Accordingly it is necessary to perform the reaction in a very feebly acid 

 solution, and to judge of the results without other precautions than a thorough 

 aeration. I have not been able hitherto to answer the question after the nature 

 of the matter, which at the isatan-splitting, most probably is set free beside the 

 indoxyl. Pressed yeast, produces in woad-extract, heated with crude isatase at 

 30 C., more alcohol and carbonic acid, than in the same extract without isatase 

 (in the proportion of 8:5), so that in the first there must certainly be formation 

 of sugar capable of fermentation. But this sugar results, probably not from the 

 isatan, but from the action of other enzymes, present in the crude isatase, on 

 glucosides or carbohydrates, present in the isatan-solution, such as myrosine on 

 myronates, and diastase on granulose. 



The process of the decomposition cannot be studied with Fehling's cupric 

 solution, as the isatan is decomposed by the alkali. 



That to Schu nek's indiglucine no value can be attached follows from i. 



In order to state the influence of heating on the isatase action, the experi- 

 ments were arranged as described elsewhere for the indigo-enzymes 1 ), with the 

 difference, that for the above reasons, alkalisation and subsequent acidification 

 are here omitted. The were finely powdered enzyme is shaken in an experiment 

 tube with the isatan solution, and in a water bath, at determined temperature, 

 heated a determined number of minutes. There are always performed two experi- 

 ments at the same time, so that a colorimetrical comparison of the produced 

 indigo is possible, e. g. at 48 C. and 50 C., or at 40 Und 60, 45 and 55, etc. 

 The best results were obtained with dilute isatan-solutions, which are brought, 

 as exactly as possible, to an acidity of 0.5 cc. normal per 100 cc. of liquid, and 

 with so little enzyme, that the complete conversion was very slowly accomplished 

 and took about half an hour. 



The optimum for the action was found at 

 48 to 50 C., but could not be determined 

 more accurately as differences of two 2 C. 

 produce no distinct colorimetrical difference. 

 At 70 C. the enzyme is completely destroyed. 

 The minimumlimit is low, far below o C., as 

 is seen in the figure. Noteworthy is the slow- 

 ness with which the intensity of action decreases 

 at decrease of temperature, and the quickness 

 with which it takes place when the temperature 

 rices. So the action at 10 and at o C. respec- 

 tively is as strong as at 60 and 60.5 C. 



On other substances but isatan isatase seems not to act; it has certainly 

 no action on indican, neither could I decompose with isatase the potassium indoxyl- 

 sulphate in horse urine. 



When judging of these experiments it must be kept in view that other enzymes 

 are present in the crude isatase, which may produce substances not indifferent 

 for the isatase action. So mention was made above of the presence of the myrosine 



10 20 30 40 50 60 70 C. 

 Action of isatase on isatan. 



') Indigofermentation p. 586. 



