12 



still much more marked than in the woad. Particularly fit for this demonstration 

 are the leaves of Pyrus communis, Trollius, Aeonitum, Asarum, Salix purpurea, Populus 

 nigra and several other species, which at necrobiosis turn of a jet black and at 

 necrosis remain green. Pear-leaves especially are recommendable for the experiment: 

 the enzyme in them is tyrosinase, the nature of the chromogene is unknown, tyrosine 

 it is not. Hence, when preparing a herbarium, the chief thing to keep such plants 

 uncoloured, is to prevent necrobiosis. This frequently happens of itself, as the 

 acid cellsap is so much concentrated in drying, that enzyme action cannot occur; 

 so in the drying of woad-leaves, where the highly sensitive isatase remains in- 

 active. In other cases, to obtain this end, it will be necessary to destroy the 

 enzyme, either by boiling water, or by poisonus vapours. 



Sometimes necrobiosis gives rise to aromatic or stimulant matters, which 

 are present in the plant itself as glucosides, from which they are set free by 

 specific enzymes at the dying of the cells. This fact is well-known regarding the 

 myronates and the myrosine of the Cruciferae, the amygdaline and emulsine of 

 the Amygdaleae, the spiraeine, gaultherine and gaultherase of Spiraea. But it holds 

 good, too, for the cumarine of Asperula odorata, which appears not in it as such, 

 but as a glucoside, which by necrosis continues unchanged and hence can be 

 removed from the plant by boiling, while there is besides in this plant a specific 

 enzyme, which by necrobiosis produces from the glucoside cumarine. This enzyme 

 is not identic with emulsine and differs likewise from gaultherase. In a quite 

 corresponding way the aromas originate from the fruit of the vanilla and the 

 roots of Geum urbanum. 



The comparative study of necrosis and necrobiosis in plants shows the way for 

 the detection of a number of new chromogenes or glucosides and specific enzymes. 



Conclusions. 



Indoxyl occurs not, as I formerly thought, in a free state in the woad but 

 as a loose compound, called by me isatan. 



Isatan is only constant in feeble acid solutions, and is obtained by extracting 

 the woad therewith. It is decomposed, under formation of indoxyl, by alkalies 

 and stronger acids, and in solutions, less acid than 1.5 cc. of normal acid per 

 100 cc., by an enzyme, isatase, which acts the most vigorously at 50 C., and 

 occurs in all parts of the woad-plant. 



Isatan is not decomposed by the indigo-enzymes nor by microbes in as much 

 as the latter do not form alkali. Isatase does not act on indican. 



Isatase is localized in the chromatophores, isatan in the protoplasm, which 

 is in accordance with the formerly described localisation of the indigo-enzymes 

 and of indican. 



If woad is extracted without acid, so that the isatase can act, or with dilute 

 alkalies, e. g. V 2 pCt. solution of dinatrium phosphate, indoxyl is produced. 



The necrobiotic stripe in partly killed woad-leaves results from the action 

 of isatase on isatan. 



