supersaturation. This occurred
to a much greater extent and much more rapidly at pH 7.43
than at pH 8.34, with intermediate behavior at pH 7.90. Any
micro-precipitation of UCB, which may contain small pro-
portions of PC, will reduce Bf and its interactions with PC
vesicles. Vazquez et al. [28] have also reported significant
reversal of UCB interaction with phospholipid membranes
with time.
In view of the above considerations, and evidence that the
uptake of UCB by PC vesicles is almost instantaneous (t1/2
= 2 msec) [29], we conclude that the "immediate" effects on
P after addition of UCB to PC are least susceptible to time-
dependent effects arising from the thermodynamic drive to
decrease supersaturation. Indeed, even the "immediate" val-
ues of P may still be subject to some aggregation effects,
particularly at pH 7.43. The qualitative trend in the "imme-
diate" decrease of P with increasing pH is consistent with
the results of Tipping et al. [12] and with our model, based
on the binding of H2B and high pKa values for UCB. The
conclusions are opposite to those given by Eriksen et al.
[16], which were based on assumptions of binding of the
dianion, B=, and low pKa values.
Other systems studied by Eriksen et al. [16] (their Table one
and Figure one-b) also show exceptionally large and rapid
decreases in binding of UCB with time at pH 7.4, interme-
diate behavior at pH 7.90, and minor effects at pH 8.34.
These systems all contained 12.8 μM UCB, mixed with var-
ious types of phospholipids ± cholesterol, at varied concen-
trations: egg PC, 150 μM + cholesterol 15 μM;
phosphatidlyethanolamine, 106 μM; phosphatidlyetha-
nolamine, 102 μM + cholesterol 10 μM; sphingomyelin, 32
μM; and phosphatidylserine, 55 μM. The qualitative inter-
pretations above apply to all of them. The data points are
limited, and some of these systems involve charge effects,
which introduce further uncertainties. In all systems, after 3
hours, UCB binding is greater at pH 8.3 than at pH 7.4. The
"immediate" results, however, show a stronger reverse
effect of pH, P = 82% at pH 7.4 vs. 25% at pH 8.3, for the
egg PC-cholesterol system as compared to the egg PC sys-
tem. They also show lower binding to sphingomyelin at the
higher pH, indicating predominant binding of H2B. This
common pattern leads us to conclude that the complex pH-
dependence of precipitation of UCB from supersaturated
solutions can lead to highly anomalous pH-dependence of
UCB interactions with phospholipids.
The study of UCB binding to gangliosides by Vazquez et al.
[30], reported little change in UCB binding going to pH 7.4
to 8.0, but a small increase in going from pH 7.0 to 7.4, sug-
gesting an important role for UCB anions. Assuming low
pKa values, the unbound dianion fraction changes only
from 0.992 at pH 7.0 to 0.999 at pH 8.0, whereas the mono-
anion fraction decreases by a factor of 10 (Table 1). There-
fore, these observations are incompatible with low pKa
values, whereas with high pKa values, both the mono- and
dianon fractions increase significantly over this pH range
(Figure 1). Other problems with this study (Additional file
1, Table S1) are that: the systems were highly supersatu-
rated with UCB, the UCB was neither purified nor pro-
tected against degradation, equilibrium was not attained
rapidly, and the pH range studied was insufficiently broad
to derive pKa values.
Zucker and Gössling used stop-flow kinetics to examine the
rates of uptake of BSA-bound UCB by cultured HepG2
cells [26]. They found an inverse correlation between pH
and the rate of UCB flip-flop, which they showed to be due
to pH effects on the rates of dissociation of UCB from albu-
min and from the membrane bilayer. They concluded that
"the identification of an inflection point at pH 8.1 is indica-
tive of a pKa value for bilirubin in this range". Although
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Mukerjee and Ostrow BMC Biochemistry 2010, 11:16
Page 8 of 12
this seemingly supports our contention that the pKa's of
UCB are above 8.0 [4], these are kinetic and not equilib-
rium data, involve BSA (which is pH-sensitive in this
range), and measure diffusion across a complex hepatocyte
membrane. Studies of binding of UCB to whole cells [31],
or natural membranes (e.g. red cell ghosts, mitochondria,
synaptosomes) [28,32-35], likewise cannot be used to eval-
uate unequivocally the role of pKa values for UCB. These
heterogeneous structures contain a mix of phospholipids, as
well as proteins and other components, which may ionize
and change conformation in the pH range studied and have
unknown effects on membrane structure.
