64 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

 A. Distribution of the Nitrogen in Three Groups. 



The differentiation of proteins by the estimation of the various 

 groups of the units was first attempted in Hofmeister's laboratory 

 by Hausmann in 1 899, who estimated amide nitrogen, diamino nitrogen 

 and monoamino nitrogen. The protein (i gramme) was hydrolysed 

 with 20 c.c. of concentrated hydrochloric acid by boiling for five hours 

 under a reflux condenser ; the solution was diluted and distilled with ex- 

 cess of magnesia, and the ammonia, which was liberated, was collected in 

 excess of standard acid ; the solution was then acidified with acid and 

 precipitated with phosphotungstic acid; after twenty-four hours the 

 precipitate was filtered off, washed with phosphotungstic acid, dissolved 

 in alkali and the nitrogen estimated in an aliquot portion by Kjeldahl's 

 method ; the filtrate was made up to a definite volume and nitrogen 

 estimated in an aliquot portion. 



Numerous objections to the accuracy of the data were raised. 

 Henderson maintained that the amount of amide nitrogen varied accord- 

 ing to the strength of the acid employed in the hydrolysis and the 

 time of hydrolysis ; Kutscher, and also Chittenden and Eustis, showed 

 that the precipitation of the diamino acids was not complete, and 

 Schulze and Winterstein found that certain monoamino acids, e.g.> 

 phenylalanine, were precipitated by phosphotungstic acid under certain 

 conditions. Hart preferred barium carbonate to magnesia for distilling 

 off the ammonia. 



Osborne and Harris, Giimbel, and also Rothera, critically ex- 

 amined the various objections. The amount of amide nitrogen was 

 not found to vary as Henderson stated; if similar conditions are 

 always maintained in the precipitation with phosphotungstic acid most 

 valuable comparative results can be obtained ; the errors of incomplete 

 precipitation of the diamino acids and precipitation of monoamino 

 acids almost compensate each other. 



Adopting Giimbel's suggestion of distilling off the ammonia in 

 vacua at 40 and Osborne and Harris' procedure, the process may be 

 carried out as follows : 



I. About i grm. of protein is boiled with about 100 c.c. of 20 

 per cent, hydrochloric acid in a 500 c.c. round bottom flask under a 

 reflux condenser until the solution no longer gives the biuret reaction, 

 usually from seven to ten hours. It is then evaporated in vacua at 

 40 to a volume of 2-3 c.c. ; the greater portion of the hydrochloric 

 acid is thus removed. 



