(509 ) 
compared as to their intensity of action on indican at different 
temperatures, for which notable differences have been found. No 
other group of enzymes is known to lead with equal ease and 
certainty to the determination of these relations as this group of 
the indigo-enzymes. 
The experiments were conducted as follows. Of solutions of about 
0.5 pCt. imdican!) 10 ec. were passed into equal test-tubes selected 
for the purpose. After heating them to the required temperature in 
a large beakerglass, arranged as waterbath, with thermoregulator and 
thermometer, the enzyme was added and the temperature kept 
constant. 
After a few hours the tubes were taken out, alcalised and the 
indoxyl oxidised by strong shaking, then acidified, by which a very 
fine, equally divided, purely blue precipitate of indigo-blue is obtained, 
which allows colorimetrically to establish the intensity of action 
with sufficient exactness. 
It proved wholly indifferent whether in these experiments use 
was made of the indican of Indigofera or of Polygonum. Evidently 
it is the same in both plants. 
Great attention should however be paid to the degree of acidity 
of the indican solutions. The most favourable enzyme action was 
observed at the rate of about 0.5 cc. normal acid per 100 ce. liquid. 
An increase of the acid to 2 cc. slackens the reaction notably ; 
likewise the addition of alkali to feebly aikaline. Acid salts, as 
kaliumbioxalate and kaliumbiphosphate, act in the same way as 
free acids. 
The quantities of the enzymes employed for the experiments 
amounted to 2—60 milligrams of finely powdered crude enzyme per 
10 cc. of the 4/, pCt. indican-solution. 
First of all was now established the maximum temperature at 
which the action of the enzymes ceases entirely, that is, where the 
enzymes are nearly suddenly destroyed. For Indigofera this maximum 
is at 75° C., but when using a great deal more enzyme a feeble 
action could still be observed at 78° C. which however quite ceased 
at 80° C. For Polygonum the maximum temperature is at 55° C., 
and in large quantities a feeble action was still observable at 60° C. 
For this determination the tubes were placed, for Indigofera, at 
12°.5, 75°, 78° and 80° C.; for Polygonum, at 52°.5, 55°, 58° and 
60° C. For both enzymes the action at the rising of the temperature 
diminishes very quickly near the maximum. In a similar way were 
') Stronger solutions give no more exact results. 
