[Vor. 5 
264 ANNALS OF THE MISSOURI BOTANICAL GARDEN 
by addition of distilled water to a volume of 750 сс., and the 
liquid finally obtained after straining and filtering through 
a Buchner funnel made up to 1 liter, plus 1 per cent toluol as 
antiseptic. 
In the preparation of the enzyme powder the melted mass 
was extracted for 18 hours with three times its volume of dis- 
tilled water, plus 1 per cent chloroform, after which it was 
strained and then filtered through a Buchner funnel. The 
solid residue was ground for 20 minutes with an equal volume 
of fine quartz sand and further extracted for 24 hours with 
2 volumes of water, after which it was likewise strained under 
pressure and filtered. The final solutions thus obtained were 
of a heavy colloidal nature. By this freezing and grating 
method, with subsequent grinding and secondary extraction, 
it is believed that the plant cells are thoroughly ruptured and 
that the extraction is likely to be as complete as it is possible 
to make it without special apparatus for comminution and 
pressure. The tissue pulp after grinding showed under the 
microscope a very thorough disruption of the cells. The col- 
loidal nature of the solutions made their filtration under pres- 
sure a slow process, so that during it, as well as during the 
time necessary for the later filtration of the enzyme precipi- 
tate, all solutions were preserved with 1 per cent toluol. 
Precipitation of the enzyme-protein complex was effected 
with 95 per cent alcohol, in the proportion of three volumes 
alcohol to one of plant extract. The precipitate obtained after 
treatment for fifteen minutes was collected under pressure 
upon filter paper, and quickly dried with an electric fan at 
room temperature, after which the papers were stored in 
glass-stoppered bottles for future use. Later in dissolving 
the dry enzyme material it was found that the greater por- 
tion on each filter was easily removed by moistening it and 
scraping the surface with a safety razor blade. A moist 
chamber or any vessel with a flat bottom was found satisfac- 
tory for the work. The residue on the filters was obtained 
after trituration in a mortar, by solution in water, and strain- 
ing through cloth. 
