Quantitative Determination Of Unknown Protein Concentrations

Quantitative determination of unknown protein concentrations using the biuret assay and BSA standard calibration Abstract The purpose of this study was to determine the protein concentration of two unknown samples labelled as x and y. This was done using the biuret assay. Quantitative analysis was performed by creating a calibration plot using Bovine Serum Albumin (BSA) at concentrations between 0 and 3mg/test volume. Absorbance was measured at 550nm using a spectrophotometer, producing a linear equation of y = 0.1723x and an R^2 of 0.9698. Unknown samples were analysed 3 times to ensure reliability. The mean concentration of unknown x was 5.60mg/ml and the standard error of the mean was 0.656mg/ml. the concentration of unknown y was 4.24mg/ml and the standard error of the mean was 0.308mg/ml. Even though the biuret method showed lower sensitivity compared to more modern assays, it provided reliable results for quantifying proteins in the clinical range. These results show the importance of albumin as a marker in diagnosing liver and renal problems. Introduction The main objective of this experiment was to determine the protein concentration of two unknown samples, X and Y using the biuret assay. Quantitative measurement is a fundamental technique in pharmaceutical biosciences. It is essential for monitoring body fluids, purifying enzymes and diagnosing clinical conditions. The theory behind this experiment is based on the biuret reaction. In alkaline conditions copper II ions from the biuret reagent form a coordination complex with the peptide bonds of the protein. This results in a characteristic colour change from light blue to deep violet. This colour change is directly proportional to the number of peptide bonds present and therefore the protein concentration too. The spectrophotometric method was used to quantify the protein. This involved measuring the absorbance of the samples at a wavelength of 550nm. A calibration plot was made using BSA, a reference protein. Plotting the known amounts of BSA against their absorbance meant a linear equation was formed. This equation meant the protein amounts in unknown X and Y could be estimated. Statistical analysis was then used to make sure the findings were precise and reliable. Methods The reagents used in this experiment included a biuret reagent which acted as the colorimetric indicator. BSA was used as the protein standard at a concentration of 5g/dm^3. Deionised water was used as the solvent for dilutions and as a blanking solution the unknown protein samples were labelled as Unknown X and Unknown Y. The main piece of equipment that was used for quantification was a spectrophotometer which was set to a wavelength of 550nm. A vortex mixer was used for ensuring homogenous reagent distribution. Test tubes and cuvettes were also used. 19 labelled test tubes were prepared. To reach a volume of 0.6ml, varying volumes of the BSA and deionised water were added to tubes 1-12. This represented a range of protein amounts from 0.5mg to 3.0mg. For the unknown samples, 0.4ml of Unknown X was added to tubes 13,14 and 15. 0.4ml of Unknown Y was added to tubes 16,17 and 18. Each was given 0.2ml of deionised water to keep a constant sample volume. Tube 19 was the blank and contained 0.6ml of water only. 0.6ml of biuret reagent was added to each of the 19 tubes. Each tube was then mixed using a vortex mixer and allowed to stand at room temperature for 15 minutes. This allowed for full colour development. The reagents were then transferred into cuvettes. The spectrophotometer was calibrated to 0 absorbance using the blank test tube. The absorbance of each standard and unknown was measured and recorded at 550nm. The data recorded was used to construct a calibration curve. This would allow the concentration of the unknown samples to be found. Results Tube Number Protein Amount (mg/test volume) Absorbance Reading 1 Absorbance Reading 2 Absorbance Reading 3 Average Absorbance 1, 2 3.0 0.464 0.486 - 0.4750 3, 4 2.5 0.331 0.444 - 0.3875 5, 6 2.0 0.360 0.349 - 0.3545 7, 8 1.5 0.310 0.314 - 0.3120 9, 10 1.0 0.265 0.284 - 0.2745 11, 12 0.5 0.149 0.150 - 0.1495 13, 14, 15 Unknown X 0.376 0.486 0.295 0.3857 16, 17, 18 Unknown Y 0.244 0.333 0.300 0.2923 19 0.0 (Blank) 0.000 - - 0.0000 Calibration plot of absorbance against protein concentration: Calculations for Unknown x: Replicate 1) 0.376/0.1723 = 2.1822mg 2.1822 x 2.5 = 5.46mg/ml Replicate 2) 0.486/0.1723 = 2.8207mg 2.8207 x 2.5 = 7.05mg/ml Replicate 3) 