BRITTON CHANCE 



When the pseudo first-order velocity constant for the donor 

 reaction {k^d) is large compared to the corresponding value for 

 the combination of enzyme and substrate {kix), then ki can be 

 computed from the rate of the over-all reaction {dx/dt) and the 

 enzyme concentration {e) : 



-t. = - ^. (7) 



xe at 



With the guaiacol test under these conditions, values of ki 

 of 0.89 X 10^ M-^ sec.-i at 20° G. and 1.03 X 10^ M"! sec.-i 

 at 30 °C. were obtained with pure horse-radish peroxidase (25). 

 These values agree very well with the value obtained in the flow 

 apparatus at 25° G. (0.9 X 10^ Af-i sec.-i (7)). 



Other studies on the over-all reaction of peroxidase do not 

 permit us to assume as small a lifetime for any unknown inter- 

 mediates in the peroxidase reaction as in the case of catalase 

 presented above. In the guaiacol assay just mentioned a life- 

 time of 2 millisec. suffices, while the work of Mann (51) at 

 higher peroxide and guaiacol concentrations indicates that 

 6 X 10~^ sec. suffices. The classical purpurogallin assay 

 method allows 10~* sec. for the breakdown of any unknown 

 intermediate (8) . 



Thus the reaction of enzyme and substrate to form complex 

 I is second order at reaction times of about 10~* sec* and, in 

 view of the similarity of the reactions of catalase and peroxidase 

 with peroxide, it is unlikely that an unknown intermediate 

 precedes complex I in either case. 



REACTION KINETICS WITH OXIDIZING AGENTS 

 OTHER THAN PEROXIDE 



George has found that compounds spectroscopically similar 



* A considerable error may be introduced into the determination of ^i 

 from the over-all reaction kinetics if there is any possibility that a direct reac- 

 tion of the oxidizing agent and the hydrogen donor takes place by any pathway 

 in addition to that identified with ki. An example of such a possibility is the 

 attempt to determine ^i for the reaction of peroxidase and hypochlorous acid 

 by using the guaiacol test (37). 



316 



