Proteins are present in the living world, irrespective of the size of the organism, since they form the structural and functional basis of cell. Under certain circumstances, it may become necessary to identify the presence of protein. Isolation of any new compound (e.g., antiviral principle) needs to be attacked primarily through identification reactions. Some of the color reactions of proteins which could be used as identification tests are given below:
To 2mL of the test solution add 2mL of 10% NaOH. Mix. Add two drops of 0.1% CuSO4 solution
|Violet or pink color
||Compounds with two or more peptide bonds give a violet color with alkaline copper sulphate solution
To 4mL of the solution which should be at neutral pH add 1mL of 0.1% freshly prepared ninhydrin solution. Mix the contents and boil for a couple of minutes. Allow to cool
|Violet or purple color
||The ninhydrin test is answered by amino acids and proteins. The formation of a complex called Rheumann’s purple due to condensation of two molecules of nin-hydrin with one molecule of ammonia from amino acid is responsible for the violet color. The a-amino group is the reactive group
To 5mL of the solution add 1mL of conc. HNO3. Boil the contents. After cooling ad excess 40% NaOH
|On adding acid, yellow color will be noticed. When NaOH is added deep orange color will develop
||The yellow color is due to the nitro derivatives of the aromatic amino acids present in the protein. The sodium salts of nitro derivatives are orange in color.
||GLYOXYLIC REACTION FOR TRYPTOPHAN
Add 2mL of glacial acetic acid to 2mL of the test solution. Then about 2mL of conc. H2SO4 carefully down the sides of the test tube. Observe the color change at the junction of the two liquids.
|Violet ring is formed at the junction
||The indole group of tryptophan reacts with the glyoxylic acid released by the action of conc. H2SO4 on acetic acid to give a purple color.
To 5mL of the solution cooled on ice add 1mL of 10% NaOH solution and 1mL of 0.02% a-Naphthol solution. After few minutes add 10 drops of alkaline hypobromide solution
|Intense red color
||The guanidine group or arginine reacts with a-Naphthol to form a bright red colored complex.
To 2mL of solution add 2mL of 40% NaOH and 10 drops of 2% lead acetate solution. Boil for a minute and cool.
||The sulphur in sulphur containing amino acids of the proteins in presence of NaOH, is changed into Na2S which forms black lead sulphide when reacted with lead acetate.
||MODIFIED MILLION’S TEST
a) To 1mL of solution add 1mL of 10%mercuric sulphate in 10% sulphuric acid. Boil gently for half a minute.
||The yellow precipitate is due to the precipitation of protein. Mercury combines with tyrosine of the protein.
|b) Cool under a tap water and add a drop of 1% NaNO2 solution and warm gently
||A red color develops
||The red color is due to reaction of the precipitate with the nitrous acid.