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 egg-

white 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

nm.
BLANK (B) 0.000
STANDARD(S) 0.209
TEST(T) 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.