(514) 
2. The formation of indigo was much stronger when a current 
of air was passed through the liquid in the presence of an alkali. 
3. The copper-reducing power of the liquid obtained by the action 
of the air and subsequent filtration was much larger than that of 
the original liquid which had not been treated with the extracted 
leaf-powder. 
4. After a short action the.liquid began to show fluorescence: 
This phenomenon was observed more readily if a current of carbon 
dioxide was passed through the liquid; no indigo is then deposited. 
5. The indigo-yielding substance could be removed by agitating 
the liquid with ether and chloroform. 1) 
6. ‘This substance was identical with the one present in the 
liquor after the so-called technical fermentation had ceased. 
7. The action was destroyed by a number of enzyme poisons. 
8. Addition of alcohol up to a certain concentration stopped 
all action, but on diluting with water action recommenced. 
9. Heating above a certain temperature definitely destroyed the 
action. 
10. Cooling below a definite temperature retarded the action as 
long as that temperature was maintained. 
The observations recorded in 3, 7, 8, 9 and 10 together with 
the solubility of the active principle in brine and glycerol put it 
beyond doubt that we are dealing here with an enzyme. 
In agreement with this I found that emulsin acts on the liquid 
in precisely the same manner. This experiment was made on the 
advice of Dr. vAN RomBURGH. 
As the enzyme may differ from emulsin I will, provisionally, call 
it ,indimulsin”. This indimulsin does not act below 5°. In a dry 
condition it gradually becomes inactive at higher temperatures, but 
its power is completely destroyed after being heated for half an 
hour to 125°. Its solution in brine becomes totally inactive at 
88—92°. 
No great change of action is observed in liquids containing 10 
per cent of alcohol by volume, but in the presence of 25 per cent 
all action ceases. Several other enzyme poisons such as mercuric 
chloride, copper sulphate, lead acetate, alkalis, acids, sulpher dioxide 
temporarily or definitely retard the complete or partial action of the 
enzyme according to their nature and quantity. 
1) Dr. van RompurGH was the first to prove that the indigo-yielding substance con- 
tained in the so-called fermentation liquor could be extracted with chloroform or ether. 
