Estimation of Protein Concentration by Spectrophotometry - Lab Report Example

Estimation of Protein Concentration by Spectrophotometry Abstract Spectrophotometry is a standard technique for measuring the concentration in solution of a substance which absorbs light, since (according to the Beer–Lambert Law) the degree of absorption is proportional to the concentration of the solute. In the case of proteins, a solution is allowed to react with the Biuret reagent (a combination of a base such as potassium hydroxide and copper II sulphate); the copper forms a complex with the peptide bonds of the protein and the resulting solution has a strong purple colour, with maximum light absorption at a wavelength of 540 nm. This technique is known as the modified Lowry method, and is useful as a diagnostic tool for medical events involving muscle damage, such as myocardial infarction. In this experiment, three samples of a protein solution at known concentrations of 1 g/dl, 3 g/dl, and 5 g/dl were used to establish a standard reference curve for absorption at 540 nm; as expected, a linear relationship between protein concentration and absorption was shown. Following this, a sample of unknown protein concentration was tested at three levels of dilution to establish its concentration; because the highest concentration of this unknown solution absorbed at a level beyond the strongest reference solution tested, its concentration was computed from the more dilute samples. The concentrations obtained for the three unknowns were 7.05 g/dl (based on the 3:5 dilution), 4.22 g/dl, and 1.56 g/dl. Introduction An assessment of the concentration of protein in solution is useful in medical diagnosis and research, particularly for assessment of muscle damage following injury or breakdown (rhabdomyolysis), myocardial infarction, or chronic illnesses such muscular dystrophy that involve damage to muscle tissue (National Human Genome Research Institute 2010). Damaged skeletal muscles release the enzyme creatine kinase and the structural microfilament protein α-actin (Amat et al. 2005). Damaged heart-muscle cells release creatine kinase as well as the protein troponin (Zimmermann et al. 1993). The concentration of such proteins in the bloodstream is an important biomarker for these and other medical problems. Blood protein concentration can also indicate how the body is responding to exercise, and thus can be used to help plan athletes’ training and diet programmes (Hargreaves & Spriet 2006). Proteins are made up of polypeptide chains: amino acids connected by peptide bonds, in which the carboxyl group of one amino acid joins with the amine group of the next amino acid, releasing a single water molecule (Alberts et al. 2004). Since the number of peptide bonds is one less than the number of amino acids in the protein, and the number of amino acids in a single protein molecule is normally quite large, the concentration of peptide bonds in a protein solution can serve as a convenient proxy for the concentration of the protein itself. Spectrophotometry is a reliable and convenient method of measuring protein concentrations in solution. Because the copper ions included in the Biuret reagent react with the nitrogen atoms of peptide bonds to form a complex with strong and specific light absorbance, the depth of colour of a particular sample treated with Biuret reagent accurately reflects the protein concentration of the sample (Wilson & Walker 2005; Zumdahl & Zumdahl 2009). Assuming all other conditions are identical, the absorbance of a sample is linearly proportional to its concentration; this is known as the Beer-Lambert Law, A = ɛc1 (where A = absorbance; ɛ = the molar extinction coefficient, determined by the compound involved and the wavelength of light used for the test; c = the concentration of the sample; and l = the length of the light path through the sample) (Bond University 2009). Since ɛ and l are constants (given that the same equipment is being used to measure absorbance of the same substance at the same wavelength), changes in A should correspond proportionally to changes in c. The goal of this experiment was to use protein solutions at three known concentrations treated with Biuret reagent to establish a standard 540-nm absorption curve, and then to test three protein solutions of unknown concentration using the same procedure in order to establish their concentration. Procedure The experiment was carried out as described in “Laboratory Class 3: Estimation of Protein Concentration”, Cell Biology Laboratory Manual BMED11-203, pages 29-33 (Bond University, 2009). Results Absorbance was measured for a sample of 5.5 ml of distilled water; a “reagent blank” consisting of 5 ml of Biuret Mix plus 0.5 ml of distilled water; three samples consisting of 5 ml of Biuret Mix plus 0.5 ml of reference 5 g/dl protein solution at 1:1, 3:5, and 1:5 dilution; and three samples of the unknown protein solution prepared in the same manner as the reference samples. With the reagent blank used to determine the zero point for absorbance measurements, the following data were obtained: Sample Mean Absorbance at 540 nm Concentration B (distilled water) -0.077 N/A RB (reagent blank) 0 0 S1 (5 g/dl reference solution) 1.121 5 g/dl (known) S2 (3 g/dl reference solution) 0.706 3 g/dl (known) S3 (1 g/dl reference solution) 0.243 1 g/dl (known) U1 (unknown, full strength) 1.401 (6.25 g/dl by procedurally invalid extrapolation based on absorbance; see discussion) U2 (unknown, 3:5 dilution) 0.964 4.22 g/dl U3 (unknown, 1:5 dilution) 0.358 1.56 g/dl Table 1: Measurements obtained and concentrations computed for unknown solutions This data was graphed as follows: Figure 1: Standard absorbance curve derived from measurement of 5 g/dl, 3 g/dl, and 1 g/dl protein solutions, along with measurement of distilled water and unknown samples. Unknown sample measurements and interpolation of unknown sample concentrations for samples 2 and 3 are shown in red. Figure 1 displays the data obtained in graphic form. Known-sample measurements are displayed as gray X’s, with the measurement for distilled water just below the X for the reagent blank. The gray line represents a best-fit line for the sample blank and the three known solutions; as expected, the relationship between concentration and absorbance for these measurements is linear. Concentrations for unknown samples U2 and U3 were obtained by interpolation based on the best-fit line; concentrations obtained were 4.22 g/dl and 1.56 g/dl, respectively. The absorbance of the undiluted unknown sample U1 was greater than that of the strongest known solution; as discussed below, extrapolation of the best-fit line to derive a concentration