Estimation of Protein Concentration - Lab Report Example

Lab Report: Protein Concentration Estimation Abstract The ability to measure the concentration of protein in solution is important in several areas of medicine and sports physiology. One of the best ways of doing so is to expose a sample of protein in solution to the Biuret reagent, which combines with a protein’s peptide bonds to form a complex with a pronounced purple colour, and then use a spectrophotometer to measure the resulting solution’s absorption of light of 540-nm wavelength. The Beer–Lambert Law predicts that the measured absorbance of such a solution will be proportional to the concentration of protein in the solution. To investigate the process of spectrophotometric measurement of protein concentration, a series of samples of known and unknown protein concentration were prepared, combined with a Biuret reagent mixture, and measured for absorbance at 540 nm. Measurement of the reference series confirmed the Beer-Lambert prediction of a linear relationship between protein concentration and light absorbance. Measurement of the unknown protein solution confirmed the existence of a “saturation effect” where this linearity breaks down at excessive protein concentrations relative to the Biuret reagent available; and measurement of the more dilute unknown samples indicated that the dilution procedure that was used to create these samples had not been carried out at quite the level of accuracy desired. Introduction Certain proteins (including creatine kinase, troponin, and others) are released into the bloodstream as a result of various forms of muscle damage – which may be the result of physical injury (Amat et al. 2005), of heart attack (myocardial infarction) (Zimmermann et al. 1993), of intense exercise (Hargreaves & Spriet 2006), of illnesses like muscular dystrophy, of taking statin drugs (Hamilton-Craig 2003), or of other medical conditions. Accordingly, tests that conveniently and reliably show the protein concentration in blood are very useful to physicians as well as to top athletes and their trainers. Proteins consist of long chains of amino acids linked by peptide bonds, in which each amino acid’s carboxyl group bonds covalently to the amine group of the next amino acid in the chain (Alberts et al. 2004). Since all these peptide bonds have the same structure regardless of which amino acids are bound, the number of peptide bonds is indicative of the amount of protein present. In order to enable the use of spectrophotometry as a means of assessing protein concentrations in solution something must be done to make the protein absorb light. This can be done by the ‘modified Lowry method’: a Biuret reagent consisting of a strong base and a source of copper ions such as copper II sulphate is combined with the sample to be tested. Under basic conditions, the copper in the reagent attaches to the nitrogen atoms of the protein’s peptide bonds, forming a complex that gives the solute a strong purple colour. The intensity of this colour, measured by the degree of absorption at 540 nm by the spectrophotometer, provides an indication of the number of peptide bonds present, and thus of the concentration of protein in the solution being tested (Wilson & Walker 2005). The relationship between the concentration of a light-absorbing solute and the degree of light absorbance measured by a spectrophotometer at a particular wavelength is described by the Beer-Lambert Law: A = ɛc1 (Bond University 2009) ‘A’ represents the measured absorption of light; ‘ɛ’ is the molar extinction coefficient, and is a function of the nature of the substance being tested and the wavelength used for the test; ‘c’ is the concentration of the solute; and ‘l’ is the length of the path of light through the solution being tested. Since ‘ɛ’ and ‘l’ are constants when a given substance is being tested using a given spectrophotometer, the only ‘real’ variables to be measured are ‘A’ and ‘c’, and the relationship between them is normally linear; this means that doubling the concentration of the light-absorbing substance in a solution will also double the amount of light absorbed. In order to derive a solute’s concentration from the amount of light absorbed, a practical value for ‘ɛ’ must be established. This is accomplished by testing a series of reference solutions at known concentrations, in order to establish a standard absorption curve at the wavelength being tested. Once this has been done, solutions with unknown solute concentration can be tested in the same manner, and assigned a concentration by interpolation with reference to this standard absorption curve. In this experiment, three known concentrations of protein in solution were used to establish a standard absorption curve at 540 nm, and three concentrations of an unknown-strength protein solution were tested in order to determine their concentrations. Two ‘blank’ samples were also used: one was simply distilled water, and the other included the Biuret reagent in order to provide a ‘reagent blank’ which was used to set the zero point for the other spectrophotometric measurements. Procedure Eight test tubes were labelled and prepared as follows: B (blank): 5.5 ml distilled water RB (reagent blank): 5 ml Biuret Mix plus 0.5 ml distilled water S1 (standard 1): 5 ml Biuret Mix plus 0.5 ml 5 g/dl protein reference solution S2 (standard 2): 5 ml Biuret Mix plus 0.3 ml 5 g/dl protein reference solution plus 0.2 ml distilled water S3 (standard 3): 5 ml Biuret Mix plus 0.1 ml 5 g/dl protein reference solution plus 0.4 ml distilled water U1 (unknown 1): 5 ml Biuret Mix plus 0.5 ml unknown protein solution U2 ( unknown 2): 5 ml Biuret Mix plus 0.3 ml unknown protein solution plus 0.2 ml distilled water U3 ( unknown 3): 5 ml Biuret Mix plus 0.1 ml unknown protein solution plus 0.4 ml distilled water All test tubes were allowed to sit at room temperature for 30 minutes after mixing, in order to allow the reaction between protein and the Biuret reagent to complete. After this, the RB sample was used to set the spectrophotometer’s zero point at 540 nm, and absorption was measured for all samples. Absorbance measurements were made in triplicate. Results Using the procedure described above, the following mean absorbances were measured: B (blank): -0.077 RB (reagent blank): 0 S1 (standard 1): 1.121 S2 (standard 2): 0.706 S3 (standard 3): 0.243 U1 (unknown 1): 1.401 U2 ( unknown 2): 0.964 U3 ( unknown 3): 0.358 Because concentrations for S1, S2, and S3 were known to be 5 g/dl, 3 g/dl, and 1 g/dl respectively, the absorbances for these three samples along with the reference blank were