440 CARNEGIE INSTITUTION OF WASHINGTON. 



At room temperatures 0.3 per cent NaOH solution extracted about 

 5 per cent of the alfalfa solids and about 7 per cent of the nitrogen. 

 The precipitate produced by adding a slight excess of HCl to this 

 extract was a mixture of protein and carbohydrates. It contained 

 nitrogen equal to only 55 per cent of protein and yielded furfurol 

 equivalent to a content of 7.54 per cent of pentosans. Although 

 only a very small part of the residual nitrogen was extracted by dilute 

 aqueous NaOH solution at room temperature, nearly all of the re- 

 mainder was readily dissolved by boiling for a few minutes with 60 

 per cent alcohol containing 0.3 per cent NaOH. This removed 

 about 17 per cent of the alfalfa solids and 39 per cent of its nitrogen. 

 This extract, which was dark brown in color, yielded a voluminous 

 precipitate containing 22.8 per cent of the total alfalfa nitrogen. 

 This doubtless consisted mostly of protein, because the dried prepara- 

 tion contained 14.4 per cent of nitrogen (ash-free) and gave charac- 

 teristic protein reactions. Unlike the corresponding product from 

 the alkaline aqueous extract, this precipitate was dissolved readily 

 in a slight excess of acid or alkali. Obviously some change was 

 caused by heating with the alkaline alcohol, otherwise this protein 

 would have been dissolved by the aqueous alkali previously employed. 

 Whether this protein exists in the plant cells as a conjugated protein 

 which is thus hydrolyzed, or as an insoluble salt-like compound with 

 some non-protein substance, possibly the pigment, which latter is 

 altered during treatment with hot alkaline alcohol and is thereby 

 rendered soluble, remains for further investigation to determine. 



The filtrate from this precipitate contained about 60 per cent of 

 the solids and 40 per cent of the nitrogen dissolved by the alkaline 

 alcohol. More than half of these solids was precipitated by normal 

 lead acetate and recovered by treating the precipitate with an excess 

 of HCl and alcohol. Although a considerable part of the extracts 

 containing this substance was not treated with lead acetate, almost 

 3 per cent of the total alfalfa solids was thus obtained. A superficial 

 examination of this substance shows it to be much like the alcohol- 

 soluble product extracted from the "colloid precipitate" with acid 

 alcohol, which perhaps is related to the flavone pigments. 



The intimate association of this brown pigment with the protein 

 is striking and we are inclined to believe that future investigations 

 will show that in the cell these are chemically combined. 



The residue, after thus extracting with the several solvents, con- 

 tained 30.3 per cent of the total dry solids of the alfalfa plant, but 

 only 5.3 per cent of its nitrogen. Since indifTusible proteins can not 

 be extracted so long as the cell walls remain intact, the small amount 

 of nitrogen found in this residue shows that the cells had been rup- 

 tured to a greater extent than we had anticipated when this work was 

 first undertaken. 



