92 PHYSIOLOGY 



TESTS FOR PROTEIN 

 A. COLOUR REACTIONS OF THE PROTEINS 



These are of importance since in many cases they are an indication of the nature 

 of the groups present in the protein molecule. 



(1) THE BIURET REACTION. When a solution of a protein is made strongly 

 alkaline with caustic potash or soda, and dilute copper sulphate added drop by drop, 

 a colour varying from pink to violet is produced. In the case of the proteoses and 

 peptones (the hydra ted proteins) the colour is pink ; in the case of the coagulable 

 proteins, violet. According to Schiff this colour is given by all compounds containing 

 the following groups : 



,CO.NH 2 



CO 

 2 <; 

 X 



.NH 2 



CO.NH 2 

 CO NH a 



I 



CO NH 2 

 and the group 



I I 



(NH 2 )C-CO NH C 



I I 



We have already seen that this grouping is typical of the protein molecule. 



(2) THE XANTHO-PROTEIC REACTION. On adding strong nitric acid to 

 a solution of protein and boiling, a yellow colour is produced which turns to a deep 

 orange when excess of caustic alkali or ammonia is added. The production of this 

 reaction points to the existence of benzene derivatives in the protein molecule, and 

 it is therefore a general test for the presence of aromatic groups. 



(3) MILLON'S REACTION. Millon's reagent is a solution of mercuric nitrate 

 in water containing free nitrous acid. On adding a few drops of this to a protein solu- 

 tion a white precipitate is produced which turns a brick-red colour on boiling. It 

 depends on the presence in the protein of a hydroxy-derivative of benzene, and is 

 determined in the protein by the tyrosine, which is oxyphenylalanine. 



(4) SULPHUR REACTION. On warming a solution of protein with caustic 

 soda in the presence of lead acetate, a black colour is produced owing to the precipi- 

 tation of lead sulphide. The depth of coloration gives a rough indication of the amount 

 of sulphur in the protein under investigation. 



(5) THE HOPKINS-ADAMKIEWICZ REACTION. It was stated by Adam- 

 kiewicz that on the addition of acetic acid and concentrated sulphuric acid to protein, 

 a violet colour was produced. Hopkins and Cole showed that the success of this reaction 

 depended on the presence of glyoxylic acid CHO.COOH as an impurity in the acetic 

 acid used. The test is therefore performed now as follows : 



Glyorylic acid is prepared by the action of sodium amalgam on a solution of oxalic 

 acid. A few drops of this solution are added to the solution of protein, and strong 

 sulphuric acid poured down the side of the tube. A bluish violet colour is produced 

 at the junction of the two fluids. This reaction is due to the presence in the protein 

 of tryptophane. 



The so-called Liebermann's reaction has been shown by Cole to be essentially a 

 modification of the above, and is due also to the presence of tryptophane. In this 

 test the protein is precipitated by alcohol, washed with ether, and heated with con- 

 centrated hydrochloric acid, when a blue colour is produced, glyoxylic acid being 

 derived from the alcohol and ether. 



