262 Biological Chemistry. 



whereas two molecules of glycine obtained by the hydro- 

 lysis of the polypeptide yield four atoms of nitrogen when 

 treated with this reagent 



2CH 2 (NH 2 ) COOH + 2HO NO = 2N 2 + 2CH 2 (OH) COOH 



A complex protein will contain only a relatively small 

 number of free amino groups capable of reacting with 

 nitrous acid. On hydrolysis, however, amino groups will 

 be set free, the more complete the hydrolysis the larger 

 the number. By measurement of the amount of nitrogen 

 set free during the course of hydrolysis (or digestion as it 

 is called) by a ferment, when an aliquot part of the 

 mixture is treated with excess nitrous acid, the amount of 

 change undergone by the protein can be ascertained. The 

 reaction with nitrous acid can be best carried out with 

 the apparatus devised by Van Slyke (see p. 246). 



The relative number of carboxyl groups set free by 

 hydrolysis can also be estimated. This cannot, however, 

 be accomplished by direct titration with a standard solu- 

 tion of an alkali and the use of an indicator in the ordinary 

 way, as an ammo-acid contains a basic (amino) as well 

 as an acidic group, and acts as an ampholyte (compare 

 p. 220). If, however, the action of the basic group can 

 be eliminated, then a hydrolysis mixture should become 

 more and more acid as the hydrolysis proceeds. The 

 elimination of the basic action of the amino group can be 

 effected by treatment with a large excess of formaldehyde. 

 Glycine, for example, reacts with formaldehyde according 

 to the equation 



CH 2 (NH 2 )-COOH + ECHO = H 2 + CH 2 (N : CH) COOH 



The methyleneimino-derivative thus obtained reacts as 

 an acid, and can be titrated with a standard solution of 

 alkali when phenolphthalein is used as an indicator. The 

 employment of this method can be illustrated by consider- 



