THE END-PRODUCTS OF PROTEIN HYDROLYSIS 133 



Diamino-monocarboxylic acids, general formula: 



, 



\ 



R.COOH 



Diaminohydroxy-monocarboxylic acids. 



Heterocyclic compounds, i. e., amino-acids containing a ring of 

 atoms, one or more nitrogen atoms being included in the ring. 



The first necessary step in the analysis of the amino-acids produced 

 by the hydrolysis of protein, consists in the separation of the diamino- 

 acids from the monoamino-acids. This may be accomplished by means 

 of phosphotungstic acid, which precipitates the diamino-acids while 

 the monoamino-acids are left in solution. The diamino-acids Arginine 

 and Histidine may be separated in the form of their silver salts, while 

 the remainder of the precipitate produced by phosphotungstic acid 

 consists of the diamino-acid Lysine. 



The monoamino-acids may be estimated by evaporating the whole 

 mixture to dryness in vacuo and then dissolving the mixture of acids 

 in alcohol, and passing into the solution dry hydrochloric acid gas 

 which catalyzes the formation of alcohol esters of the amino-acids. 

 These esters are volatile and may be separated into fractions each 

 containing only a small number of acids, by means of fractional dis- 

 tillation in vacuo. The esters in each fraction are then reconverted 

 into free acids and alcohol by hydrolysis, and the individual acids 

 separated and estimated by special methods adapted to the peculiar 

 properties of each of the acids present in the particular fraction con- 

 cerned. The difficultly soluble acids, Tyrosine, Cysline and Diamino- 

 trioxydodecanic Acids are separated from the digest before ester ification. 



This method, due to Emil Fischer, is laborious and inaccurate, but 

 it greatly surpasses the methods which were formerly in use, and 

 which did not even permit a partial separation of the various mono- 

 amino-acids in a protein digest. The method permits the accurate 

 quantitive determination of only five acids, namely, the diamino-acids 

 histidine, arginine and lysine, and the monoamino-acids tyrosine and 

 cystine. The estimates of the other acids are only approximate, and 

 must be regarded as minimum values, since it has been found that in a 

 known mixture of amino-acids it is only possible to account for about 

 two-thirds of the nitrogen, by Fischer's method. For many purposes, 

 in which a knowledge of the total proportion of nitrogen present in the 

 form of monoamino-acids suffices, Fischer's method has now been 

 largely superseded by the method of Van Slyke which is described 

 below (p. 144), but if we desire to ascertain approximately the quantity 

 of the individual monoamino-acids contained in a protein digest, 

 Fischer's method, or modifications of it, still affords the only available 

 procedure. 



