29 



When the fresh leaf of the tobacco is rapidly dried at about (j(P C. 

 (14U- F.) aud theu moistened again and ke])t in a moist atmosphere, the 

 veins and their finest ramifications turn brown in about half an hour, 

 while the mesophyll and epidermal cells remain green even after a 

 week. Further investigations on this point will be made later. In 

 the fresh leaf, both oxidizing enzyms, the oxidase and the peroxidase 

 occur in the ribs and veins as well as in the parenchyma, the indica- 

 tions being tiiat they are more abundant in the ribs than in the i)aren- 

 chyma. The bundle sheath aud sieve tissue give the most intense 

 reaction on the oxidase, while the reaction on the peroxidase sets in 

 quickly and with about uniform intensity in all the cellular tissues. 

 The growing point and youngest leaves contain an esj)ecially large 

 quantity of the oxidase. A section through the stalk shows oxidase 

 only in the sieve tissue and bast parenchyma, while peroxidase also is 

 contained in the i:)ith.^ Both enzyms are found in the root, the former 

 more in the central and the latter in the perii^herar parts aud also in 

 the flower. The stigma of the pistil and the stigmatic fluid also show 

 strong reaction upon oxidase. 



The two oxidizing enzj^ms are also contained in the young tobacco 

 plants. Several dozen of these, measuring on an average not more 

 than 3 to 4 cm. from the tii> of the root to the plumula, were rubbed in 

 a mortar with a little water and some sand. The filtrate gave a very 

 intense reaction for oxidase,- and after this was destroyed by warming 

 to 70^ C. an intense reaction for peroxidase also. 



A colorless clear solution of the tobacco peroxidase can be obtained 

 in the following manner: A number of Iresh tobacco leaves are well 

 crushed in a mortar, with the addition of sand and some dilute alcohol 

 of 30 per cent. This mixture is pressed and the turbid liquid directly 

 mixed with three times its bulk of strong alcohol. After standing two 

 hours the mixture is thrown upon a filter and the filter contents, after 

 being washed with some alcohol, extracted with about four to six times 

 its bulk of water at the ordinary temperature, heated for a minute to 

 70^ C. (158- F.), and filtered. This clear, colorless filtrate gives no 

 indication of the oxidase, but a very intense reaction for peroxidase. 

 When this solution is compared with that of the juice of fresh tobacco 

 leaves it is easy to decide what result is caused by the oxidase alone. 



To determine w hether the tobacco oxidase bears more resemblance 

 to the tyrosinase or the laccase a few drops of freshly ex^jressed juice 

 of normal tobacco leaf were added to 2 cc. of a cold saturated tyrosin 

 solution, but even after four hours no characteristic darkening of the 



^In order to observe the localization of the peroxidase small pieces of the tissue 

 are treated with strong, but not absolute, alcohol for three minutes at 70^ C. (158- F-). 

 Thus the oxidase is killed and can not interfere with the tests for the peroxidase. 



-The indophenol reaction did not turn out satisfactorily, only a weak violet-blue 

 color resulting. On the addition of hydrogen peroxide, however, an intense blue 

 reaction was at once obtained. 



