19 
This filtering, however, proceeds so slowly that a brown coloration is | 
meanwhile developed by oxidation. By mixing the juice, however, 
with an equal quantity of 90 per cent alcohol, or by rubbing the lam- 
ina in a mortar with an addition of 50 per cent alcohol—whereby pro- 
teins and other matters are precipitated—a larger amount of clear fil 
trate ready for the test is soon obtained. 
It is often preferable to dry the leaf at a temperature of from 40° 
to 45° C., grind it to a fine powder, extract this for a short time with 
cold water, and apply the tests to the filtrate. It need hardly be men- 
tioned that the juices of the pith or of the main ribs of the leaves can 
serve directly for the tests. They filter easily and do not darken as 
readily as the juices from the lamina. 
In regard to the guaiac blue, it may be mentioned that it sometimes 
fades out soon after its formation. This may be due to tannin and 
other reducing compounds; it seems not to be due to another enzym. 
ARE THE TOBACCO OXIDASE AND PEROXIDASE KINDS OF PROTEINS? 
These enzyms belong neither to the coagulable albumins nor to the 
nucleoproteids. The latter are precipitated by dilute acids, but since 
the juice of green tobacco leaf is of moderate acid reaction, it can not 
contain ordinary nucleoproteids in solution. Even upon the addition 
of 1 per cent of acetic acid to the juice, no nucleoproteids are precipi- 
tated. It, as Spitzer has recently claimed, the peroxidase of mammalia 
is a nucleoproteid, we must assume a considerable difference between 
the peroxidase of mammalia and that of tobacco, and probably of other 
vegetal sources. 
The following tests show, further, that oxidase and peroxidase do not 
belong to the coagulable albumins. The freshly expressed and filtered 
juice of the tobacco leaf yields upon heating to 54° C. a copious coagu- 
lum of albumin, while the filtrate therefrom gives a strong reaction for 
oxidase and peroxidase. On further heating this filtrate to 65° ©. for 
a second, another but a very insignificant coagulation is observed. The 
filtrate of this precipitate still yields both reactions, but it may now be 
heated to the boiling point without giving acoagulum. Further, when 
green tobacco leaves are dried at a temperature of 50° C., pulverized, 
and extracted with ten times the weight of cold water for thirty min- 
utes, a filtrate of weak acid reaction is obtained which contains mere 
traces of albumin. Nevertheless, it gives the peroxidase reaction with 
guaiac, even after heating for a second to 85° C. (not to 90° C.). This 
solution was heated for a few seconds to a temperature of 66° C. and 
the slight turbidity removed by filtration, after which the cooled filtrate 
was saturated with ammonium sulphate, whereby a dark precipitate 
was formed. This contained the oxidase and peroxidase and a part of 
the brown coloring matter of the original extract, while the filtrate 
yielded no trace of the guaiac reactions. After being freed as far as 
possible from the adhering liquor, by pressing between sheets of filter - 
