158 



H. H. Mann— T/ie Enzymes of the Tea Leaf. 



[No. 2, 



(calculated on the dry matter) the difficulty was especially great in the 

 present case. The method finally adopted for isolating and at a latter 

 date for estimating the amount of oxidising enzyme present was as 

 follows: — JO grams of fresh (or 6-6 grams of withered leaf ) were ground 

 np in a mortar till they formed a pulp, and in each case 5 grams of hide 

 powder (pure for analysis) were added and the mass again ground 

 thoroughly together. 50 cuhic centimeters of water were now thoroughly 

 incorporated with the mixture and the whole left for two hours. At 

 the end of this time the mass was filtered quickly through cloth, with 

 pressure, and the residue washed twice with water. It was found that 

 practically the whole of the extractable part of the oxidising enzyme 

 was thus removed. The liquid obtained was now mixed with four 

 times its volume of alcohol, which precipitated the whole of the enzyme. 

 After settling thoroughly, the precipitate was filtered again through 

 cloth, and to the residue 25 to 30 c. c. of water were added. The whole 

 of the enzyme was thus obtained in a small volume which on filtration 

 gave a clear liquid in which various tests could be made. 



The standard test for oxidising enzymes or oxidases is that with 

 guaiacum resin. If an alcoholic solution of this resin be mixed with a 

 liquid containing one of the class of substances under discussion, a blue 

 colour varying in intensity with the quantity of enzymes present will 

 appear after two or three minutes. With the solution from tea leaf 

 prepared as above, this reaction was obtained immediately, and if 

 further a drop or two of Hydrogen Peroxide were added the reaction 

 became very much more intense. It was hence at first supposed that 

 two enzymes were present, the one giving a blue colour without 

 Hydrogen Peroxide, the other only producing the reaction in its pres- 

 ence. If this were the case they ought surely to have different resis- 

 tances to heat, and by this means one ought to be able to separate them. 

 This was found, however, not to be the case. A solution in water 

 containing the oxidases was exposed to various temperatures in each 

 case for three minutes, with the following results : — 





Rr-action with Guaiacum Rrsin. 



• Temperature. 



Without FTydrooren Peroxirle. 



With Hydrogen Peroxide. 



60°C. 



Just as intense as before heating. 



Jnst as intense ns before 

 heating. 



70°C. 



Do. 



Do. 



8o.8rc. 



Slight decrease in intensity of 

 reaction. 



Reaction distinctly lower 

 in intensity. 



SS-SB^C. 



Reaction practically disappeared. 



Reaction almost dis- 

 appeared. 



