Estimation of protein by biuret method.

Principle of the test
Uria on heating forms biurate
Biuret gives a violet co lour with alkaline copper sulphate solution. This test is answered in general by substances containing 2 or more peptide linkages. Thus when proteins are prated with biuret reagent a pint to purple co lour is obtained. The intensity of the co lour development is proportional to the no. of peptide linkages, proteins gives a purple-violet co lour, proteases and peptones a pink co lour and peptides a much pale pink. However the test is not absolutely specific for peptide bond since any compound containing two carbonyl group (–CONH2 ), joined through a single atom of mtrogen or carbon also will give a positive test.

Requirements: –
1) std. protein solution ( 5 mg / ml )
2) Bitnet reagent.
3) Sample protein solution.
4) Distilled water.

Procedure: – Prepare a series of test tubes for blank standard and sample solution as follows.

Mix the solution in each test tube thoroughly and maintain the tubes at room temperature for ten minutes. Measure the extinction at 540 nm against the blank.
Plot the graph of extinction against concentration and determine the concentration of protein in the given sample solution by extrapolation.

Preparation of biuret reagent:
Dissolve 3 gms. Of cuso4 5H2o and 9.0 gms of Na-k-fortunate in 500 ml of 0.2 N NaOH. Add 5 mgs of KI and make up the volume to one liter with 0.2 N NaOH solution.


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