( 497) 
ningen, and kindly procured by Mr. van LOOKEREN CAMPAGNE. 1 
also received from Mr. HAZEWINKEL of Klaten, Java, perfectly well 
preserved extracts of Indigofera in tins, together with crude enzyme 
prepared from this plant. 
2. Preparation of the Enzymes. 
For this preparation I followed the method pointed out before 
(le. pag. 124). The plants are rubbed fine in a mortar under 
alcohol and during the rubbing the alcohol is a few times renewed. 
In the beginning alcohol of 96 pCt. is taken, which is sufficiently 
diluted by the juice of the plant, but afterwards some water is added 
as otherwise the chlorophyll-pigment cannot be completely extracted 
from the granules. I suppose this must be explained by the strong 
water-attracting power of the alcohol, which produces from the 
protoplasm a proteid, impervious to the chlorophyll pigment and 
possibly to the alcohol itself, but which, by water, becomes again 
permeable. In this operation the indigo-enzyme is precipitated 
in the cells and this occurs so quickly that the indican, which is 
soluble in alcohol has disappeared before its decomposition can set 
in. As by this method the chlorophyll is completely extracted by 
alcohol, a colourless product is obtained, which, after drying, first 
at 37° C. and then at 55° C., is a snow-white powder, directly, or 
after further pulverising, fit for enzyme experiments. In stoppered 
bottles I have kept such preparations for months without observing 
any decrease of activity !). 
As, in the preparation of the indigo-enzyme from Polygonum tinctor ium 
decomposition of the indican occurs much more easily than with 
Indigofera, it is necessary, in order to get colourless preparations 
from this plant, to procecd with greater precaution and to kill the 
protoplasm more quickly. This is done by taking only a small 
quantity of leaf substance at a time for the rubbing in the mortar 
so that the alcohol can penetrate in a few seconds. With Indigo- 
fera much larger quantities of leaves may be taken, without fear of 
obtaining preparations coloured by indigo. 
As I could not point out by the ammoniac-experiment, the presence 
of free indoxyl in Polygonum leaves, I thought at first that the 
1) The loss of activity in enzyme preparations may be compared to the loss of 
germinating power in plant-seeds. If they are kept in complete absence of water, 
both, the activity of enzymes and the germinating power of seeds, will last an 
unlimited length of time, 
