Qualitative test for proteins and amino acids by using Biuret test methods

Biuret Test

Chemical requirement: Protein solution, sodium hydroxide, copper sulphate, Millon’s reagent, ninhydrin.

Apparatus 

Burette stand, heating mental, Test tube, Pipette, beaker, burette.

Principle 

Proteins are compounds consisting of amino acids. Proteins exhibit colloidal properties and there for do not diffuse through an intact animal membrane. Hydrolysis, with acids or alkalis, breaks the protein into constituent amino acids.

Procedure Biuret test

To 2-3 ml of protein solution in a test tube add an equal volume of 10% sodium hydroxide solution. Mix thoroughly and add 0.5% copper sulphate solution drop by drop until a purplish- violet color is produced.

(Blue precipitate of copper hydroxide is formed if excess copper sulphate is added).

Biuret reaction is due to the peptide linkage O+C-N-H of due to two carbamyl groups (CONH2). Hence, it is positive with all proteins.

CONH2
|
NH
|
CONH2

|
Biuret

Ninhydrin reaction 

To 5 ml of dilute protein solution (ph between 5 and 7) add 0.5 ml of a 0.1% solution of ninhydrin. Heat to boiling for one or two minutes and allow cooling. A blue colour appears.This test is given by all amino acids except proline and hydroxyproline. Proline and hydroxyproline react with ninhydrin and give yellow colour.

Millon’s reaction

To 5 ml of protein solution add 3-4 drops of Millon’s reagent and mix. Heat to boiling point over a small flame. Proteins like egg albumin. Gives white precipitate which gradually turns red on heating. Proteoses and peptones give red colour. If no colour develops, add 2-3 more drops of Millon’s reagent and heat again. However, large quantity of Millon’s reagent gives yellow colourwhich is not a positive reaction. This reaction is due to the presence of the hydroxyphenyl group in protein molecule e.g. Tyrosine.