II 



as before, come to the conclusion that such is not the case. My primitive un- 

 certainty was caused by the very unequal acceleration of the oxidation of indoxyl 

 solutions by different powders spread on the surface. So the oxidation is some- 

 what furthered by the crude enzyme of woad, and very strongly, by that of 

 ImUgofera leptostachya, but by boiling, the crude enzymes are by no means deprived 

 of this property. By a minute comparison of the behaviour of crude indoxyl 

 solutions prepared from isatan and indican, with purified ones 1 ), I ascertained 

 that, both in the crude enzymes and in the crude indoxyl solutions, there are 

 present soluble and insoluble chemical compounds, which influence the quickness 

 of the indoxyl oxidation, but which are not destroyed by enzyme poisons and by 

 heating, and which accordingly have not the nature of enzymes. 



Crude isatase has neither an oxidizing action on pyrogallol, hydrochinon, 

 and guajac emulsion. 



Though thus oxydase is wanting in the crude isatase, there is present in it, 

 as in all such like powders, prepared at random from higher plants, peroxydase 

 (leptomine of Rac ib o rski) 2 ), that is the enzyme which, in the presence of 

 hydrogen peroxyd, colours guajac emulsion blue. But indoxyl is by no means 

 oxidized by it to indigo. 



6. Nekrosis. and Nekrobiosis. 



Living tissues can die off in two ways : by necrosis, that is the dying of 

 the protoplasm with simultaneous destruction of the enzymes, and by necrobiosis, 

 in which the protoplasm dies, but the enzymes remain active. The phenomenon, 

 formerly described by me as the blue stripe in partly killed woad-leaves, on 

 the confine of the living and the dead portions, which both retain their green 

 colour, reposes accordingly on necrobiosis. The action of isatase on isatan explains 

 this phenomenon satisfactorily and renders my former hypothesis of alkali for- 

 mation at the dying of the protoplasm superfluous. 



The simplest way to perform the experiment is to kill the tip of a young 

 woad-leaf in a Bunsen flame, or in the vapour of boiling water, then to allow 

 the leaf to remain at ordinary temperature, by which in the said part alone indigo 

 precipitates. If the chlorophyll pigment is extracted with alcohol, then both the 

 living and the dead parts become colourless, the portion between them blue. 

 The phenomenon ist best distinguished in young woad-leaves; in older leaves 

 with a higher acid percentage, it is hardly to be observed because the acid renders 

 the isatase inactive. 



In various other plants, too, nekrobiosis causes formation of pigments. If 

 these pigments are brown or black, and if the experiment is performed in the 

 usual way with the leaves of these plants, then the coloured stripe may become 



') Besides from woad I prepared indoxyl by decomposing in a closed bottle a 

 4 pCt. indican solution with indigo enzyme at 60 C. Moreover Mr. H. ter Meulen 

 had the kindness to prepare for me in the Chemical Laboratory of the Polytechnical 

 School indoxyl solutions in chemical way. The purified indoxyl was always obtained 

 by ether extraction. 



2 ) Berichte der Deutsch. Botan. Gesellsch., Bd. 16, pag. $2, 119, 1898. 



