ESTIMATION OF SERUM PROTEIN BY BIURET METHOD:

■ Introduction: The biuret test is given by three substances which contains groups joined either
directly or through a single atom of nitrogen and carbon. Similar substances which contain -CONH2
group, -CSNH2 group or -C(NH)NH2 group or -CH2NH2 group may also respond to this test. Now
protein substances containing the necessary group (e.g. oxamide) also respond to this test. The end
reactions of biuret test is developed upon formation of cu- biuret compound.
■ Principle: Proteins which contain peptide linkages form a complex with copper in alkaline medium
giving a bluish violet colour and this reaction is called the Biuret reaction. The intensity of this colour
is proportional to the number of peptide linkages present and thus a measure of the concentration of
proteins. This reaction is given by all substances containing 2 or more peptide linkages. The
substances containing -CSNH2, -CH2NH2, -C(NH)NH2 group also gives this reaction. This test is
apparently due to the coordination of cupric ions (cu+2) with unshared electron pairs of peptide
nitrogen atoms, two from each of the adjacent peptide chains and oxygen of water to form a coloured
coordination complex that is known as biuret complex. Higher proteins give a purplish violet colour
and lower proteins give a rose pink colour.

■ Reagent Preparation:

i. 0.9% NaCl.

ii. Bovine Serum Albumin ( BSA): 100 mg of BSA was dissolved in 20 ml of 0.9% NaCl solution, such
that 1 ml of BSA solution contains 10 mg of albumin.

iii. Biuret stock solution: 4.5 g of Na-K tartarate was dissolved in 400 ml of 0.2(N) NaoH, 15g of
cuso4, 5H2O was added to it and stirred to dissolve completely, 5g of KI was dissolved in the
solution and the volume was made 1 litre with 0.2(N) NaOH.

iv. Working reagent: 200 ml of biuret stock reagent was diluted 1 lt with 0.2 (N) NaoH containing 5g
of KI.
■ Procedure: In a series of test tubes, BSA solution, 0.9% NaCl, biuret reagent and serum were taken
in following manner:

Test Tube No. BSA solution (ml) 0.9% NaCL (ml) Biuret reagent (ml)
1 (standard) 0.2 0.8 5
2 (standard) 0.4 0.6 5
3 (standard) 0.6 0.4 5
4 (standard) 0.8 0..2 5
5 (standard) 1.0 0.0 5
6 (Blank) 0.0 1.0 5
7 (standard) 1.0 0.0 5
8 (standard) 1.0 0.0 5

The solution in the test tubes were mixed well and was allowed to stand for 20 mins. Optical Density
(O.D.) of solutions were measured at 530 nm after setting the photoelectric colorimeter to 0 with the
blank.
■ Result:

Test Tube No. Optical Density

1
2
3
4
5
6
7
8

■ Calculation:

The total amount of protein in serum is given by the following formula-

(O.D of unknown)/(O.D of standard) ×{ Conc. of standard (mg)}/{volume of sample(ml)} × 100 × dil. factor