16 
The presence of diastase in the tobacco leaf was demonstrated by 
the writer as follows: Four large healthy leaves were well crushed in 
a mortar with some sand and pressed. ‘The filtered juice was precipi- 
tated with alcohol, and the precipitate, after being washed with alcohol 
of 90 per cent and pressed between filter paper, was digested with 10 
c.c. of highly diluted starch paste at 60° C. (140° F.). The iodine test, 
applied to a drop of the liquid on a white porcelain plate, showed that 
the starch gradually diminished and disappeared after two hours. 
Leaves kept for eight days in darkness and near death by starvation 
did not, when so examined, show any diastase content. This would 
probably also prove the case with leaves from the curing barn. The 
destructive effect of oxidase on diastase (Woods) comes into play here. 
PROTEOLYTIC ENZYM IN THE TOBACCO LEAF. 
The decrease of protein in the tobacco leaf, while curing is going on, 
can be ascribed only to a proteolytic enzym. The amount of it, how- 
ever, is probably small, since the coagulable albumin contained in 
the juice of tobacco leaves decreases very slowly; even after several 
weeks leaves taken from the barn may show some of it. It may 
be possible that the oxidizing enzyms injure this enzym, as they do 
the diastase, and thus prevent an accumulation beyond mere traces © 
in the fresh leaf. Many authors have encountered difficulties in 
attempting to prove the presence of proteolytic enzyms in green leaves 
and fruits. One of the causes of this failure may be that blood fibrin, 
or coagulated egg albumen, has been used for the test; while certain 
proteolytic enzyms may exist which can easily peptonize dissolved 
albumen without being capable of dissolving fibrin or coagulated 
albumen. Indeed, there may exist a great variety of proteolytic 
enzyms. Thus, Bourquelot and Hérissey have shown an enzym to exist 
in fungi which is capable of easily digesting casein, while it can not dis- 
solve fibrin. The writer’s experiments with casein and fibrin, in 
attempting to prove the presence of a proteolytic enzym in the fresh 
tobacco leaf, were not conclusive, no visible solution of these proteins 
taking place when digested for several days at 40° C. The method of 
Fermi and Buscaglioni also proved unsuceessful. These authors recom- 
mend a gelatin solution that solidifies at the ordinary temperature. The 
liquid to be tested is, in the presence of an antiseptic substance, left 
upon the gelatin in the test tube for several days. The presence of 
even a trace of proteolytic enzyms will soon be indicated by the lique- 
faction of the gelatin below the line of demarcation. The principal 
objection which may here be raised is that proteoiytic enzyms may 
exist which can act on gelatin only at a higher temperature—that is, 
above the temperature 20° to 22°, at which the usual gelatin prepara- 
tion liquefies by melting. 
In the following experiment the test recommended by Martz was 
adopted. ‘Ten fresh and 10 starved ‘eaves, of as nearly the same size 
