EXPERIMENT 5 QUANTITATIVE DETERMINATION OF PROTEIN

Introduction:
5.1 Biuret Assay The quantitative estimation of protein concentration can be carried out by several methods. One of the methods that commonly used is Biuret assay. Biuret assay is a test that can be used to determine the protein concentration between 1 to 20 mg/mL. This reaction is dependent in part on peptide bonds and not solely on amino acid moieties. The biuret assay is based on the ability of Cu (II) ions to form a violet-coloured chelate complex with peptide bonds (-CONH-groups) in alkaline. In this experiment, the biuret assay were test on standard solution of Bovine Serum Albumin (BSA) and also an unknown protein albumin solution with unknown concentration called solution X.

Procedure:
For this experiment, the materials needed are standard solution of Bovine Serum Albumin (BSA) in 10mg/ml, protein albumin solution X with unknown concentration and biuret reagent. The biuret reagent is prepared by dissolving 3g of copper sulphate (CuSO4.5H2O) and 9g of sodium potassium tartrate in 500ml of 0.2M NaOH. Then, add 5g of potassium iodide (KI) and make up to 1 litre with 0.2M NaOH. Firstly, the experiment was started with a series of concentrations of albumin was prepared according to the table given. Test tubes 1. 2. 3. 4. 5. 6. Solution X (mL) 0 0.2 0.4 0.8 1.2 1.6 Distilled water (mL) 2.0 1.8 1.6 1.2 0.8 0.4 Biuret reagent (mL) 8.0 8.0 8.0 8.0 8.0 8.0 Absorbance (A540) 0 Protein concentration (mg/mL) 0

After that, test tubes was shaked after adding the biuret reagent and was incubated for 10 minutes at 37oC water bath. Next, the test tubes was left to cool at room temperature and the absorbance was read at the wavelength of 540nm. Test tube 1 was used as blank. Finally, the concentration of protein albumin in each tube was calculated and a graph of absorbance against albumin concentration was plotted.

Data And Calculation:

Test tubes 1. 2. 3. 4. 5. 6. Albumin (mL) 0 0.2 0.4 0.8 1.2 1.6 Distilled water (mL) 2.0 1.8 1.6 1.2 0.8 0.4 Biuret reagent (mL) 8.0 8.0 8.0 8.0 8.0 8.0 Absorbance (A540) 0.000 0.040 0.055 0.113 0.204 0.232 Protein concentration (mg/mL) 0.0000 -4.8620 -1.2805 1.5835 3.3886 3.0733

By using regression equation from standard calibration curve, which is y = 0.0652x + 0.0717, we can calculate the diluted protein concentration in solution X. Therefore, diluted protein concentration in solution X = (y - 0.0717) / 0.0652, where y = absorbance value. For test tube 1, diluted protein concentration in solution X x = (y - 0.0717) / 0.0652 = (0.000-0.0717) / 0.0652 = -1.0997mg/mL

For test tube 2, diluted protein concentration in solution X x = (y - 0.0717) / 0.0652 = (0.040 - 0.0717) / 0.0652 = -0.4862mg/mL

For test tube 3, diluted protein concentration in solution X x = (y - 0.0717) / 0.0652 = (0.055 - 0.0717) / 0.0652 = -0.2561mg/mL

For test tube 4, diluted protein concentration in solution X x = (y - 0.0717) / 0.0652

= (0.113 - 0.0717) / 0.0652 = 0.6334mg/mL

For test tube 5, diluted protein concentration in solution X x = (y - 0.0717) / 0.0652 = (0.204 - 0.0717) / 0.0652 = 2.0291mg/mL

For test tube 6, diluted protein concentration in solution X x = (y - 0.0717) / 0.0652 = (0.232 - 0.0717) / 0.0652 = 2.4586mg/mL

To calculate the actual protein concentration (mg/mL), we must first calculate the dilution factor and multiple with diluted protein concentration (mg/mL).

For test tube 1, No dilution factor, actual protein concentration (mg/mL) is zero since no solution X is added.

For test tube 2, Dilution factor = 2.0/0.2 = 10 Protein concentration = -0.4862 x 10 = -4.862mg/mL

For test tube 3, Dilution factor = 2.0/0.4 = 5

Protein concentration = -0.2561 x 5 = -1.2805mg/mL

For test tube 4, Dilution factor = 2.0/0.8 = 2.5 Protein concentration = 0.6334 x 2.5 = 1.5835mg/mL

For test tube 5, Dilution factor = 2.0/1.2 = 1.67 Protein concentration = 2.0291 x 1.67 = 3.3886mg/mL

For test tube 6, Dilution factor = 2.0/1.6 = 1.25 Protein concentration = 2.4586 x 1.25 = 3.0733mg/mL

Discussion:
The biuret assay is a chemical method used for detecting the presence of peptide bonds. In the presence of peptides, a copper (II) ion forms violet-colour coordination complexes in an alkaline solution. The Biuret reaction can be used to assay the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law. The Biuret reagent is made of sodium hydroxide (NaOH) and hydrated copper (II) sulfate, together with potassium sodium tartrate. The reagent turns from blue to violet in the presence of proteins, blue to pink when combined with short-chain polypeptides. The Biuret assay is reliable for amounts above 0.5 mg. It is slower and less sensitive compare to Folin-Lowry method which gives more accurate values in g/mL. The biuret method demonstrates less protein-to-protein variability than UV absorbance, and it requires the use of relatively large sample sizes. The biuret assay consumes much more material than Folin-Lowry method. The biuret is a good general protein assay for batches of material for which yield is not a problem. As compare with Folin-Lowry method, the Biuret can be scaled down for smaller cuvette sizes, consuming less protein. Proteins with an abnormally high or low percentage of amino acids with aromatic side groups will give high or low readings, respectively. For solution x we typically obtain a linear

relationship between absorbance and amount protein over a range of 0.5 to 20 mg protein same like its standard solution of Bovine Serum Albumin. The Biuret assay are not been reliable for amounts below 0.5 mg. Bovine Serum Albumin (BSA) is used as standard because of its stability, its lack of effect in many biochemical reactions, and its low cost. BSA is also rich of amino acid in respond to the reaction.