Interactions of UCB with Dodecyl Maltoside Micelles
(Additional file 2, Table S2)
Non-ionic long-chain alkyl-saccharides, such as octylgluco-
side (C8G) or dodecylmaltoside (C12M), are readily purified
and form micellar systems that are suitable for studies of
the influence of pH on the uptake of UCB. Binding proba-
bly involves interactions of the -OH groups of the saccha-
ride with the ionized -COO- groups of UCB, and induces
marked changes in the ellipticity of the bound UCB [13].
Kano et al. [13] showed that solubilization of UCB (20 μM)
in 1 mM C12M micelles (their Figure five) generated no cir-
cular dichroism effects from pH 4.5 to 6.2. (Note that their
legend for Figure five misidentifies the saccharide as C8M,
octylmaltoside; the text indicates it is C12M that was stud-
ied.) Kano's Figure three shows that UCB does not interact
with C8G below the c.m.c. of 22 mM. The same would be
expected of C8M, whose c.m.c. value should be similarly
high, but the concentration of the alkyl-saccharide used in
their Figure five is 1 mM, well below the presumed c.m.c.
of C8M.) Above pH 6.2, the observed ellipiticity increased
steeply with rising pH up to about pH 8.5, and leveled off to
a constant value from pH 8.8 to 10. Since the conformation
of the non-ionic C12M is unaffected by pH, their titration
curve reflects increased binding of UCB dianion (B=) as the
proportion of unbound dianion, fB=, increases with increas-
ing pH.
The rise in ellipticity with pH has a mid-point of about pH
7.5. Reference to our Figure 3 shows that this is compatible
with high pKa values of 8.12 and 8.44 and a UCB distribu-
tion ratio K (bound/free) between 10 and 100. If the low
pKa values of 4.2 and 4.9 are invoked, the midpoint pH
value would have to be lower than about 5, as discussed
above. Moreover, at pH 6.5, the fraction of UCB dianion,
fB=, would already be 97.5%, yet the observed ellipticity is
near zero at that pH. In addition, the steep rise in ellipticity
observed above pH 6.5 would not be expected. Thus, only
the higher pKa values are compatible with the observed
Interactions of UCB with Cyclodextrins (Additional file 2,
Table S2)
Kano et al. [14] and Lightner et al. [36] studied CD of UCB
in solutions of β-cyclodextrin (β-CDx) as a function of pH.
Both studies used purified UCB and were conducted within
minutes of mixing UCB with the cyclodextrins; Kano's
studies also took precautions to minimize oxidation and
photo-oxidation of the pigment. In each study, the molar
circular dichroism absorption coefficient (Δε) was minimal
at pH 6.0 and increased steeply as pH increased above pH
7.0, indicating that UCB dianions are the primary interact-
ing species. Lightner's systems [36] were even more super-
saturated than Kano's and measurements were provided
only at integral pH values from 6 to 10, rendering Kano's
data [14] preferable for analysis.
In the study by Kano et al. of UCB binding to β-CDx [14],
no ellipticity was observed at pH 5.5, but Δε increased sig-
moidally as pH increased from 5.5 to 9.5, with a steep slope
between pH 7.0 and 8.5, and no further change between pH
9.5 and 10.8 (their Figure 3). Chiral recognition effects,
registered at several pH values with the protonated 1-
amino-β-cyclodextrin as the host molecule, (their Figure
three [14]), support the conclusion that predominantly UCB
dianion was bound.
