10 



and diastase in the crude isatase preparations, and below I will refer to the presence 

 of peroxydase. 



5. Extraction of indoxyl from the woad-leaves. 



Once acquainted with the chief properties of isatase and isatan, it is possible 

 at will to extract isatan or indoxyl from the woad. Though in my former com- 

 munication I spoke already of the indoxyl extraction, my being unacquainted with 

 isatase prevented me from doing this with perfect clearness. 



As alkalies produce indoxyl from isatan the extraction of woad-leaves therewith 

 will at every temperature produce indoxyl. But by the presence of alkalies the 

 indoxyl becomes so very oxidisable and then passes at the air so quickly into 

 indigo, that the air, ever present in the leaves, causes a great portion of the 

 indoxyl to get lost. On the other hand, neutral or feebly acid solutions oxidize 

 much more slowly; it is true that also in these finally all the indoxyl passes into 

 indigo, but such solutions keep unchanged for hours at room temperature and 

 are fit for studying the properties of the indoxyl. 



The chief point for obtaining such neutral or feebly acid indoxyl solutions 

 from woad-leaves, is during the extraction to furthei the isatase-action, conse- 

 quently to do the very thing which I formerly indicated as essential for the 

 indoxyl extraction from indican plants, where all depends on the action of the 

 indigo-enzymes. With woad this can best be effected by keeping the extraction 

 temperature between 45 and 50 C., and by addition of chalk or of a salt of 

 feebly alkaline reaction, partly to neutralize the acid of the leaves. Thus a good 

 result is obtained by entirely filling a wide-mouthed stoppered bottle with young 

 woad-leaves, and pouring over them a Va pCt. dinatrium-phosphate solution (Na~ 

 H PO 1 + 12 H 2 O), heated at about 50 C., removing the air as much as possible, 

 closing the bottle and allow it to stand at 40 C. for 24 hours. By decantation 

 and pressing the leaf matter, boiling and filtering, all the indoxyl is obtained in 

 an amphoteric solution, which is somewhat brownish, but is excellent for indoxyl 

 experiments. The presence or absence of undecomposed isatan is observed by 

 precipitation with lead acetate, whereby the indoxyl remains dissolved. The indoxyl 

 can also be shaken out with ether and in the remaining liquid sought with isatase 

 for isatan. Not decomposed isatan remains also in the filtrate, when the indoxyl 

 is allowed to oxidize at the air and the indigo-blue is filtered off. 



The ether solution of the indoxyl, obtained by shaking it out of the extract, 

 can be evaporated at low temperature at the air, by which the indoxyl is left 

 behind as a liquid soluble in water, which can be coloured by different impurities. 

 Though the watery solution of this purified indoxyl is inconstant at the air, 

 its oxidation to indigo-blue proceeds slowly enough for studying the influence 

 \\ich different substances exert on this process. 



Various circumstances have induced me to put anew the question, whether 

 in this oxidation an oxidizing enzyme is active 1 ). After much doubt I have finally, 



') Mr. Brcaudat erroneously asserts (Compt. rendus T. 127, p. 769, 1898 and 

 T. 128, p. 1478, 1898) that in the extracts of Isatis indigo-white occurs, which, by an 

 oxydase is turned into indigo-blue. 



