DISTRIBUTION OF NITROGEN 97 



II. Estimation of the Different Groups of Amino 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 grams of protein, or better 6 grams 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 I c.c. or 2 c.c. (enough to contain O'l gram of protein) are withdrawn 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 (15-20 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 

 containing 0-2 gram 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. 1 The method of distilling in vacuo is to be preferred 

 as the same apparatus is repeatedly employed in the other estimations. 



The distillation in vacuo is performed in the apparatus shown in 

 % 5 (P- 9&)> or in some arrangement by which the ammonia vapour 

 passes into the standard acid. 



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 



j y. Biol. Chem., 8, 427. 

 PT. I. 7 



