T. B. Osborne and A. J. Wakeman 249 
The filtrate from the last precipitate was made neutral to litmus 
with hydrochloric acid and evaporated until a large part of the 
lactose was removed by successive crystallizations. The mother 
liquor (780 cc.) was saturated with ammonium sulfate, the pre- 
cipitated proteins were dissolved in water, and an equal volume 
of saturated ammonium sulfate solution was added which pre- 
cipitated much of the protein. This fraction was dissolved in 
water and alcohol added to 74 per cent by volume. The pre- 
cipitate produced when dried over sulfuric acid weighed 2.3 gm., 
Fraction I. 
The alcoholic filtrate from Fraction I was treated with an excess of 
strong alcohol and a little ammonium acetate solution added, 
which caused a practically complete precipitation of the rest of 
the protein. This was washed by centrifuging with 92 per cent 
alcohol and then centrifuged with alcohol of 70 per cent (by 
volume) in which much dissolved. After repeating this treat- 
ment three times a turbid, colloidal solution resulted. On add- 
ing five or six drops of a strong solution of ammonium acetate 
some protein was precipitated. This, Fraction II, was filtered 
out, and when dried over sulfuric acid weighed 0.55 gm. The 
clear brown filtrate (200 cc.) was poured into a liter of absolute 
alcohol, a few drops of ammonium acetate were added, and the 
flocculent precipitate was centrifuged out. When dried over sul- 
furic acid this fraction, No. III, weighed 1.8 gm. 
To determine whether Fraction III which was soluble in rela- 
tively strong alcohol contained the alcohol-soluble protein the 
anaphylaxis reaction was used. The results obtained by H. G. 
Wells were as follows: | 
Sensitizing Dose. Intoxicating Dose. Reaction. 
Alcohol-soluble protein.’ = Alcohol-soluble protein. Died in 15 min. 
“ce “é ; “ec ce (74 ce 10 “cc 
Fraction I. * None. 
c¢ I ; ce iz ce 
Alcohol-soluble protein. Fraction I. . 
“cc (<¢ 3 ce 12 <é 
Fraction III. s i 3 
“ el < Is ¢ 
Ly Ebe Alecohol-soluble protein. © Severe. 
(73 “ce 
43 ITT. Died in 5 min. 
