32 



in 20 cc. of water and 0.5 gram of nicotine tartrate added. This mix- 

 ture was digested for two days at from 50° to 60° C. (122o to 140° F.) 

 in a large iiask, holding about 500 cc. of air, to enable oxidation to 

 go on. An addition of a small amount of thymol prevented bacterial 

 growth. A small U tube, holding 10 cc. of dilute chemically fjure sul- 

 l^huric acid of 0.2 i:>er cent, was attached to the flask. The examina- 

 tion of this acid after two days with ^essler's reagent indicated that 

 ammonia was present, but the colorimetric comi^arison showed that 

 the amount was hardly more than 0.1 milligram. However, the amount 

 of ammonia formed but not volatilized was much larger, as indicated 

 by the strong reaction obtained after the addition of a little potassium 

 carbonate to the mixture and warming for a short time in order to liber- 

 ate the ammonia in the form of carbonate from other less volatile salts.^ 

 Thus there can be no doubt that the tobacco peroxidase can attack 

 nicotine Avith formation of ammonia, but this i)rocess is exceedingly 

 slow. Indeed, the sweating, lasting fully eight weeks, can diminisli 

 the nicotine content on an average only by about one-third.-^ 



What the x)roducts of destruction of nicotine are besides ammonia 

 can be determined only when the purified enzyms and a pure nicotine 

 salt serve in large quantities for the exj^eriment. It may be men- 

 tioned, however, that the writer has examined in vain an aqueous 

 extract of fermented tobacco for nicotyrin and nicotinic acid — known 

 oxidation i^roducts of nicotine. 



The writer has now fully established the presence of oxidizing 

 enzyms in the tobacco leaf.^ That such enzyms can exert a powerful 

 action upon certain compounds, leading even to the formation of car- 

 bonic acid, is known.^ Oxidations produce heat, hence it can safely be 

 inferred that the so-called tohsicco fermentation consists in the activity 

 of oxidases, while the curing of tobacco consists in the combined work 

 of oxidases, diastase, and peptase. As the use of the term "fermen- 

 tation" might lead in this case to an entirely erroneous conception, the 

 writer proposes "oxidizing enzymosis" or "oxidizing euzymation" as 

 correct scientific designations. 



There has already been mentioned an interesting case of oxidase 

 action in a technical branch, viz, the preparation of the Japanese lac. 

 Furthermore, in the manufacture of the natural indigo bacteria are 

 not concerned (Molisch), but simply an oxidizing enzym (Breaudat). 



1 The oxidase might have exerted a more powerful action on the nicotine tlian the 

 peroxidase. 



2A control experiment was made with a colorless peroxidase solution (p. 29) upon 

 highly diluted free nicotine at the ordinary temperature, in order to observe whether 

 a brown solution is produced by a change of the nicotine, but the mixture remaiued 

 colorless after one day under these conditions. However, it may be mentioned that 

 nicotine, when exposed a long time to air and light, will turn brown. 



^ He has already pointed out (p. 15) that there is sufficient access of -air possible 

 to enable oxidation in the tobacco piles. 



•* The further inference is certainly justified that certain basic compounds might 

 thus give up their nitrogen in the form of ammonia. 



