339 



as otherwise the chlorophyll-pigment cannot be conipletely extracted froni the grami- 

 les. 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 per- 

 meable. 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 conipletely extracted 

 by alcohol, a colourless product is obtained, which, after drying, tirst at 37" C. and 

 then at 55» C, is a snow-white powder, directty, 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 Polygoniint tiiictoriiim decom- 

 position of the indican occurs much more easily than with Indigofera, it is necessary, 

 in order to get colourless preparations from this plant, to proceed with greater pre- 

 caution and to kill the protoplasma 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 Indigofera much larger quantities of 

 leaves may be taken, without fear of obtaining preparations coloured by indigo. 



As I could not point by the ammoniac-experiment, the presence of free indoxyl in 

 Polygonum leaves, I thought at first that the difference was to be explained by ad- 

 mitting that the enzyme of Pülygonum is more soluble in water than that of Indigo- 

 fera and so, during the extraction could perhaps in higher concentration act on the 

 indican. But the experiment showeil that this is not the case. Neither can the acid 

 reaction of the juice of Polygonum, caused by kalium bioxalate. account for this dif- 

 ference, as the addition of this salt, kalium biphosphate, or of a little acid, to the 

 materials used for the preparing of the enzyme from Indigofera, produces no 

 change in the course of the phenomena. The addition of asparagine is likewise with- 

 out effect. Nor is the explanation to be found in the relation of both enzymes to the 

 temperature. I have so come to the conclusion that in Polygonum part of the indican 

 is decomposed by the direct action of the living protoplasm itself. This part is 

 however small, and by quickly immersing in boiling water the protoplasm is killed 

 before it causes decomposition. 



In the preparation of indigo-enzyme from Phajus grandiüorus nothing particular 

 is observed. But we saw before that the decoction method produces no indican but 

 indoxyl from this plant. 



As the figure below shows that the enzyme of Phajus becomes inactive already 

 at a lower temperature (67" C.) than that of Indigofera (75° C), I must admit that 

 also in the leaves of Phajus katabolism exists together with enzyme action and 

 that also in the geaves of Phajus katabolism exists together with enzyme action and 

 that, at the immersion in boiling water, simultaneously with the dying af the proto- 

 plasm, this katabolism causes a vigorous indican decomposition^). Hence Polygonum 



') The loss of activity in enzyme preparations may be compared to the loss of 

 germinating power in plant-seeds. It 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. 



') In § 3 P- 513. will be demonstrated tliat all the indican is localiscd in the rotoplasm. 



22* 