used to construct a best-fit linear standard absorption curve. This curve was then used to compute concentrations for U2 and U3 based on their measured absorbance. The concentrations computed were 4.22 g/dl for U2 and 1.56 g/dl for U3. As discussed below, extrapolation of the reference curve beyond the values provided by reference samples is unreliable; such being the case, the absorbance of 1.401 measured for U1 cannot be trusted to provide an accurate measure of that sample’s concentration. Instead, the concentration of U1 was computed based upon the concentrations of U2 and U3 and the degree to which these samples had been diluted relative to U1. A graph was constructed to display the results of this experiment: Figure 1: Standard absorption curve for protein solutions of 0 g/dl, 1 g/dl, 3 g/dl, and 5 g/dl, as well as distilled water and three unknown samples. Reference measurements, as well as both “blank” measurements, are shown as grey X’s; and the standard absorption curve is shown as a grey line. Discussion As predicted by the Beer-Lambert Law, the standard absorption curve for the three reference samples and the reagent blank is linear; the minor variations from perfect linearity are probably due to less-than-perfect precision of the dilution process. The distilled water sample absorbed less light than the reference blank sample, indicating that the reagent mixture absorbed some 540-nm light even without protein present. What is being measured by the spectrophotometer in the modified Lowry procedure is not the protein itself, but rather the number of peptide bond-copper complexes that have been formed by the reaction between protein and the Biuret reagent. Normally, the limiting factor for the formation of these complexes is the amount of protein present, so that the number of bound complexes is proportional to the amount of protein. However, if the amount of protein is such that there are more peptide bonds present than copper ions to react with them, the limiting factor for the reaction is the availability of the copper rather than the number of peptide bonds; in this case the reaction cannot proceed to completion and test will not yield an accurate reading of protein concentration; in effect, what is being measured is the amount of copper rather than the amount of protein. One of the reasons for using several strengths of reference solution to establish a reference curve is to ensure that the amount of reagent being used is adequate to provide a linear measure of protein concentration within a given range; if this linearity is not seen at higher concentrations of the reference sample, the experiment must be re-run with greater availability of Biuret reagent. In this experiment, it was seen that while the series of reference samples showed good linearity, sample U1 appeared to be sufficiently concentrated to exceed the resolving power of the reagent supplied. Its absorbance of 1.401 would have indicated a concentration of around 6.25 g/dl based on the extrapolated reference curve, but based upon the more reliable measurements of U2 and U3 and the known dilution protocol that produced these samples, the true concentration of U1 was between 7.03 and 7.80 g/dl. The difference of 0.75 to 1.55 g/dl between the observed absorbance of U1 and its computed concentration represents exhaustion of the Biuret reagent added to that sample. Like the reference samples S2 and S3, unknown samples U2 and U3 should have had a concentration ratio, based on the dilution protocol, of exactly 3:1. However, the spectrophotometric results gave a concentration of U3 that was significantly greater than one third of the concentration of U2. Lacking a reliable third measurement for the absorbance of the unknown protein solution, it is impossible to know which of these two samples provided an anomalous reading; but the most likely cause of the discrepancy between expected and actual results for U2 and U3 is an inaccuracy in the dilution of one or both of these samples. Further, as mentioned above, the uncertainty created by this discrepancy meant that the concentration of U1 could be estimated only with a 10% range, since at least one of the two samples being used to compute U1’s concentration had not been diluted to exactly the correct strength. Questions 1. Within the range of values tested (0 to 5 g/dl), the reference samples demonstrated an almost perfectly linear relationship between protein concentration and light absorption at 540 nm. 2. The distilled water sample absorbed less 540-nm light than the sample containing Biuret Mix. This means that if the water sample had been used to zero the spectrophotometer, all the other readings would have been too high since they would have measured the absorbance due to the concentration of protein as well as absorbance by the components of the Biuret Mix itself. The reagent blank provides an acceptable zero point for the measurements of the samples containing protein solution. 3. Samples U2 and U3 should have been diluted to exactly 60% and 20% of the concentration of sample U1, with a ratio of U2 to U3 of exactly 3:1. However, the spectrophotometric measurement did not confirm the latter ratio; the measured absorbances of U2 and U3 were in the ratio 2.7:1. This indicates that one or both of these samples was not diluted accurately. 4. The 30-minute waiting time after mixing protein solution with Biuret reagent allows the copper-peptide reaction to proceed to completion, particularly with more concentrated samples where there is not a large excess of copper ions available to react with peptide bonds. 5. As described above (see ‘Discussion’), extrapolation of the reference curve is not a reliable procedure since it is possible that measurements of unknown samples with higher absorbance than the highest reference absorbance have been reduced by exhaustion of the Biuret reagent. In such cases, the measured absorbance will reflect a lower value than the actual concentration of protein, since not all peptide bonds will have been converted to a colored copper-peptide complex. 6. Blood levels of creatine kinase are measured to help establish a number of conditions involving muscle damage, including heart attack, physical injury (e.g. muscle tears), and damage due to statin drug usage. 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. Hamilton-Craig, Ian, 2003, ‘Statins and muscle damage’, Australian Prescriber 2003;26:74-5. Retrieved from http://www.australianprescriber.com/magazine/26/4/74/5/ . Hargreaves, M. & Spriet, L. 2006, Exercise Metabolism, 2nd ed, Human Kinetics, USA. 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