31 



trace of coloration is perceptible. The addition of a little hydrogen 

 peroxide to both will increase the difference of coloration still more. 



All these tests were made ar from IS'^ to 20° C. (04.4^ to 68^ F.). A 

 number of other compounds were also tested, such as arbutin, guaiacol, 

 and toluidine, but no decisive reaction was obtained within twenty -four 

 hours at the ordinary temperature. 



There may exist great differences in the amount of tobacco oxidase 

 and tobacco peroxidase produced in different varieties of the tobacco 

 plant and under different conditions. The quantity of each may even 

 differ in the upper leaves fully exposed to the sun and the lower leaves 

 growing mostly in the shade. There may also be formed compounds 

 in certain varieties of tobacco that will more (juickly destroy the 

 enzyms during curing, or fermentation, than in other varieties. Thus 

 considerable difference was goticed in comparing a sample of tobacco 

 from Connecticut with one from Florida. In the fermentation of the 

 former the tobacco peroxidase was almost completely destroyed, while 

 in that of the latter a considerable part was still intact. Moreover, 

 neither the fermented nor the cured Connecticut leaf contained any 

 tobacco oxidase, although it was found in a greenhouse specimen of the 

 fresh leaf. 



It is interesting to note that the best way of bringing the oxidizing 

 enzym to the fullest action i^ossible is that i^racticed in the curing of 

 the Perique tobacco.^ The rolls, or twists, of the tobacco leaves are 

 subjected to a pressure of about 7,000 pounds per square foot to bring 

 the juice from the interior of the cells to the surface. After twenty- 

 four hours the tobacco is taken out and aired a few minutes, which 

 causes a darkening to set in. In this way the juice is reabsorbed by 

 the tissues, whereupon the pressure is again applied. This operation 

 is repeated daily for ten consecutive days, and at longer intervals 

 thereafter. A very dark product is thus obtained, but it is not strong, 

 as the oxidation of the nicotine has been carried very far. 



One of the most interesting features of the sweating of tobacco is 

 the destruction of a part of the nicotine, this part yielding up its 

 nitrogen probably as ammonia, which is indeed a product of the sweat- 

 ing. It was, of course, of importance to prove that the oxidizing 

 enzyms contained in the tobacco leaf can decompose nicotine, and for 

 this purpose 50 grams of cured tobacco from Connecticut which had 

 not yet been subjected to fermentation and showed a strong reaction 

 for peroxidase, but none for oxidase, was thoroughly moistened with 

 water. After two hours 250 cc. of alcohol of 50 per cent was added 

 and the mixture allowed to stand for two days. The liquid obtained 

 by pressing was now mixed with one and a half times its volume of 

 absolute alcohol and the brown-colored precipitate washed upon the 

 filter with some alcohol. After pressing between filter paper, the pre- 

 cipitate, containing a large proportion of the peroxidase, was dissolved 



' Farmers' Bull. No. 60, U. S. Dept. of Agr. 



