Solution Tension and Toxicity in Lipolysis. 263 
amount of indicator was used in each titration (one drop from an ex- 
pansion stopper). The amount of lipolytic activity was measured by 
the number of c.c. of M-20 KOH required to make the preparation 
neutral after digestion. Suitable controls were carried in each test 
and will be described. The indicator was of course added at the close 
of the incubation period. 
Sources and control of error. — The control preparations were exactly 
like the others, except in one particular, namely, the enzyme solution 
used had been boiled over the free flame. Boiling was continued 
only a few seconds, as the activity is practically destroyed at a lower 
temperature. That this treatment was adequate is shown by the fact 
that the controls were neutral after digestion, which means that the 
butyrate was beyond doubt also neutral. 
The individual digestive mixtures were made up in the following 
manner: Six were used for each concentration of the toxic agent, of 
which three were controls. To each vial were first added 2 c.c. of the 
toxic solution of a concentration twice as great as was intended for 
the test. Then to the three controls were added for each individual 
2 c.c. of the doz/ed enzyme solution. Then to the other three individ- 
ually were added 2 c.c. of the uxdoiled enzyme solution. A total vol- 
ume of 4 c.c. was thus present in each individual of every test. 
Then, to each individual were added 0.10 c.c. of neutral butyrate. 
This order of procedure was strictly followed in every test. After the 
incubation the difference in acidity between the controls and the others 
as found by titration was attributed to the activity of the enzyme. In 
the concentration of the enzyme used there was no initial acidity, 
and the boiling left the reaction neutral, so there was no error 
from those sources. If the butyrate had happened to be slightly acid, 
the same amount would have been added to each vial. The butyrate 
was measured accurately from a 1 c.c. pipette graduated to one-hun- 
dredths. To ascertain the full activity of the enzyme a set of six 
mixtures was prepared, in which distilled water replaced the toxic 
agent. 
conditions. — Since it seemed more important to eliminate 
: ace factors than to provide conditions for the maximum 
activity of the enzyme, all of the tests were carried out with the 
_Feaction neutral. Some of the toxic solutions have an initial acidity 
_as, for example, zinc nitrate M-64 is so acid that 3.40 c.c. of M-20 
KOH are required to bring 4 c.c. of it to neutral reaction. In such 
cases the natural acidity of the reagent was not neutralized. It can 
