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EMERSON: BLACKENING OF BAPTISIA TINCTORIA 623 
alcohol and left for an hour or two, to precipitate chlorophyl, 
proteins and other matters; this precipitate, dissolved in dis- 
tilled water, was filtered again, and this solution used for the 
experiments. 
Kinds of extracts made.—I\t was found that as soon as the 
pounded leaves were exposed to the air they turned from green to 
black in less than half an hour, whereas those not so much exposed 
kept green. So two kinds of extracts were made, one in which 
the pounded leaves were at once covered with water, when only 
those near the top became black, another in which the pounded 
leaves were left for twenty-four hours to get as black as possible 
in the air, and then extracted as usual. The first method gave 
a dark brown solution which kept growing darker so that it was 
difficult to see the color-reactions ; the second gave a clear amber- 
colored extract, free from the products of oxidation, easily show- 
ing the slightest reaction to color tests, but otherwise acting like 
the first. In order to get purer enzyme solutions, a portion of the 
Original solution was treated with three times its bulk of alcohol 
and filtered, dissolving the precipitate in water, and this process 
was repeated, thus securing second and third extractions much 
lighter and clearer and rather more sensitive than the first. 
In general 95 per cent. alcohol was used for the first treat- 
‘Ment, and 80 per cent. for the second and third. Although some 
absolute alcohol was also used for the first extraction and gave 
“much heavier precipitates, it was too expensive to use in the 
necessary quantities. There was little difference in the amount 
of precipitate between 95 per cent. and 80 per cent. alcohol. 
Tests for enzymes. — When the extracts were ready they were 
tested by taking about ten cubic centimeters in a tube and adding 
a few drops of solution of gum guaiac (2 grams of guaiac in 100 
Cubic centimeters of absolute alcohol) ; then the solution turned 
an opalescent blue, becoming slightly darker when shaken, and 
aclear blue on adding 95 per cent. alcohol, which dissolves the 
€xcess of guaiac. This showed that an oxidase was undoubtedly 
Present. Upon the addition of one drop of hydrogen peroxide, 
the solution turned a very deep blue, and this color would last 
Several hours without fading. This second reaction suggested the 
Presence of a peroxidase. These two tests were used in all the 