At pH 10.8, an affinity constant of 23 L/mol was reported
for UCB binding to β-CDx, indicating that 4.67 μM, or
18.7% of the total [UCB] (25 μM), was bound to 10 mM β-
CDx at that pH. Assuming that the UCB binding is con-
fined to B=, and that binding of UCB is proportional to the
concentration of unbound, monomeric B=, the amount of
UCB bound at lower pH values can be calculated using Δε
values relative to Δε at pH 10.8. It is then possible to calcu-
late Bf and, akin to modeling the data on binding of UCB to
PC as described above, to apply Equation four of Hahm et
al. [4] to calculate the unbound, monomeric dianion con-
centration, [B=], at each pH, assuming different pKa values.
Assuming the high pKa values of 8.12 and 8.44 and the
dianion dimerization constant of 0.26 μM-1 [4] in calculat-
ing [B=]f from Bf, the four data points at pH values of 10.8,
9.5, 8.65 and 8.2 showed an excellent proportionality
between Δε and [B=] using the equation Δε = Q × [B=]f,
where the constant, Q = 1.099 ± 0.018 (n = 4, r = 0.974,
S.D. = 0.17). Curve A in Figure 5 shows the results of fit-
ting all the data to the above equation, Δε = 1.099 × [B=].
Curve A gives a reasonable fit for the experimental Δε at
the five highest pH values (7.83 and above) and, of course,
the very low pH of 5.5, where Δε = 0. Bf was below calcu-
lated solubilities at both pH 10.8 and 9.5, but there was
likely stable supersaturation of the unbound UCB phase at
the three lower pH values, based on previous observations
[7], as discussed earlier. The rapid decrease in Δε below pH
8.65 is primarily due to the rapid decrease in the fraction of
unbound monomeric UCB that exists as the dianion (fB=).
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Mukerjee and Ostrow BMC Biochemistry 2010, 11:16
Page 9 of 12
The systematic discrepancies between the data and Curve A
in the pH range of 7.3 to 6.9 (Figure 5) can be ascribed to
many factors that are difficult to evaluate, including self-
aggregation of the more highly supersaturated phase of
unbound UCB (e.g. R = 369 at pH 7.0).
In comparison to the reasonable representation of the Δε
data using pKa values above 8.0, the results obtained are
very different (Figure 5, Curve B) if the lower pKa values
of 4.2 and 4.9 [3,17-20], are used to calculate [B=] from Bf.
The proportionality constant K, relating Δε to [B=] was
determined by fitting the data at the pH values of 9.5 and
10.8, where the solutions are undersaturated with unbound
UCB. K was then used to calculate Δε from the estimated
[B=] at other pH values. Since the dianion fraction (fB=) of
unbound UCB would be close to unity at pH 7.0 or above,
and have the value of 0.79 even at pH 5.5, little variation is
expected in [B=] and, therefore, in Δε over the alkaline pH
range. This is shown by Curve B in Figure 5, which does
not fit Kano's Δε vs. pH data [14]. This indicates that the
low pKa values are highly incompatible with the experi-
mental observations. The same conclusions apply to the
data of Lightner et al. [36], which show similar pH depen-
dence of binding at alkaline pH.
