180 PHYSIOLOGY OF NUTRITION 



by oxidation alter the composition of the aporrhegmas. The end product of 

 these processes is ammonia, which is then used in the synthesis of asparagin. 1 



The following method is employed in the quantitative study of the various 

 nitrogenous substances that have been mentioned in the preceding paragraphs. 2 

 The total nitrogen content is determined from one portion of the material, and 

 the protein nitrogen is determined from another portion, the difference between 

 these two quantities being the amount of the non-protein nitrogen. For the 

 determination of the separate nitrogen compounds, the plants to be studied are 

 extracted with water and the extract is precipitated with lead acetate. The 

 precipitate contains proteins, pigments and other compounds, while the crys- 

 talline nitrogenous substances are in the filtrate. The filtrate is treated with 

 mercuric nitrate, which precipitates asparagin, glutamin and allantoin; also, 

 in part, xanthin, hypoxanthin, guanin, arginin, tyrosin. The precipitate is 

 suspended in water, treated with hydrogen sulphide and the mercuric sul- 

 phide thus formed is filtered out. The filtrate is neutralized with ammonia 

 and concentrated by evaporation, after which it is allowed to stand for some 

 time. Crystals of the nitrogenous compounds separate out and may be further 

 dealt with by suitable methods. If no material is precipitated by mercuric 

 nitrate, then the plant extract is treated with lead acetate and filtered, the 

 filtrate being treated directly with hydrogen sulphide. The lead sulphide is 

 filtered off, the filtrate is neutralized with ammonia and then concentrated by 

 evaporation over a water bath. 



The method of Sachsse 3 is used especially in the determination of asparagin 

 and glutamin. This procedure depends upon the fact that these amides break 

 down, upon being boiled with weak hydrochloric acid and water, into amino 

 acids and ammonia, as is illustrated by the following equation. 



(Asparagin) 



NH 2 .CO.CH 2 .CHNH 2 .COOH + H 2 = 



(Aspartic acid) (Ammonia) 

 COOH.CH 2 .CHNH 2 .COOH + NH 3 . 



Half of the asparagin nitrogen is thus split off. The ammonia nitrogen is 

 then determined, according to the usual methods, and the number thus obtained 

 is doubled, to give the asparagin nitrogen. The same method is of course also 

 available for the determination of glutamin nitrogen. 



For microchemical identification of asparagin the method of Borodin 4 is 

 employed. The sections to be studied are mounted in alcohol under a cover 

 glass and the alcohol is allowed slowly to evaporate out at the margin of the 

 cover. If asparagin is present it crystallizes during this process. The crystals 



» Butkewitsch, Wl., Das Ammoniak als Umwandlungsprodukt stickstoffhaltiger Stoffe in hoheren 

 Pflanzen. Biochem. Zetisch. 16: 411-452. 1909. Prianischnikow, D., and Schulow, J., Ueber die syn- 

 thetische Asparaginbildung in den Pflanzen. Ber. Deutsch. Bot. Ges. 28: 253-264. 1910. 



2 Abderhalden, Handbuch. [See note 1, p. 158.] 



a Sachsse, Robert, Ueber eine Methode zur quantitativen Bestimmung des Aparagins. Jour, prakt. 

 Chem., N.F. 6: 1 18-127. 1873. 



4 Borodin, 1878. [See note 3, p. I74-I 



