380 CARNEGIE INSTITUTION OF WASHINGTON. 



nitrogen found before hydrolysis. After hydrolysis the proportion of free- 

 amino nitrogen is about 50 per cent greater than before, which may, or may 

 not, mean that peptides are present in the juice. 



Examination of the basic nitrogen by Kossel's method for separating argi- 

 nine, histidine, and lysine showed that about 16 per cent was precipitated by 

 silver nitrate and baryta, but no evidence of the presence of arginine or lysine 

 was obtained. The color reaction with diazobenzene sulphonic acid, as well 

 as the small precipitate produced by HgS0 4 , indicated the presence of a 

 little histidine, but as both of these reactions are given by other nitrogenous 

 substances, they cannot be accepted as proof that histidine is actually present. 



The filtrate from the silver nitrate-baryta precipitate yielded no lysine 

 picrate, but a relatively considerable amount of a much more soluble picrate, 

 which has not yet been obtained in sufficient quantity for indentification. 



The freshly expressed juice of the alfalfa plant is strongly colored and its 

 color for the most part remains in solution after adding 53 per cent of alcohol. 

 This solution, when concentrated, appears black by reflected light, but clear 

 ruby-red by transmitted light. A part of this color is removed by shaking 

 with iso-amyl alcohol, and this is presumably present in the free state. When 

 the acidified aqueous layer is again shaken with iso-amyl alcohol a somewhat 

 larger quantity is removed, probably set free from salt-like combination. 



When the aqueous layer is gently hydrolyzed by boiling with very dilute 

 hydrochloric acid an insoluble product separates, in highly hydrated clumps, 

 which dissolves in absolute alcohol with a characteristic deep brown color. 



By shaking the filtrate from this latter product with normal butyl alcohol, 

 or with iso-amyl alcohol, a further considerable amount of substance is 

 extracted having the color characteristic of that previously extracted by amyl 

 alcohol. The color and properties of this coloring matter resemble those 

 characteristic of flavones, but as yet we have not obtained convincing chemical 

 evidence that these alfalfa coloring substances actually belong to this group. 

 Such data as we have secured show that these flavone-like substances are 

 present in relatively large amount, chiefly combined with protein in the in- 

 soluble fractions of the alfalfa and with other substances, as yet unidentified 

 in the soluble fractions. Further investigations of these interesting products 

 are in progress, which, if successful, may contribute facts of importance .to a 

 knowledge of the chemistry of the cell. This latter surmise is supported by the 

 fact that the colorless cells of the yeast plant, after hydrolysis with dilute acid, 

 yield colored solutions when shaken with normal butyl alcohol which can 

 not be distinguished by the eye from those similarly obtained from the alfalfa 

 press juice. This observation indicates that these colored substances are not 

 necessarily concerned in the photosynthetic processes of the leaf. 



Besides these colored substances, amyl, or butyl, alcohol extracts from the 

 products of hydrolysis of the solids of the alfalfa juice a large amount of 

 hydrochlorides of basic substances, possibly largely amino-acids, which ren- 

 der the isolation of colored substances in a state of purity a matter of great 

 difficulty. However, a beginning has been made in dealing with the complex 

 mixture of compounds contained in the juice of this leaf, and it now appears 

 to be only a matter of time and patience to obtain an insight into the chemistry 

 of the plant cells which promises to yield much that is new and interesting. 