corresponding to such a measurement is not reliable. Discussion For the samples of known protein concentration, the relationship between concentration and 540-nm absorbance was linear to a close approximation, as predicted by the Beer-Lambert Law. The absorbance at full strength was slightly below what would be expected from the absorbances at 3:5 and 1:5 dilution; this could be the result of imprecision in the dilution process, or it could represent the beginning of the “saturation point” reached when all the copper in the Biuret reagent has reacted with the peptide bonds in the sample. The blank (distilled water) sample was shown to have lower absorbance than the reagent blank; this demonstrates that a “pure” blank is not suitable to establish a zero-point for spectrographic measurements. Had the reference curve been plotted using the blank sample as its starting point, the measurements obtained for the reference samples would have deviated further from the best-fit line. The absorbance of a treated sample is limited both by the number of peptide bonds and by the availability of copper ions to react with these bonds; accordingly, a given amount of Biuret reagent can be used to assess only a finite amount of protein. As a result, the best-fit line obtained for reference solutions can be used to interpolate concentrations for samples with absorbances within the range of the reference values obtained; but using this line to extrapolate concentrations of samples with absorbances beyond the range of reference observations is unreliable. It was observed in this experiment that extrapolating in this manner would have yielded a concentration of 6.25 g/dl for the full-strength unknown solution, while computation of the concentration of the full-strength unknown based on the measurements obtained from the more dilute unknown samples yielded a significantly higher concentration of between 7.05 and 7.8 g/dl. In order to measure the protein concentration of this sample directly, the experiment would need to be re-run with the inclusion of reference samples of higher concentration; because of the saturation factor, it would be advisable in this case to increase the concentration of the Biuret mix as well, and perform a full set of new measurements of all reference and unknown samples. According to the experimental protocol, the concentrations of samples U2 and U3 should have a ratio of exactly 3:1. However, the ratio based on measurements of these samples’ absorbances was in fact 2.7:1; this is most likely the result of one or more errors made in the dilution process. Because of this discrepancy, the concentration of the full-strength unknown solution can only be estimated within an approximately 10% range as described above, depending on whether U2 or U3 is used as the measurement standard for estimating the concentration of U1. Questions 1) The results from the reference samples do show a linear relationship between protein concentration and 540-nm light absorption, at least up to the 5 g/dl concentration of the highest-strength reference solution. The small variances from a linear best-fit curve could be due to minor measurement or dilution errors, or to the beginning of saturation with the 5 g/dl reference solution. 2) Because the “pure blank” of distilled water had an absorbance significantly below that of the reagent blank, it would not be suitable for constructing a reference curve for interpolating the concentrations of unknown protein solutions. The purpose of using a reagent blank is to ensure that all measured differences in absorbance are due to the presence of protein in the sample being tested, not to other differences in the sample. 3) With reference to the original unknown solution U1, solutions U2 and U3 were diluted at ratios of 3:5 and 1:5, respectively; the concentrations of U2 and U3 should thus have a ratio of 3:1. Since the protein concentration of U1 could not be accurately measured based on its absorbance (due to the saturation effect), it is impossible to judge the accuracy of the dilutions of U2 and U3 with reference to U1. However, the fact that the measured concentrations of U2 and U3 had a ratio of 2.7:1 indicates that there were inaccuracies somewhere in the dilution process. 4) After combining the protein solutions with the Biuret reagent, 30 minutes was allowed in order for the reaction between the copper ions in the reagent and the nitrogen in the protein’s peptide bonds to proceed to completion. This is required especially when the protein solution is relatively concentrated relative to the amount of copper available, since as the copper is depleted the reaction proceeds more slowly. 5) No. Because both the number of peptide bonds in the sample being tested and the number of copper ions in the regent added to the sample are both limiting factors in the formation of the copper-nitrogen complex that absorbs 540-nm light, the concentration of samples that absorb more than the highest reference value cannot be accurately inferred from their degree of absorbance. Above a certain point, we are, in effect, measuring the amount of copper in the reagent rather than the amount of protein in the sample. 6) Blood concentration of creatine kinase is one of the standard diagnostic indicators for muscular dystrophy. References Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Ralf, M., Roberts, K. & Walter, P. 2004, Essential Cell Biology, 2nd ed, Garland Science, Spain. Amat, A. M., Boulaiz, H., Prados, J., Marchal, J. A., Puche, P. P., Caba, O., Serrano, F. R. & Aranega, A. 2005, ‘Release of α-actin into serum after skeletal muscle damage’, British Journal of Sports Medicine’, vol.39, no.11, pp.830-834, [Online], Available: http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1725075&blobtype=pdf [2009, July 22]. Bond University 2009, Cell Biology Laboratory Manual, Bond University, Australia. Hargreaves, M. & Spriet, L. 2006, Exercise Metabolism, 2nd ed, Human Kinetics, USA. National Human Genome Research Institute (National Institutes of Health), 10 June 2010, “Learning About Duchenne Muscular Dystrophy”. Available online at http://www.genome.gov/19518854 . Wilson & Walker 2006, Practical and Techniques of Biochemistry and Molecular Biology, 6th ed, Cambridge University Press, Cambridge. Zimmermann, R., Baki, S., Dengler, T. J., Ring, G. H., Remppis, A., Lange, R., Hagl, S., Kubler, W. & Katus, H. A. 1993, ‘Troponin T release after heart transplantation’, British Heart Journal, vol. 69, no. 5, pp. 395-398, [Online], Available: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1025100 [2009, July 22]. Zumdahl, S. S. & Zumdahl, A. S. 2009. Chemistry, 7th ed, Houghton Mifflin Company, Boston New York. Read More