From the mean fit of the Δε data at pH 9.5 and 10.8 to the
concentrations of [B=] calculated using pKa values of 8.12
and 8.44 and the dianion dimerization constant of 0.26 μM-
1, it is possible to calculate Δε from [B=] at lower pH val-
ues, where total solution concentrations are above solubil-
ity, by assuming that all solutions are at true saturation, with
the excess [B=] forming a suspension. For such systems,
[B=] = 0.051 × 10(2pH-16.56). Here, 0.051 μM is the solubility
of H2B and 16.56 = pKa1 + pKa2 [4,6]. The calculated curve
C (Figure 5) shows a steep drop in Δε below pH 9.3. Curve
C has little resemblance to the experimental data, suggest-
ing that, at most pH values, a stable supersaturation was
probably important in these relatively short-term experi-
Interactions of UCB with Bile Salts (Additional file 3, Table
Taurine-amidates of bile salts, which have pKa values near
1.0, are the only bile salts which are fully ionized through-
out the pH range from 4 to 10 [37], and are thus most suit-
able to assess the roles of pH and pKa values on UCB
binding. Rapid solvent partition of purified UCB from
undersaturated chloroform solutions into buffered aqueous
solutions of 50 mM taurocholate (TC) (at least 80% in
micelles), at ionic strength = 0.15, was performed over the
pH range of 6.0 to 9.5 [1,21]. The data were fitted to the
equation log P = log Po + [1 + 10(pH-A) + 10(2pH-B)], where
Po is the partition ratio for H2B, and the values for A and B
were 7.36 ± 0.5 and 14.08 ± 0.05 respectively.
Solubilities of UCB in 50 mM TC [21] and in water [4]
were calculated from the fitted P data and an estimated sol-
ubility of UCB in chloroform of 1.14 mM [9]. (As argued
before [4,6], a better estimate of the solubility of UCB in
chloroform is 0.88 mM, which will decrease the estimated
UCB solubilities in 50 mM TC and water by 23%, but not
affect the shape of the titration curves.) Using the high
aqueous pKa values of 8.12 and 8.44, the total and unbound
concentrations of each UCB species in 50 mM TC was cal-
culated from the solubilities in 50 mM TC/chloroform [21]
and water/chloroform [21] respectively; their difference
equaled the concentration of the bound species. From these,
the distribution ratios (bound/free) of the three UCB species
were calculated to be 1.41 for H2B, 12.9 for HB-, and 730
for B=, indicating predominant binding of B= [21], as con-
cluded previously by Kano et al. [38]. From the relative
contributions of bound B=, HB- and H2B, the apparent pKa
values of UCB associated with the TC were estimated: 7.16
± 0.5 for pKa1, 6.69 ± 0.5 for pKa2 and 13.85 ± 0.05 for the
sum of pKa1 and pKa2, which is derived with somewhat
greater precision.
The relationship of these pKa values, lower than the aque-
ous values of 8.12 and 8.44, to micelle-water distribution
ratios for H2B, HB- and B=, has been discussed in our com-
panion paper [2]. pKa values in the bile salt systems in the
range of 6 to 7 have also been reported from studies using
micellar electrokinetic capillary chromatography [39,40].
Since these authors did not determine micelle-water distri-
butions, these were average values for the whole system,
but appear to be compatible with Hahm's pKa values in 50
mM TC derived by solvent partition [21]. Utilizing essen-
tially the same equations [1,40] relating changes in pKa
values resulting from uptake in micelles and micelle-water
distribution ratios for H2B, HB- and B= mentioned above
[21], the pKa values of 6 to 7 in 50 mM TC are thus com-
patible with the high pKa values in water [4].
CD studies have been performed with unpurified UCB in
50 mM solutions of various bile salts [15,41,42]. In many
cases, the UCB concentrations used appear to be above sat-
uration. For 50 mM sodium taurocholate (TC), calculated
Bf was above aqueous phase saturation at pH below 7.5
when total UCB was 4.6 μM and at pH below 8.3 when
total UCB was 34 μM. Bile salts, however, have been
shown to promote extensive, stable supersaturation of UCB
[8,9]. In systems of UCB in 50 mM TC [15], the observed
ellipticities were reasonably proportional to the two UCB
concentrations, even though the calculated solubility in 50
mM TC was exceeded at several pH values. Similar propor-
tionalities are seen over higher ranges of concentration of
sodium glycocholate [15] and sodium deoxycholate [41] at
pH values of 7.0 and above.
