35 
quence. But there are also conditions in the usual treatment of cigar 
tobaceo which may injure the catalase. Thus no trace of it was found 
in one sample of sweated tobacco from Connecticut of 1897. In most 
cases, however, catalase is retained in the sweated cigar tobacco for a 
much ionger time than either oxidase or peroxidase. 
The comparative tests for a and / catalase were carried out in the 
following way: The air-dried samples were very finely powdered after 
removal of the midrib. After remaining twenty-four hours in an exsic- 
cator 2 grams were extracted for five hours with 40 ¢. c. of water at the 
ordinary temperature with frequent shaking, and then filtered through 
filter paper. Twenty cubic centimeters of the filtrate were mixed with 
5 c. c. of neutralized commercial hydrogen peroxide. The development 
of oxygen by the soluble or 6-catalase commences a few seconds after 
mixing. Then the extracted tobacco powder was, after washing, sus- 
pended in 30c. c¢. of water and 10 ¢. c. of hydrogen peroxide added. 
The oxygen here developed was due to the action of the insoluble 
or a catalase. The proportion of the developed oxygen gradually | 
decreased in all cases after a short time, since the hydrogen peroxide 
in the concentration applied oxidized and killed the enzym rather soon. . 
The reaction was promoted by moderately shaking the flask. 
In those cases in which after a rapid development of oxygen gas an 
early stop was noticed a second dose of hydrogen peroxide was added, 
which was repeated as cften as found necessary until the reaction had 
been carried on for a given time. 
It will be noted from the method of testing just described that the 
data relating to 6-catalase are obtained from 1 gram of tobacco; those 
relating to a-catalase from 2 grams. 
Thus far no data are known that would enable us to calculate the 
absolute weight of catalase from a certain volume of oxygen developed 
in a given time by the decomposition of hydrogen peroxide. But quite 
generally we might make the following distinction as to relative quan- 
tities. The amount of a-catalase is small when under the above- 
described conditions less than 50 ¢. ce. oxygen are developed in fifteen 
minutes by 2 grams of tobacco; it is moderate when between 50 and 
200 c. ec. are developed, and high when the volume rises above 200 ¢. ¢. 
As to /-catalase the amount may be called small when less than 20 
ce. c. of oxygen are developed in fifteen minutes by 1 gram of. tobacco; 
moderate, at 20 to 100 ¢.c., and high, when the volume rises above 100. 
