Determination Of Protein Content

DATE :

EXPERIMENT 4 : DETERMINATION OF PROTEIN CONTENT

Introduction:

Proteins are the fundamental constituent of the body. They are the major organic constituent of protoplasm and extra-cellular material. Different kinds of proteins are specific for different functions. Proteins may function as– enzyme, hormone, receptor, etc. All proteins are constructed from the same ubiquitous set of 20 amino acids, covalently linked with in a linear sequence, which folds, form a 3-D structure, and are further modified to attain the active conformation.

Biuret Test is a method for quantitative assay of proteins. Biuret is obtained by heating Urea at 180o C, which gives positive test with Biuret reagent. Biuret contains two peptide linkages and gives purple/violet colour in the reaction with alkaline CuSO4 solution. Proteins, which are polypeptides, react in the same way as Biuret. Thus for positive result, the presence of atleast two peptide linkages (CO-NH) are necessary. The Cu+2 of CuSO4 forms a co-ordination complex with the four nucleophillic Nitrogen, provided by the peptides. The Biuret reagent contains alkaline solution of CuSO4 with either Na-K-tartarate or Na Citrate to prevent precipitation of Cu+2 in hydroxide form. This is quantitative because the intensity of colour depends on the number of peptide bonds.

Preparation of BSA Standard Solution A set of BSA Standard solution was prepared with known amount of protein and was subjected to Biuret Test. The O.D. was taken at 540nm. The value of the O.D. was used to determine the concentration of protein for both Egg-white and Gramflour.

Protocol : 1) A BSA stock was prepared with a conc. of 20mg/ml.

2) 4 sets of standard was prepared from the stock and Biuret Reagent was BSA

Amount of BSA

Water

Biuret Reagent

Blank

0.0ml

–

2ml

5ml

Std-I

0.1ml

2mg

1.9ml

5ml

Std-II

0.2ml

4mg

1.8ml

5ml

Std-III

0.3ml

6mg

1.7ml

5ml

Std-IV

0.4ml

8mg

1.6ml

5ml

added according to the table below.

3) Spectrophotometric reading was taken at 540nm after 30mins. of incubation.

1. Determination of Protein content in Egg-White In this experiment, amount of protein Albumin in liquid eggwhite was determined. Liquid egg-white contains 10-11% protein of which albumin (ovalbumin and conalbumin) constitutes 67%.

Materials Required : • • • • • •

Egg (Egg-white) 0.1 (N) NaOH solution Distilled Water Biuret Reagent Volumetric flask, stirrer, beaker Spectrophotometer, Quartz cuvette

Procedure : 1) 5gm egg white was taken in a pre-weighed beaker. 2) The volume was made upto 100ml by adding 0.1(N) NaOH. 3) 0.5ml of egg-white solution was taken and 5ml of Biuret reagent was added and incubated for 10mins. 4) Spectrophotometric reading was taken at 540nm.

RESULTS AND CALCULATIONS: TEST TUBE

ABSORBANCE AT 540

BLANK (B) STANDARD(S) TEST(T)

nm. 0.000 0.209 0.100

Concentration of protein in the suppied egg white sample = (Absorbance of Test/Absorbance of Standard)xConcentration of Standard = (0.100/0.209)x20 mg/mL = 9.569 mg/mL So, 1mL of test solution contained 9.569 mg protein. So, 0.5mL of test solution contained (9.569x0.5) mg protein = 4.784 mg protein. Now,100mL of the original sample solution contained 35gm of egg white. So, 0.5mL of the original sample solution contained (35/100)x0.5 gm of egg white. = 0.175gm of egg white. Thus, 0.175gm of egg white contained 4.784 mg of protein. So, 1gm of egg white contained (4.784/0.175)mg of protein = 27.34 mg of protein.

Discussion 1) The violet colour of the Biuret Assay is due to the complex formation between Cu++ ion of Copper Sulfate solution and the Nitrogen of peptide bonds. Usually Cys, Trp, Tyr are reducing Cu++ to Cu+. 2) This experiment gives an idea about the protein content of the Egg both of which are consumed by humans. Therefore it gives an idea about protein intake by humans via egg.