pigment is removed. After filtering and drying the crude isatase is obtained as 

 a white, feebly acid powder in which, of course, all substances not soluble in 

 alcohol are present, hence, all the other enzymes of the woad too. As the enzyme 

 is quite insoluble in water it can be purified by extraction with destilled water, 

 by which the other enzymes, at least those that are soluble, disappear. Solvents 

 for the isatase itself I have not yet found. 



As the woad, like the cabbages, is very rich in gypsum, the crude isatase 

 contains so much of it that to remove it with destilled water is troublesome. 

 I have therefore, in order to answer the question, whether in the action of isatase 

 on isatan perhaps a sulphate is produced, as in the splitting of kalium myronate 

 by myrosine, prepared in the following way isatase free from gypsum. Woad 

 leaves cut fine were rubbed down in destilled water, then pressed out, and the 

 remaining matter extracted with water until the filtrate proved free from sulphuric 

 acid. Then the chlorophyll pigment was removed by alcohol and the remaining 

 matter dried and powdered. 



Though the thus obtained preparation is poor in enzyme, because this is 

 localized in the chlorophyll granules, which during the pressing of the leaves are 

 for the greater part also pressed out, it is still sufficient to bring about a strong 

 isatan decomposition. As was to be expected, sulphates were not thereby set free. 



The isatase is spread through the whole woad-plant ; it occurs as well in 

 the growing parts as in full-grown roots, stemps, leaves, and flowers. So the 

 distribution is another than that of the isatan, which is wanting in all full-grown 

 parts, and is the more accumulated in growing roots, stems, and leaves, the 

 younger they are. Another distribution also than that of the indigo-enzymes in 

 the indican plants, which are only found in the parts rich in indican. 



On the other hand the distribution of the isatase within the cell itself, 

 corresponds with that of the indigo-enzymes: both are localized in the chroma- 

 tophores. The isatan has also, in the cell, a localisation corresponding with that 

 of the indican, for in as much as can be inferred from micro-chemical experi- 

 ments, both are found in the living protoplasm of epidermis, mesophyll and other 

 parenchymatous tissues. For establishing the localisation of isatan and isatase in 

 the cell, the same way can be followed which I formerly pointed out for detecting 

 the indican and the indigo enzymes 1 ). 



As regards the isatan, for this end, not too thin microscopic sections of young, 

 vigorously growing stems or leaves are put in a boiling mixture of hydrochloric 

 acid and isatine; by the acid indoxyl is separated, which produces, with the isatine, 

 red crystal needles of indigo-red, localized in the protoplasm. More difficult to 

 observe, but still, I think, quite convincing is the precipitation of indigo-blue, as 

 small granules, in the living protoplasm, when the sections, in a living state, are 

 put in a mixture of boiling hydrochloric acid and ferrichlorid. Remarkable is the 

 strong accumulation of isatan in the epidermis cells, and especially in the hairs 

 found on the young leaves. 



The localisation of isatase in the chromatophores can be demonstrated in 

 two ways. Either little bits of the easily loosening epidermis of woad-leaves, or 



') Indigofermentation p. 579. 



