HORSE-BADISH PEROXIDASE 423 



{cf. 2079, p. 179) have drawn attention to the fact that the PZ vahies of 

 Willstatter and Kuhn were far higher than any reported by later workers. 

 For pure crystalhne horse-radish peroxidase (and also for paraperoxidase), 

 Theorell {277 Tj) gives a PZ value of 900-1^200, while Willstatter found one 

 value of 4700 and Kuhn and co-workers (lOlG), a PZ of 3400, for a preparation 

 containing only 0.009% hemin, i.e., 1/15() of the hematin content of pure 

 peroxidase. The tendency has been to dismiss the values of Willstatter and 

 Kuhn as erroneous (cf. Theorell, 277S). If we transform them into molecules 

 of hydrogen donor oxidized per liter per second by one mole of enzyme, we 

 find, from Kuhn's values, a value of about 3 X 10* moles liter-' sec.-', 

 which agrees much better with the values found by Chance {424-) for the 

 oxidation of leucomalachite green (3 X 10^ to 4 X 10") or of ascorbic acid 

 (1.8 X 10*), than the values for pyrogallol oxidation by the pure enzyme 

 (about 10^); Willstatter {3094) found pyrogallol to be oxidized more rapidly 

 than leucomalachite green. This suggests that a mediator for the oxidation 

 of pyrogallol may be removed during the purification. On the other hand, 

 the necessity of a mediator for the peroxidative oxidation of ascorbic acid, 

 claimed by Szent-Gyorgyi {1375,2725, cf. also 2743), is not evident from the 

 experiments of Chance (424) or Keilin and Mann {1502). 



In an interesting investigation Chance (422,424) has shown that 

 peroxidase action with a low concentration of hydrogen peroxide 

 proceeds as postulated by the Michaelis-Menten theory of enzyme 



action : 



ki k, 



E + S^ES-^E + S' 



k: 



according to the equation : 



k, ki 



(Fe) + H2O2 ;^ (Fe • H2O2) + H2D -^ (Fe) + 2 H2O + D 



k2 



H2D = hydrogen donor = leucomalachite green. The rates of for- 

 mation and breakdown of the hydrogen peroxide compound with and 

 without hydrogen donor were measured directly by a modified 

 Hartridge-Roughton flow technique (423,426). The shift of the Soret 

 band caused by the formation of the hydrogen peroxide compound 

 was measured by means of mirror oscillograph recordings, compen- 

 sation being made for a rather high absorption of malachite green in 

 this region by using two filters with maxima of transmission at 370 

 and 430 mn, respectively, the transmissions of which were affected 

 equally by the malachite green absorption. The formation of the 

 malachite green was measured at 570 m/u. The following values were 

 found: ki = 1 X 10^ moles liter"^ sec."\ k2 = 0.2 sec."\ k2/ki (true 

 dissociation constant) = 2 X lO'^ M, k3/(H2D) = 1.8 X 10^ moles 

 liter"^ sec."' for ascorbic acid, 3 X 10^ moles liter"' sec."' for leuco- 



