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eaves which has not been completely extracted by alcohol. +) If 
now lime-water is added cautiously so as not to interfere with the 
action of the enzyme, but just sufficient to keep the reaction alkaline, 
a filtrate is obtained which does not give the isatin-soda reaction 
unless the liquid is previously acidified. As already stated, this 
reaction also fails when an excess of Nas HPO, is added to the 
acid indoxyl solution. 
Whether on adding isatin to an acid solution of indoxyl a con- 
densation product may be formed (on account of the acidity) which 
yields indirubin in the presence of alkalis, of whether isatin acts 
on a possibly existing lime-indoxyl compound in a different manner 
is still an open question. 
If we like to make ourselves independent of this phenomenon in 
a simple and practical manner, this may be done by adding a 
decent quantity of ferrous sulphate, which causes the end reaction 
to be acid without affecting the action of the enzyme. On filtering 
and extracting the residue with alcohol, the indican is obtained in 
solution. The FeSO, should be allowed to act upon the indican 
for 20 minutes. If in this experiment the leaf powder is used and 
if the same has not been extracted too much with alcohol, the liquid 
yields afterwards a filtrate which is quite free from iron provided a 
sufficiency of the powder has been used; this is capable of absorbing 
10—20 per cent of its weight of FeSO, That the Fe SO, has 
not caused any reduction may be seen from the fact that on shaking 
with milk of lime, filtermg and neutralizing, a solution of indican 
is obtained which is perfectly identical with the one to which 
Fe SO, has not been added. 
The variations in the reactions of the fermentation liquid before 
and after oxidation are also very interesting. VAN ROMBURGH pre- 
tended it to be acid, vAN LOOKEREN and VAN DER VEEN pretented 
it to be alkaline. But as the first named tested the liquid immediately 
after the fermentation and the others did so after the oxidation, 
the difference in opinion is easily explained. 
1 also found that in the first ease the liquid was acid. This 
liquid is now, however, treated with air and in consequence one 
of the dissolved substances (indoxyl) is oxidated to indigo, a substance 
which is quite indifferent to litmus. Now, a good deal depends 
on the question: how does a solution of pure indoxyl behave towards 
bases and acids. 
!) 20 grams of the enzyme powder treated at the ordinary temperature with 2000 cc. 
of water and a little chloroform yielded a liquid requiring 20 ce, of lime-water for 
neutralization, 