0.295/0.1723 = 1.7121mg 1.7121 x 2.5 = 4.28mg/ml Mean concentration for x = (5.46 + 7.05 + 4.28)/3 = 5.60mg/ml Calculations for Unknown y: Replicate 1) 0.244/0.1723 = 1.4161mg 1.4161 x 2.5 = 3.54mg/ml Replicate 2) 0.333/0.1723 = 1.9327mg 1.9327 x 2.5 = 4.83mg/ml Replicate 3) 0.300/0.1723 = 1.7412mg 1.7412 x 2.5 = 4.35mg/ml Mean concentration for y = (3.54 + 4.83 + 4.35)/3 = 4.24mg/ml Statistical analysis for Unknown x: Mean = 5.60mg/ml Standard deviation = 1.14mg/ml Standard error of the mean = 0.656mg/ml Statistical analysis for Unknown y: Mean = 4.24mg/ml Standard deviation = 0.533mg/ml Standard error of the mean = 0.308mg/ml The determination of unknown proteins was achieved by comparing the absorbance of samples x and y against a BSA calibration plot. The calibration plot showed a strong linear relationship between the amount of protein and absorbance at 550nm. It produced an equation of y = 0.1723x with an R^2 value of 0.9698 this tells us that the biuret assay was accurate within the tested range. The unknown protein samples were analysed 3 times for precision. Using the equation and a dilution factor of 2.5, the final concentrations were calculated. Unknown x: The absorbance readings led to a mean concentration of 5.60mg/ml. the standard deviation of 1.14mg/ml and standard error of the mean of 0.656mg/ml indicate a high statistical variation. Unknown y: the absorbance readings led to a mean concentration of 4.24mg/ml. the standard deviation of 0.53mg/ml and standard error of the mean of 0.308mg/ml meant that this sample showed more consistency than unknown x. Both of the mean absorbances were in line with the linear range of the calibration plot, validating the quantification of the samples. The experimental blank stayed at an absorbance of 0.00 which confirmed that there wasn’t any interference from the biuret reagent during the spectrophotometric measurement. Discussion The experiment determined that unknown x had a mean protein concentration of 5.60mg/ml, while unknown y was lower at 4.24mg/ml. these values were in line with the calibration curve, confirming that the absorbance measured at 550nm was directly proportional to the protein amount. The biuret assay has many limitations. It requires a high amount of protein to produce a colour change which makes it less sensitive than the Bradford or Lowry assays. Secondly, the biuret reagent reacts with the peptide backbone instead of the specific amino acid side chains. This makes it susceptible to interference from certain buffers. In this experiment, a limitation was the high statistical variance in unknown x (a standard error of the mean of 0.656mg/ml. This was likely due to an outlier in the second replicate which may have came from pipetting inaccuracies or incomplete mixing of the reagent. In future experiments, increasing the number of replicates or the using automated pipettes could improve the accuracy of the results. BSA was used in this assay due to its high stability and similarity in structure with Human Serum Albumin (HSA). In bioscience, albumin is an abundant protein in plasma and plays a key role in keeping oncotic pressure and transporting essential ligands including hormones, fatty acids and drugs. The concentration and function of albumin are indicators of disease development. Hypoalbuminemia is associated with liver disease. Albumin levels are also a marker for kidney dysfunction. Albumin in urine is often the first sign of diabetic nephropathy. Research indicates that glycation of albumin in diabetic patients can impair its ligand binding capacity, contributing to more complications. Conclusion In conclusion, the biuret assay gave a reliable method for determining protein concentrations in unknown samples. Unknown x and y were accurately quantified even though there was some experimental variance. This practical showed the importance of spectrophotometric analysis in clinical diagnostics and the key role albumin plays as a marker for health. References Okutucu, B., Dinçer, A., Habib, Ö. and Zihnio?lu, F. (2007). Comparison of five methods for determination of total plasma protein concentration. Journal of Biochemical and Biophysical Methods, 70(1), pp.145-147. Gornall, A.G., Bardawill, C.J. and David, M.M. (1949). Determination of serum proteins by means of the biuret reaction. Journal of Biological Chemistry, 177(2), pp.751-766. Mindovix (2024) Comprehensive Insights into Bovine Serum Albumin. (online) Available from: https://mindovix.com/articles/insights-into-bovine-serum-albumin/ (Accessed 13th April 2026).