936 
This might give a good explanation of the symbiose experiment, 
supposing that Actinomyces produces homogentisinic acid from tyrosin 
and that the symbiotic bacterium oxidises this acid to melanin. Taken 
for granted that these two processes are due to two separate enzymes, 
this conception may be called ‘the two enzymes theory” of the melanin 
production. 
In order to obtain more certainty regarding the correctness of 
this suppositon, [ made some experiments with the soda salts of the 
homogentisinie acid (C,H,O,) and compared the results with the 
conversion of the calcium and soda salts of the gentisinie acid 
(C,H,O,). Both substances I owed to the Chemical Laboratory of the 
Technical University, the homogentisinie acid as lead salt, which 
I converted into the soda salt, the gentisinic acid in free state. Both 
behave towards microbes in a corresponding way, but the gentisinie 
acid oxidises more greater difficulty. 
I also received from Professor PrEKELHARING the lead salt of homo- 
gentisinic acid, prepared from urine, but this could not be distinguished 
from the other. 
At the preparation with these substances of neutral or feebly 
alkaline agar plates, on which the oxidising microbes were to be grown, 
the difficulty arose that already during the heating at the air a brown 
colour appeared, which was not the case when cold. It could, 
however, with certainty be stated that, as was expected, Actinomyces 
produced no pigment from these acids; on the other hand, the 
symbiotic bacterium gave a dark brown colour, which may finally 
run into jet-black. As this bacterium produces some alkali, it 
might seem doubtful whether this alkali might be the cause of the 
more intense pigment production, or if any oxidising enzyme, produced 
by the bacterium, were active in this case. By cautiously neutralising 
the existence of an oxidase, which diffuses in the agar to a relatively 
great distance from the bacterial colony, could be ascertained. It is 
clear that the thus found enzyme might be called ‘‘homogentisinase”. 
It will be seen by and by that it also occurs in higher plants and 
perhaps corresponds to the common laccase. 
The formerly described Microspira tyrosinatica (le) living in the 
sea and in sewagewater, oxidises tyrosin directly to melanin without 
intervention of any other organism. That this is done here also by a 
vigorously active tyrosinase is easily shown with the form living in 
the sea, the bacterium, when killed by chloroform, being still able to 
cause the melanin reaction. | think it is proved now, that also in this 
case the tyrosinase consists of two enzymes, as it is possible with 
Microspira to oxidise the homogentisinie acid to a dark pigment. 
a 
