28 



tobacco peroxidase, occur in the fresh as well as in the recently fer- 

 mented Florida tobacco leaf.^ The former enzym is, however, much 

 more sensitive to heat than the latter, being killed at from 65° to 66^ 

 C. (149° to 151^ F.), while the latter is killed only at from 87° to 88° 

 C. (188.60 to 190.40 y.). 



Dried tobacco leaf (not cured) was finely pulverized, and 1 gram of 

 the powder left with 20 grams of water for one hour at the ordinary 

 temperature. A part of the filtrate was heated for three minutes to 

 550 C. (1310 F.). To about 2 cc. of this liquid were then added a few 

 drops of tincture of guaiac,' whereupon a blue coloration appeared in 

 a few minntes, exactly as in the control case. A second portion was 

 now heated to 65° C. (149° F.) for three minutes, and the test ap])lied 

 after cooling, but only a slight trace of a blue color was noticed after 

 ten minutes. Evidently most of the tobacco oxidase was destroyed at 

 that temperature. 



The killing temperature of the tobacco peroxidase was determined in 

 a similar manner. However, Lere reaction is still obtained with great 

 intensity after the solution is heated for three minutes to 80° C. 

 (1760 F.), but very feebly after heating for one minute to 87° 0. (I880 F.). 



Another reaction for oxidizing enzyms is the so-called indophenol 

 reaction, consisting in the i)roduction of a blue color when an alkaline 

 solution of r^naphthol with paraphenylendiamine is acted upon by an oxi- 

 dase. This reaction must, however, quickly set in and with great inten- 

 sity, otherwise no reliable conclusion can be drawn. Cured and fer- 

 mented tobacco from Florida did not show tbis reaction in a marked 

 manner,-^ but it set in at once upon the addition of a little peroxide of 

 hydrogen. The latter alone will not produce this result in the absence 

 of an oxidizing enzym. 



In the manner above mentioned the writer's investigations have 

 shown that dark tobacco two years old, from Quiiicy, Fla., yielded no 

 reaction for tobacco oxidase, but still a moderate one for tobacco perox- 

 idase, while a sample of light-colored tobacco four years old from the 

 same source yielded not the slightest reaction either for the oxidase or 

 the peroxidase. Evidently these enzyms themselves are gradually 

 changed. From these observations it may be inferred that the cold 

 sweat, or after-fermentation, might thus proceed for about two years 

 and end by the gradual dying off of the oxidase and peroxidase. 



1 French savants were the first to call attention to this difference between the 

 oxidizing enzyms. The names oxidase and peroxidase, proposed by a French savant, 

 are not specific names, but gronp names. There may exist among various oxidases 

 and peroxidases as many differences as there are among protein bodies. Hence it is 

 entirely unjustifiable, at this stage of our knowledge, to introduce one specific name 

 for all peroxidases, as one author has done. 



2 In all these cases freshly prepared guaiac tincture (1:.50) was employed, as old 

 guaiac tincture is unreliable and with peroxide of hydrogen alone will sometimes 

 yield a greenish coloration. 



3 Only a slow and weak reaction was thus develojied. 



