8 



microscopic sections of stems or leaves, all in a living state, can be put in a 

 neutrally reacting woad-decoction, rich in isatan, and heated to ca. 45 C. After 

 some minutes already the chromatophores begin to colour blue; the intensity of 

 colour increases some time, to reach its limit in an hour or so. 



The blue-colouring of the colourless chromatophores of epidermis and stem- 

 pith, is here distinctly to be observed, so that, particularly the fragments of the 

 first, become very interesting preparations. 



The localisation of the isatan in the protoplasm, of the isatase in the chro- 

 matophores, renders their inter-action in the living cell possible without any in- 

 fluence of the acid cell-sap. At the death of the cell, this state will suddenly 

 change and the acidity of the cell-sap determines wheter the isatase can act or 

 not on the isatan. 



In no other plant but the woad I have hitherto been able to detect isatan. 

 I had expected its presence in some short-valved Cruciferae. So in Capsella bursa 

 pastoris, where, in case the root-neck is much hurt, a trace of indoxyl can be 

 pointed out, but here also the enzyme is wanting. Likewise it wants in the indican 

 plants. Also all microbes examined are devoid of isatase. 



4.. Action of isatase on isatan. 



The action of isatase on isatan is, as observed before, only possible in neutra 

 or amphoteric and very feebly acid solutions. In alkaline solutions the obser- 

 vation becomes uncertain, because the alkali itself splits off indoxyl. If the acidity 

 amounts to 1.5 cc. of normal acid per 100 cc. of the isatan solution, the action 

 is much weakened, and at ca. 1.8 cc. of normal acid, there is no more decom- 

 position of isatan at all, which is noteworthy as this percentage of acidity is 

 reached in the cell-sap of older woad-leaves. This does not however exclude isatan- 

 decomposition by the enzyme in the living cell, as the process can be limited to 

 the protoplasm, in accordance with the localisation described. 



As the action of the isatan is judged after the formation of indigo-blue, 

 two chemical processes are involved in it, isatan splitting and indoxyl-oxidation. 

 If the experiment is performed with free access of air, for instance in a thin 

 layer of the isatan solution, with the enzyme floating on it, the indoxyl changes 

 directly into indigo; but if the isatan is decomposed with imperfect access of air, 

 for instance, in the depth of an experiment tube, then it is necessary, during the 

 experiment itself, to render the oxidation of the indoxyl as complete as possible 

 by agitation with air, which does not however always succeed wih sufficient 

 quickness, and so limits the accuracy of the experiment. Of course the liquid can- 

 not be alkalized, because then not only the indoxyl formed by the isatase would 

 become visible, but also the indoxyl set free by the alkali from the isatan not 

 decomposed by the isatase. If the object is to observe the isatase action at a 

 determined temperature, then the enzyme cannot be destroyed at the end of the 

 experiment by heating, but this must be effected by some enzyme poison, as for 

 instance sublimate. 



Addition of acid to render the colour of the indigo-blue more pure must 

 likewise be avoided, in order not to decompose isatan. 



