DATE :
EXPERIMENT 4 : DETERMINATION OF PROTEIN CONTENT
Introduction:
Proteins are the fundamental fundamental constituent of the body. body. They are are the 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 180 o 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 bo th 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
Wa t e r
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 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 Concentration of protein in the the suppied egg white sample sample Standard)xConcentration of = (Absorbance of Test/Absorbance of Standard)xConcentration Standard
= (0.100/0.209)x20 mg/mL = 9.569 mg/mL So, 1mL of test test solution contained contained 9.569 mg protein. protein. So, 0.5mL of test solution contained (9.569x0.5) mg protein = 4.784 mg protein. Now,100mL of the original original sample solution solution contained 35gm of egg white. So, 0.5mL of the original sample solution 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 (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+. 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.
