A spectrophotometer is an instrument that measures the transmittance or absorbance of a sample as a function of the wavelength of electromagnetic radiation [1]. Hence, spectrophotometric methods are widely used to measure the concentration of a solute in a solution, and in this case, protein by determining its absorbance at 595nm [2]. A spectrophotometer consists of many parts. In short, the main components of the spectrophotometer are:
A source that emits a broad band of electromagnetic radiation.
A dispersion device that selects a wavelength (or, more correctly, a waveband) from the broadband radiation of the source.
A sample area.
One or more detectors to measure the intensity of radiation.[1]
The uses of spectrophotometer include measuring of concentration of substances (by determining its absorbance at a peak wavelength), detection of impurities, monitoring dissolved oxygen content in freshwater and marine ecosystems as well as respiratory gas analysis in hospitals [2]. As the demand for spectrophotometer increases, simple and inexpensive alternatives had been developed, such as the Arduino-based spectrophotometer with white LED as a light source in place of the standard UV-visible spectrophotometer [3].
Estimation of Protein Concentration
Before the unknown protein concentration in various solutions can be determined, a standard curve of absorbance at different known protein concentrations must be plotted. In this experiment, a range of protein concentrations are obtained by simple dilution from a 10µg/mL BSA stock solution; after which their respective absorbances are read from a spectrophotometer. This is followed by the plotting of the standard curve. From there, the concentration of protein in each sample solution can be obtained by substituting the solution’s absorbance value into the equation of straight line.
The colorimetric assay used in this experiment is the Bradford protein assay method. Developed by Dr Marion Bradford in 1976 [4], the basic concept in the procedures is the binding of Coomassie Blue G-250 to the basic amino acids in a protein [5], causing a change of colour of solution into blue [6] with its peak absorbance at 595nm [5].
Materials & Methods
Bradford Reagent, Bovine Serum Albumin (BSA), apple juice sample, test tubes, cuvette, micropipette, tips.
The experiment is carried out according to the procedures described in the practical manual.
Discussion
In this experiment, the Bradford protein assay method is used to measure protein concentration as it is inexpensive and hence it is easily available [4]. Another advantage of this method is that results, which is the absorbance of a certain solution can be determined rapidly and reproduced precisely [6]. The dye-binding process takes approximately two minutes to complete [6], and this is advantageous as the absorbance of the solution mixture of BSA and Bradford reagent must be measured within sixty minutes. Also, the visible light spectrophotometer only takes a few seconds to give its readings.
However, measuring protein concentration using the Bradford method also has its disadvantages. The main limitation of the Bradford assay is its incompatibility with most detergents, routinely used to solubilize membrane proteins [4]. Although detergents can be removed by gel filtration or dialysis, or through precipitation with calcium phosphate or acetone, these methods can lead to sample dilution or sample loss, with only 70% or less of the initial protein amount which is recovered [4].
By comparison, my calculated results do not agree with the nutritional facts shown in Figure 1.2 in the practical manual. Among the reasons for this discrepancy is that the cuvette (to be put into the spectrophotometer to measure absorbance) is reused many times and cleaning of apparatus may not be thorough because only small amount of distilled water is used. Hence, some protein residues from solutions tested previously may still be present in the cuvette.
All in all, the Bradford protein assay method is not recommended for quantitative analysis of protein content in food as this method is very susceptible to interference from substances which results in overestimation of protein content [7]. Instead, the amino acid analysis method is preferred [7].
References
Mohammad, Karim & Zekry, Abdelhalim & Abouelatta-Ebrahim, M.. (2015). LED Based Spectrophotometer can compete with conventional one. International Journal of Engineering & Technology. 4(2). 10.14419/ijet.v4i2.4504.
Aryal S. Spectrophotometer- Principle, Instrumentation, Applications | Instrumentation | Microbe Notes [Internet]. Microbe Notes. 2019 [cited 13 October 2019]. Available from: https://microbenotes.com/spectrophotometer-principle-instrumentation-applications/
Dani Nandiyanto A, Zaen R, Oktiani R, Abdullah A, Riza L. A Simple, Rapid Analysis, Portable, Low-cost, and Arduino-based Spectrophotometer with White LED as a Light Source for Analysing Solution Concentration. TELKOMNIKA (Telecommunication Computing Electronics and Control). 2018;16(2):580.
Johnson, M. (2019). Protein Quantitation. [online] Labome.com. Available at: https://www.labome.com/method/Protein-Quantitation.html [Accessed 13 Oct. 2019].
Knight M, Chambers P. Problems Associated with Determining Protein Concentration: A Comparison of Techniques for Protein Estimations. Molecular Biotechnology. 2003;23(1):19-28.
Valipour Nouroozi R, Valipour Noroozi M, Ahmadizadeh M. Determination of Protein Concentration Using Bradford Microplate Protein Quantification Assay. International Electronic Journal of Medicine. 2015;.
Mæhre H, Dalheim L, Edvinsen G, Elvevoll E, Jensen I. Protein Determination—Method Matters [Internet]. MDPI. 2019 [cited 15 October 2019]. Available from: https://www.mdpi.com/2304-8158/7/1/5/htm