NUTRITION. 341 



not enough to permit of its identification. It contained nitrogen equal to 2.2 

 per cent of the total nitrogen of the original solution. Since 90 per cent of the 

 nitrogen of the second silver-baryta precipitate was recovered in the arginine 

 picrolonate, it is evident that besides the unidentified base obtained from the 

 HgS04 precipitate and arginine there is little, if anything else, among the 

 products of hydrolysis thus precipitable by silver. 



The filtrate from the silver-baryta precipitate, which should contain lysine, 

 was found to contain also stachydrine in much larger proportion than lysine. 

 The nitrogen in the stachydrine was equal to 5.4 per cent of the nitrogen in the 

 filtrate from the 53 per cent alcohol precipitate and that of the lysine to 1.16 

 per cent. These bases were separated by means of HgCl2, the fraction 

 obtained at acid reaction consisting almost wholly of the mercury salt of 

 stachydrine, while that precipitated at alkaline reaction contained the mercury 

 compound of lysine. Both of these substances were identified as picrates. 



The presence of stachydrine thus established confirms Steenbock's early 

 discovery of this betaine among the constituents extracted by water from 

 alfalfa hay. Whether all of the stachydrine found by us exists in the free state 

 in alfalfa-juice is not demonstrated by our experiments. Steenbock's work 

 indicates that at least a part of this base is free, unless all that he isolated was 

 liberated by enzymes during curing of the hay or its extraction by water. 



The fact that practically all of the nitrogen precipitated by HgCl2 at slight 

 acid reaction was identified as stachydrine shows that significant quantities of 

 other bases thus precipitable were not present. 



These results show that, so far as basic substances are concerned, the greater 

 part of the products of hydrolysis of the constituents of the alfalfa-juice are 

 quite unlike the products of hydrolysis of protein, and that Van Slyke's 

 method, which was devised to determine the relative proportions of the amino 

 acids yielded by proteins, gives results which are wholly misleading when 

 applied to green plants. 



The attempts to isolate the apparently abundant coloring matters which 

 can be extracted with amyl or butyl alcohol from the hydrolyzed juice of the 

 alfalfa-plant showed that a relatively large proportion of nitrogenous sub- 

 stances is simultaneously extracted by these alcohols from the strongly acid 

 solutions. It seemed probable that this nitrogen might belong largely to 

 hydrochlorides of amino acids, but as yet aspartic acid is the only amino acid 

 which we have been able to identify among these nitrogenous substances. 

 Experience has convinced us that precipitation with metallic salts will enable 

 us to obtain fractions from which products of hydrolysis may be obtained, and 

 also to gain an insight into the possible origin and relationships of the various 

 products whose presence has been revealed by our previous work with the 

 alfalfa-juice. Although marked progress in this direction has been made, it 

 is too soon to venture definite statements concerning the data thus far 

 obtained. In dealing with such a complicated mixture we feel that a detailed 

 statement concerning this part of our work will, at the present time, contribute 

 too little that is definite to justify its publication. 



During the past year we have been fortunate to have the cooperation of 

 Dr. A. C. Chibnall, whose previous experience in studying the proteins of 

 the leaf with Professor S. B. Schryver, of London, was of value in supplement- 

 ing our own experience with alfalfa. 



