74 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



II. Estimation of the Different Groups of Aniino Acids. 



The estimation of the several groups of amino acids present in a 

 protein is effected by the following series of operations : 



1. Hydrolysis. 3 grammes of protein, or better 6 grammes for 

 duplicate analyses, are dissolved in 10 or 20 parts of 20 per cent, 

 hydrochloric acid and boiled in a tared flask under a reflux condenser. 



After six or eight hours the hydrolysis is stopped. Portions of 

 I c.c. or 2 c.c. (enough to contain cri gramme protein) are withdrawn 

 with a pipette and diluted to 10 c.c. In these portions the amount of 

 amino nitrogen is determined, the reaction being allowed to proceed 

 for five minutes standing and then for one minute with shaking. Under 

 these conditions the same proportion of ammonia (i 5-20 per cent.) is 

 decomposed in each determination. 



The hydrolysis flask is weighed and the hydrolysis continued for 

 another period of six or eight hours, when amino nitrogen is again 

 determined. 



Hydrolysis is continued until the amino nitrogen is constant. 



The object of weighing is to ascertain if the solution has become 

 concentrated by loss of vapour and to allow of a correction for a de- 

 crease of the volume. 



2. Estimation of Total Nitrogen. The products of hydrolysis are 

 transferred to a measuring flask of 100 c.c or 250 c.c capacity. Total 

 nitrogen is estimated by Kjeldahl's method in an aliquot portion con- 

 taining 0*2 gramme of protein. All the subsequent estimations are 

 based upon this value. 



3. Amide Nitrogen. Since cystine is very easily decomposed by 

 boiling with magnesia at 100 the determination of ammonia must be 

 carried out in vacua at 40, or at room temperature by the aeration 

 method of Denis (J. Biol. Chem., 8, 427). The method of distilling 

 in vacua is to be preferred as the same apparatus is repeatedly em- 

 ployed in the other estimations. 



The distillation in vacua is performed in the apparatus shown in 



fig- 4- (P- 75> 



The Claisen flask and receiver are of I litre capacity, the guard 

 flask of 200 c.c. 



The hydrolysed solution is placed in the double-necked flask and 

 diluted to 200 c.c; 100 c.c of alcohol are added to prevent frothing 

 and then an excess of a i o per cent, suspension of calcium hydrate, as 



