248 BACTERIOLOGICAL AND ENZYME CHEMISTRY 



order to determine the amount of indigotin in the cake indigo, 

 and of indican in the original plant, older processes all giving 

 conflicting results. The methods finally devised were briefly 

 as follows : 



For the determination of indigo, one gram of indigo was 

 converted into a tetrasulphonate by means of fuming sulphuric 

 acid, and the tetrasulphonate precipitated as potassium salt by 

 addition of potassium acetate. The precipitated salt can be 

 filtered off, dissolved in water and oxidised by potassium 

 permanganate of known strength. 



The determination of the indican in the leaf depends on 

 the fact that, when brought into contact with a substance 

 known as isatin, a pure crystalline compound known as 

 indirubin is formed, by the combination of indoxyl and isatin, 

 according to the following equation : 



C 8 H 7 ON + C 8 H 5 2 N = C 16 H 10 2 N 2 + H 2 



Indoxyl Isatin Indirubin 



It was found that the best method of extracting the 

 indican from the leaf was by means of acetone. 



Armed with these exact methods of analysis, Perkin and 

 Bloxam have been able to show that the yield of indigo 

 obtained in the native process by no means corresponds 

 with the theoretical yield which should be obtained on the 

 basis of the indican present in the leaf. Several by-products 

 are present in natural indigo, particularly indigo brown, the in- 

 vestigation of which indicates that it is formed by a secondary 

 reaction from indican. Their examination of a specimen of 

 leaf from an indigo-yielding plant from Sumatra has shown 

 that, under certain conditions, twice as much indigo may 

 be present, as in the best leaf from Java. 



The decomposition of indican they agreed to be due to 

 the action of an enzyme present in the leaves, rather than 

 to the activity of bacteria. In this they confirm the opinion 

 of other investigators, notably Beyerinck, Bergtheil, and 

 Rawson. They agree with Beyerinck that the enzyme is 