Since background information is available for 50 mM TC
[21] and TC is expected to show no dependence upon pH,
Page 10
Mukerjee and Ostrow BMC Biochemistry 2010, 11:16
Page 10 of 12
we have attempted a semi-quantitative interpretation of the
pH effects for the ellipticity study of 4.6 μM and 34 μM
UCB in 50 mM TC [15]. We assumed stable supersatura-
tion in these short-term experiments (30 min or less) at pH
values of 7.23 and higher. Table 2 presents ellipiticity val-
ues at 458 nm, estimated from their published graphs. The
three measurements at 4.6 μM UCB have been multiplied
by the factor, 34/4.6 to render them comparable to measure-
ments at 34 μM UCB. The ellipticities are large and nega-
tive at the two lowest pH values of 7.23 and 7.35, become
positive at pH about 8.1, and then show higher positive val-
ues as pH increases to 9.1 and above. This general trend
was exhibited by all the bile salts studied [15,41,42].
As noted earlier, equations relating partition coefficients to
pH for 50 mM TC [21] and water [4] allow the calculation
of the fractions of total UCB solubilized by the TC as H2B,
HB- and B=. The pH-dependence of the ellipticity values at
458 nm suggest strongly that the interactions with TC
aggregates of the solubilized HB- cause negative elliptici-
ties whereas interactions of the solubilized B= cause posi-
tive ellipticities. Table 2 shows the estimated fractions of
total UCB (34 μM) that are bound to TC as HB- and B=. As
expected, as pH increases, the fraction of total UCB that is
bound to TC as HB- (fsHB-) decreases and the fraction bound
as B= (fsB=) increases. In order to test our model, we use the
equation below in which the constants A* and B* are mea-
sures of the effectiveness of (fsHB-) and (fsB=) in determining
the value of 103θ:
The experimental ellipticity values in Table 2 are well
described by this equation: n = 7, r = 0.992, SD = 1.1, A* =
-98.7 ± 5.6, B* = 5.24 ± 0.54. Figure 6 shows that, at total
UCB concentrations of 4.6 and 34 μM, the experimental
ellipticities are in reasonable concordance with calculated
ellipticities. Note that the two most positive ellipticity val-
ues for 4.6 μM UCB, at pH 8.13 and 9.12, and for 34 μM
UCB at 9.35 and 11.60 were measured in unsaturated solu-
tions. The significant changes in ellipticity at these high pH
values are clearly due to significant changes in (fsHB-) and
(fsB=) over this alkaline pH range. Remarkably, the mea-
sured ellipticity increased significantly, by about 2%,
between pH 9.35 and 11.60, which is matched by a calcu-
lated increase in ellipticity of about 4%. Since, if low pKa
values are assumed (Table 1), fB= is already close to unity at
pH 8.5 (0.9997), and the changes in ellipticity at pH values
above 9.0 are qualitatively incompatible with the low pKa
values for UCB proposed by others [3,17-20]. The model
proposed here for representing the circular dichroism val-
ues of UCB in 50 mM TC appears to be new and is likely to
be useful also for other bile salt systems.
The analyses and interpretations presented above show that
the high pKa values of 8.12 and 8.44 [1,4-6] are far superior
to low pKa values of 4.2 and 4.9 [3,17-20] in rationalizing
experimental data for the effects of pH on interactions of
UCB with PC vesicles, cyclodextrins, and micelles of dode-
cyl maltoside and bile salts. The approaches outlined also
demonstrate and take into account the possibly important
roles of self-association of B= and pH-dependent supersatu-
ration effects. They support further the conclusions of our
companion paper [2] that analyzes the effects of pH on var-
ious properties of UCB alone in aqueous and organic sol-
vent systems. That paper demonstrated that only our
solvent partition studies [4,5] met all the requirements for
valid experiments when using a poorly-soluble, unstable
compound, such as UCB. Together, the two present papers
clearly indicate that the pKa values of UCB are well above
the pKa values of simple carboxylic acids (usually about
4.5). Theoretically these remarkably high values may result
from the combined interactions of three factors that result
from the unique, complex internal hydrogen-bonding of
UCB, as presented elsewhere [5].
