1.901.] H. H. Uiiun-^Thr Enzymes of the Tea Leaf. J50 



There is therefore no difference in the sensitiveness of the substan- 

 ces producing the two reactions to heat, and both are destroyed by an 

 exposure for three minutes in aqueous solution to 83 to 85° C. and th.e 

 destruction commences below 80° 0. One may therefore say, — and the 

 conclusion is confirmed by the result of a large number of other attempts 

 (to be afterwards referred to ) to isolate the two apparently different 

 enzymes, — that we have no evidence of the presence of more than one 

 ferment oxidising guaiacum in the tea juice. I am inclined to attribute 

 the difference in reaction to the presence of part of the fei'ment in the 

 juice as zymogen or pro-enzyme, which is brought into definite action 

 by the Hydrogen Peroxide. The distinction between the reactions with 

 and without Hydrogen Peroxide, therefore, remains a convenient one, 

 and I have kept it up throughout the present work. 



To estimate the amount of these enzymes 5 c c of the clear liquid 

 prepared as above, were mixed with an equal volume of alcohol, and 

 then 10 drops of a solution of guaiacum resin in alcohol added, and the 

 colour measured in a Lovibond's tintometer. The measurement must 

 always be made at the same length of time after the addition of 

 guaiacum tincture. After the intensity of the colour has been noted, 

 •5 c.c. of a 10 volume solution of Hydrogen Peroxide were added and the 

 colour again measured. The intensity of colour gives a rough measure 

 of the relative amount of enzyme in the several cases, and has been 

 utilised for this purpose throughout the present work. It depends on 

 the fact that the colour given is all but absolutely a pure blue, and 

 lience one can neglect the amount of any other colour which may be pre- 

 sent in the liquid, and merely take the intensity of the blue as showing 

 the relative amount of the oxidase. 



It seems almost impossible to prepare the enzyme pure in a dried 

 condition. If the clear solution prepared as above be reprecipitated 

 with alcohol, a mass is produced very active towards guaiacum solution, 

 etc., but if an attempt is made to dry the precipitate at a low tempera- 

 ture its oxidising power rapidly diminishes, and when dry there is 

 hardly any reaction left. The whole of the reactions had therefore to be 

 studied in the solution prepared as above, which in addition to the enzyme 

 contained a certain proportion of gummy, pectic and saline matters. 



The oxidase was very sensitive to the action of acids. A solution 

 of the enzyme was immediately rendered absolutely ineffective in a 

 solution containing '4 per cent, of Sulphuric Acid. '04 per cent, had, 

 however, only very slight effect after 2 hours. 3 per cent of Acetic Acid 

 destroyed the ferment entirely in 2 hours. By Alkalies it was less affected 

 but still was rapidly destroyed. 3 per cent. Ammonia nearly destroyed all 

 action after 4| hours. Caustic Potash of the same strength had little 

 J. 11/21 



