NUTRITION. 439 



to extract the water-soluble constituents almost completely within so 

 short a time that autolytic changes were reduced to a minimum. 



After extracting with water, the chlorophyll, fats, phosphatides, etc., 

 were extracted by cold alcohol and ether and so obtained uncontami- 

 nated with water-soluble substances which also might be soluble in 

 these solvents. 



Dilute NaOH solution was then used to extract the residue, but it 

 was found that only a small part of the nitrogenous substances were 

 removed, although this solvent readily dissolves nearly all known types 

 of protein. After thus extracting with cold aqueous alkali, nearly all 

 of the residual nitrogen was removed by boiling for a new minutes 

 with 60 per cent alcohol containing 0.3 per cent NaOH. 



After these successive extractions the residue, consisting chiefly of 

 fibrous structures, contained only 5 per cent of the total nitrogen of 

 the original plant. It is thus clear that, if indiff usible protein can be so 

 completely removed as to leave so small a proportion of the nitrogen 

 undissolved, practically all of the cells were ruptured and their con- 

 tents thereby rendered available for further critical study. 



Over 40 per cent of the solids of the entire plant were soluble 

 in water, about one-fifth of which was in colloidal solution and 

 could be precipitated by adding about 18 per cent, by weight, of 

 alcohol. This product, which we shall call the "colloid precipitate," 

 contained 11 to 12 per cent of nitrogen, about nine-tenths of which 

 was almost certainly protein which comported itself towards acids, 

 or alkalies, in such an unusual way as to suggest a type of protein 

 hitherto unrecognized except in the spinach leaf. The balance of 

 this precipitate consisted chiefly of calcium salts of phosphoric and 

 organic acids, among the latter being one or more coloring substances, 

 which in the free state were readily soluble in strong alcohol. Possibly 

 these latter are related to the flavone pigments. 



Besides this protein in colloidal solution, which is thus precipitated 

 by quite dilute alcohol, only a little protein could be coagulated by 

 boiling and acidifying the filtrate from which the alcohol had been 

 previously removed by concentrating. By saturating the filtrate 

 from the coagulum with (NH4) 2SO4 a small precipitate with properties 

 characteristic of proteoses was obtained, but the amount of protein 

 of this type present in the juice is at the most relatively small. 



It is thus evident that the proteins of the plant juice present a 

 wholly different picture from that given by those of any physiologically 

 active tissue with which we are at present familiar. 



After the water-soluble constituents had been removed, strong 

 alcohol extracted only about 6 per cent of the total solids of the alfalfa 

 plant. All of the chlorophyll was present in this extract, together 

 with other substances, the nature of which we have not yet attempted 

 to determine. 