We do not claim that the high pKa values of 8.12 and 8.44,
derived from our solvent partition study of UCB [4], are
exact. The accuracy of these model-derived high pKa val-
ues of UCB is given on page 1131 of that paper. These val-
ues (mean ± S.D.) are: pK'1 = 8.12 ± 0.23; pK'1 + pK'2 =
16.56 ± 0.10; pK'2 = 8.44 ± 0.33. That paper also notes that
"the analysis yields a less accurate estimate of the individ-
ual pKa values than of their sum, pK1 + pK2". The errors in
these pKa values are insignificant when compared with the
difference of 7.5 pH units between the sum (pK1 + pK2) of
these high pKa values, and the sum (9.1) of the low pKa
values of 4.2 and 4.9 proposed by others [3,17-20], with
which our high pKa values were compared.
It should thus now be clear that such high pKa values not
only acceptably explain the experimental findings on the
effects of pH on the properties and interactions of UCB, but
that the often quoted low pKa values [3,17-20], are incom-
patible with the experimental data. The implications are not
trivial, since they invalidate older, but still widely held,
interpretations and modeling of the binding and cytotoxic-
ity of UCB [43-47] that were based on the assumptions that
pKa values were below 5.0 and that B= was, therefore the
dominant species of unbound UCB in the physiological pH
range (Table 1). More recent reviews are available [1,48,49]
that are based on the realistic pKa values above 8.0, and the
consequent predominance of H2B among the unbound UCB
species at physiological pH values (Figure 1).
List of Abbreviations Used
UCB: unconjugated bilirubin; H2B: UCB diacid; HB-: UCB
monoanions; B=: UCB dianion; fH2B: fHB-, and fB=, rela-
103q =+
Page 11
Mukerjee and Ostrow BMC Biochemistry 2010, 11:16
Page 11 of 12
tive fractions of the unbound monomeric UCB species; F:
[UCB]bound/[UCB]total; Bf: unbound (free) UCB concentra-
tion; R: the UCB saturation ratio = Bf/estimated solubility
of UCB at a given pH; K: distribution ratio = mols bound/
mols unbound (free); PC: phosphatidylcholine; c.m.c., criti-
cal micellar concentration; C12M: dodecylmaltoside; C8M:
octylmaltoside; C8G: octylglucoside; β-CDx: β-cyclodex-
Additional material
Authors' contributions
Both authors were equally involved in the conceptualization and writing of this
paper, and both have read and approved the final manuscript. JDO performed
the literature search and PM developed the mathematical models.
Author Details
1School of Pharmacy, University of Wisconsin, 777 Highland Ave., Madison, WI,
53705-2222 USA and 2GI/Hepatology Division, Dept. Medicine, Box 356424,
Univ. Washington School of Medicine, 1959 NE Pacific St., Seattle, WA 98195-
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Additional file 1 Studies of interactions of UCB with phospholipids.
Details of the three publications that were considered, including the
degrees of supersaturation with UCB, the analytical methods used, the
charateristics of the binding curve, the experimental problems, and the
Additional file 2 Studies of interactions of UCB with alkyl saccharides
and cyclodextrins. Details of the three publications that were considered,
including the degrees of supersaturation with UCB, the analytical methods
used, the charateristics of the binding curve, the experimental problems,
and the citation.
Additional file 3 Studies of interactions of UCB with bile salts. Details
of the seven publications that were considered, including the degrees of
supersaturation with UCB, the analytical methods used, the charateristics of
the binding curve, the experimental problems, and the citation.
Received: 26 March 2009 Accepted: 29 March 2010
Published: 29 March 2010
This article is available from: http://www.biomedcentral.com/1471-2091/11/16 © 2010 Mukerjee and Ostrow; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
BMC Biochemistry 2010, 11:16
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doi: 10.1186/1471-2091-11-16
Cite this article as: Mukerjee and Ostrow, Interactions of unconjugated bili-
rubin with vesicles, cyclodextrins and micelles: New modeling and the role of
high pKa values BMC Biochemistry 2010, 11:16


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