That pigment production in this case is difficult in liquid media, whereas 

 Microspira tyrosinatica, which I described earlier '), produces it as readily in liquid 

 as in solid media, is perhaps owing to the general propriety of Actinomyces to grow 

 but slowly in solutions, probably in consequence of the little tension of the dissolved 

 oxygen. Microspira, on the other hand, is as a true water microbe, evidently better 

 adapted to that tension. 



Theory of the melanin formation 2 ). 



In physiological chemistry it is generally accepted that at the tyrosin reaction 

 from the tyrosin first originates homogentisinic acid, ammonia and carbonic acid after 

 the formula 



C 9 HnNO 3 + O 3 = C 8 H 8 4 + NH 3 + CO 2 



Tyrosin Homogentisinicacid 



and that only afterwards by a new oxidation the homogentisinic acid is converted 

 into melanin. 



This might give a good explanation of the symbiose experiment, supposing that 

 Actinomyces produces homogentisinic acid from tyrosin and that the symbiotic bac- 

 terium 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 supposition, 

 I made some experiments with the soda salts of the liomogentisinic acid (CgHsOO 

 and compared the results with the conversion of the calcium and soda salts of the 

 gentisinic acid (C 7 H 6 O4). Both substances I owed to the Chemical Laboratory of 

 the Technical University, the homogentisinic 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 gentisinic acid oxidises more greater difficulty. 



I also received from Professor Pekelharingthe lead salt of homogentisinic 

 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, pro- 

 duced 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. 



') These Proceedings, XIII, 1066. 



) For the literature see Czapek, Biochemie der Pflanzen. Bd. 2, p. 462 and 478. 

 1905. Abderhalden, Physiologische Chemie, p. 362 and 365, 1909. 



