334 



The answer for Polygoniiin and Indigofera lies partly at hand. By the tempera- 

 ture of the boiling-point, the indigo-enzyme has been killed, so the indican caii no 

 more be deconiposed. If slowly dying, on the contrary, the indigo-enzyme can become 

 active and indoxyl is formed '). But the explanation df the second part of the pro- 

 cess, that is the transformation of indoxyl into indigo, — at the same time the only 

 point which for woad, as an indoxyl-plant, requires our attention, — is less clear. 

 I think that the course is as foUows. In slowly dying leaves the indoxyl changes into 

 indigo-blue, because, in this form of death of the cells, some alkali originates. In 

 suddenly killed leaves, on the other hand, alkali-formation does not occur, they do not 

 grow blue, and the indoxyl disappears in another way. 



If in the leaves of indigo-plants the presence of an oxidase, acting on indoxyl, 

 could be demonstrated, this would certainly explain quite well the action of higher 

 and lower temperatures. But, as I said, I could not convince myself of its existence, 

 so that I am necessarily led to the alkali-hypothesis. 



The cause of the great lack of indigo-blue which, as above observed, diminishes 

 the value of the »alcohol-experiment«, lies in the fact, that during the slowly dying tf 

 the leaves at the air, a considerable quantity of indoxyl is lost in an unknown way. 

 And in this circumstance I see one of the reasons why, in woad-leaves, there is pro- 

 duced so much more indigo by the »ammoniac-experiment« than by the »alcohol-ex- 

 periment«, because in the former the leaves die almost instantly, whilst the latter 

 requires much more time. 



With Indigofera, as said above, the »alcohol-experiment« produces hanlly any 

 indigo. I have therefore tried to substitute for it a better one, which is effected in 

 the following way, and by which, also excellent results are to be obtained with 

 Polygonum. 



At the direct action of ammoniac, indican-plants form nn indigo at all, for thereby 

 not önly the protoplasm is killed, but the indigo-enzyme, too, is so quickly destroyed 

 that it cannot decompose the indican. But we can, beforc exposing to the alkaline 

 vapour, decompose the indican and free the indoxyl, by making the plants die by 

 complete exclusion of air, but which in this case should occur in such a way, that the 

 indoxyl remains within the plant itself. Indican-plants turn then into »dead indoxyl- 

 plants« and can in this condition, quite like the living woad, be subjected to the 

 »ammoniac-experiment« with a very good result. 



The simplest way by far to reach the doublé aim of killing the plants by ex- 

 clusion of air and leaving the indoxyl in the cells, is by entirely plunging them into 

 mercury, whereby asphixion foUows with surprising quickness, the protoplasm becom- 

 ing permeable and the indigo-enzyme and the indican mixing together. .^t a proper 

 temperature *)the indican is then decomposed after a few hours and the freed indoxyl 

 remains in the leaf, albeit not exclusively in the cells in which originally the indican 

 was localised. The leaf is then taken out of the mercury, ammoniac-vapour is allowed 

 to act upon it, and at last the chlorophyll is extracted by boiling with alcohol and 



') Also a slow death of the leaves by drying or by frost rcnders tbc protdiilasni 

 permeable and the indigo-enzyme active. 



") The influence of temperature on the actiim nf the indiso-cnzynu- is inUnstiiië, 

 I hope on another occasion to return to it. 



