ANALYSIS AND CHARACTERIZATION OF PROTEINS 145 



residual mixture of products is then treated with phosphotungstic acid, 

 which results in the precipitation of the diamino-acids, namely Cystine, 

 Arginine, Lysine and Histidine, a determination of sulphur yields a 

 measure of the cystine-content. Arginine has the property of yielding 

 one-half of its nitrogen in the form of ammonia on boiling with alkali. 

 The quantity of ammonia developed on boiling the precipitate with 

 alkali therefore affords a measure of the content of arginine. 



The total nitrogen in the phosphotungstic-acid precipitate is now 

 determined, and from it is subtracted the calculated proportion of 

 nitrogen which is contributed by the cystine- and arginine-content. 

 The residual nitrogen is derived from lysine (=x) and histidine (=y). 

 On treatment with nitrous acid lysine yields a volume of free nitrogen 

 corresponding to the whole of its nitrogen-content (=x), while histidine 

 yields a volume of free nitrogen which corresponds to one-third of 

 its nitrogen-content (=Jy). The amino-nitrogen content of the 

 precipitate is therefore determined, by the nitrogen-yield on treatment 

 with nitrous acid, and after subtracting the amino-nitrogen yield due to 

 arginine (= one-fourth of the total nitrogen in arginine) and of cystine 

 (= the whole of its nitrogen content) the residual amino-nitrogen 

 evidently represents the whole of the lysine nitrogen plus one-third of 

 the histidine nitrogen, or x-fjy. But the determination of the total 

 nitrogen in the phosphotungstic-acid precipitate, and the subtraction 

 therefrom of the cystine and arginine nitrogen, has already given us a 

 measure of the total nitrogen contained in the lysine and histidine, 

 that is, of x-f-y. Subtracting, therefore, the amino-nitrogen yield due 

 to these two amino-acids from the proportion of the total nitrogen 

 which they contribute, the difference evidently corresponds to two- 

 thirds of the histidine nitrogen, from which the contents of histidine 

 and lysine may be readily computed. 



In the filtrate from the phosphotungstic-acid precipitate the total 

 nitrogen and the amino-nitrogen are separately determined. The 

 difference yields a measure of the nitrogen contained in pyrrolidine 

 (proline and oxyproline) or indole (tryptophane) rings. 



For the determination of the nitrogen evolved from amino -groups 

 on treatment with nitrous acid Van Slyke has devised a very convenient 

 apparatus which permits the rapid and accurate determination of amino- 

 groups in very small quantities of material. For descriptions of this 

 apparatus the reader is referred to Van Slyke's original articles, or to 

 current laboratory-handbooks. 1 



During the hydrolysis of proteins by hydrochloric acid a small 

 amount of a very deeply colored precipitate separates out. The 

 nitrogen-content of this precipitate is the so-called "humin" nitro- 

 gen. This appears to be derived from a portion of the tryptophane 

 and, in the presence of a sufficiency of carbohydrate, which acts as a 

 catalyzer for the formation of humin, the yield, of humin-nitrogen is 



1 For example R. H. A. Plimmer: Practical Organic and Biochemistry, London, 

 1915, p. 146. 

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