8 Fractionation of Phosphotungstic Acid Precipitate 



completely negative results. The acetone-insoluble (curative frac- 

 tion) did not contain phosphorus. 



2. These preHminary results suggested repetition of the work on 

 a larger scale. An evaporated alc. extract from loo k. of dry yeast, 

 which was semifluid and amounted to 2,580 gm., was extracted in 

 three portions, each with 4 1. of 10 percent sulfuric acid sol. Each 

 portion was macerated for 24 hr. and then shaken on a machine for 

 10 hr. After standing, oil collected at the top; the clear, dark, 

 colored liquid was siphoned off and finally filtered through a wet 

 folded filter. The oil was washed with 4 1. of water, in each case, 

 and the second extract again siphoned off. The combined extracts, 

 which amounted to 24 1., were precipitated with 50 percent phospho- 

 tungstic acid sol. After standing over night the precipitate was 

 separated by filtration, washed several times with 5 percent sulfuric 

 acid sol., and finally dried. The total amount of precipitate, 2,237 

 gm., was ground in a mortar with 2.5 1. of dry acetone and the fine 

 Suspension shaken for 1 5 min. The insoluble part was separated by 

 filtration, ground in a mortar with acetone, and the liquid filtered. 

 The insoluble residue weighed 124 gm., subsequent treatment with 

 acetone diminishing this quantity to 114 gm., which amounted to 

 5.1 percent of the total phosphotungstate precipitate. 



Acetone-insoluble fraction. The material obtained by the fore- 

 going process (114 gm.) was treated in a mortar with 400 gm. of 

 neutral lead acetate and shaken for 2 hr. The lead phosphotung- 

 state was separated by filtration and twice treated in a mortar with 

 small quantities of lead acetate to complete the decomposition. The 

 combined filtrates and washings were freed from the excess of lead 

 with hydrogen sulfid and concentrated in vaciio (temp. of the bath, 

 26-3 i°C.). During the evaporation a cotton-wool-like substance 

 separated out. After complete evaporation the residue was entirely 

 crystallin, colorless and curative in very small doses. The action 

 was similar to that of the original yeast, but only when all of the 

 residue was given. To the residue a little water was added and the 

 liquid filtered. The quantity of insoluble substance was 7.8 gm. 

 When recrystallized from water, 5.35 gm. were obtained in the form 

 of prisms. These were dried at iio°C., in vacuo; they darkened 

 on heating above 250° and did not melt at 320° (uncorr,). 



