122 THE PROTEINS AND PROTEASES [CH. 



which proteins can be detected. These are illustrated in the following 

 experiment. 



Eocpt. 121. Tests for proteins. Weigh out about 10 gms. of dried peas (Pisum) y 

 grind them in a coffee-mill and then add 100 c.c. of water to the ground mass. 

 Allow the mixture to stand for an hour. Filter, and make the following tests with 

 the filtrate (see p. 133). 



(a) The xanthoproteic reaction. To a few c.c. of the protein solution in a test-tube 

 add about one-third of its volume of strong nitric acid. A white precipitate is 

 formed (except in the case of proteoses, peptones, etc.). On boiling, the precipitate 

 turns yellow, and may partly dissolve to give a yellow solution. Cool under the tap, 

 and add strong ammonia till the reaction is alkaline. The yellow colour becomes 

 orange. The precipitate is due to the fact that metaprotein (see p. 127) is formed 

 by the action of acid on albumins or globulins, and this metaprotein is insoluble in 

 strong acids. The yellow colour is the result of the formation of a nitre-compound 

 of some aromatic component of the protein, such as tyrosine, tryptophane and 

 phenylalanine. 



(6) Millon's reaction. To a few c.c. of the protein solution add about half its 

 volume of Millon's reagent 1 . A white precipitate is formed. On warming, the preci- 

 pitate turns brick-red, or disappears and gives a red solution. The white precipitate is 

 due to the action of the mercuric nitrate on the proteins. The reaction is character- 

 istic of all aromatic substances which contain a hydroxyl group attached to the 

 benzene ring. The aromatic complex in the protein to which the reaction is due is 

 tyrosine. 



(c) The glyoxylic reaction (Hopkins and Cole}. To about 2 c.c. of protein solution 

 add an equal amount of "reduced oxalic acid" 2 . Mix the solutions, and then add an 

 equal volume of concentrated sulphuric .acid, pouring it down the side of the tube. 

 A purple ring forms at the junction of the two liquids. If the liquids are mixed by 

 shaking the tube gently, the purple colour will spread throughout the solution. The 

 substance in the protein molecule to which the reaction is due is tryptophane. If 

 carbohydrates are present in the liquid to be tested, the colour is not good, owing to 

 blackening produced by the charring with the strong sulphuric acid. 



(d) The biuret reaction. To a few c.c. of the protein solution add an excess of 

 sodium hydrate and a drop of a 1 % solution of copper sulphate. A violet or pink 

 colour is produced. The reaction is given by nearly all substances containing two 

 CONH groups attached to one another, to the same nitrogen atom, or to the same 

 carbon atom. The cause of the reaction with proteins is the presence of one or more 



1 This reagent is made by dissolving 30 c.c. of mercury in 570 c.c. of concentrated 

 nitric acid and then adding twice its bulk of water. It contains mercurous and mercuric 

 nitrates, together with excess of nitric acid and a little nitrous acid. 



2 Reduced oxalic acid is prepared as follows : (a) Treat 500 c.c. of a saturated solution 

 of oxalic acid with 40 gms. of 2/ sodium amalgam. When hydrogen ceases to be 

 evolved, the solution is filtered and diluted with twice its volume of distilled water. The 

 solution contains oxalic acid, sodium binoxalate and glyoxylic acid (COOH-CHO). 

 (6) Put 10 .gms. of powdered magnesium into a flask and just cover with distilled water. 

 Add slowly 250 c.c. of saturated oxalic acid, cooling under the tap. Filter off the insoluble 

 magnesium oxalate, acidify with acetic acid and dilute to a litre with distilled water. 



