29 
for twenty-four hours. To the filtered extract an equal volume 
of ether was added, which caused a heavy brown semi-liquid 
substance to separate out. This substance was, by treatment 
with cold absolute alcohol during several days, converted into 
a hard white brittle mass, which, on being suspended in cold 
water became soft and spongy, and again hardened, when placed 
in alcohol. By thus alternately washing with water and with 
alcohol, the preparation was purified as far as possible, and 
ultimately, after the last treatment with alcohol, it was washed 
with ether, dried in a vacuum at room temperature, and ground 
to powder. 
Estimation of Nitrogen in Protein.— 1 gm. protein was 
boiled with 20 c.c. concentratd sulphuric acid (nitrogen free) and 
potassium sulphate till the liquid was quite clear, when potassium 
permanganate was added in excess and the ammonia estimated 
by distillation with caustic soda. 
RESULTS.—Ex pt. 1. 15°9 per cent. nitrogen. 
Expt. 2. 16 ” ” 
These results are slightly lower than those obtained by 
Kjeldahl, whose protein contained 17°25 per cent. nitrogen. 
This powder was then used for testing the proteolytic activity 
of the fungi or preparations of these in the following manner :— 
In each case three flasks were employed, each containing 
accurately the same quantity of protein, enzymatic preparation, 
and water. To one of these flasks tannic acid and a trace of 
sodium acetate were added at once, whilst the other two were 
placed at 50° C. for twenty hours, when they each received the 
same quantity of tannic acid solution and sodium acetate as the 
first one. 
The effect of the tannic acid is to precipitate the proteids, 
proteoses, and peptones present so that the filtrate will only 
contain nitrogenous compounds of a simpler constitution than 
these. The amount of nitrogen found in the filtrates from the 
flasks, which were kept at 50°C., compared with that contained 
in the filtrate from the flask that was precipitated at once, will 
be an indication of the degree of proteolytic activity, and the 
results obtained expressed in c.c.’s of deci-normal ammonia may 
be used for comparison if the conditions as time, temperature, 
concentration, etc., are kept uniform. 
Preliminary Experiments.—In order to test the methods, 
the following three determinations were carried out, using the 
preparations obtained in the manner already described from :— 
Polyporus sulphureus, P. squamosus, Merulius lacrymans. 
In each case *5 gm. of the enzymatic preparation was dis- 
Pas ie aa 
