410 IX. HEMATIN ENZYMES, II 



according to spectrophotometric estimation. The values for the 

 latter were calculated from the relative affinities given by Keilin and 

 Hartree (1499), on the assumption that the dissociation constant of 

 the cyanide catalase is 8 X 10^^ M. The problem of accounting for 

 this divergence will be discussed below and in Section 4. Phenyl- 

 hydroxylamine and o-aminophenol {2528) as well as p-hydroxylamino- 

 benzene sulfonamide (2535) are also strong catalase inhibitors. 



An inhibition of catalase by carbon monoxide has been claimed by 

 Calif ano (391). Several authors were unable to confirm this (cf. 

 2079, p. 156); it is now clear that pure catalase is not inhibited by 

 carbon monoxide, but Keilin and Hartree (1490,1^99) found a light- 

 sensitive inhibition by this reagent in the presence of such substances 

 as glutathione, cysteine, and azide. 



2.4. Estimation and Enzyme Kinetics* 



Estimation. Highly purified catalase is best determined oxidimet- 

 rically by the method developed by von Euler and his collaborators 

 and extended by Zeile and Hellstrom (3166). For impure prepara- 

 tions the iodometric method of Jolles (14.23) has been recommended. 

 Finally, the oxygen development can be determined manometrically : 

 although this method is the only one available for the estimation of 

 catalase in some tissues, it cannot be recommended for pure catalase 

 (cf. below). 



If the disappearance of hydrogen peroxide is estimated oxidimet- 

 rically it is found that the reaction does not follow a strictly mono- 

 molecular course with regard to hydrogen peroxide. This is largely 

 explained by a slow destruction of the enzyme by hydrogen peroxide, 

 accompanying its catalatic action. 



Sizer (2570) has claimed that the initial rate of development of oxygen 

 from hydrogen peroxide, measured manometrically, is of zero order. His 

 claifti that he measured a more truly initial stage of the hydrogen peroxide 

 decomposition than is measured by the oxidimetric methods is not correct. 

 The deviation of his results from those obtained by oxidimetric methods is 

 probably due to a fault in the manometric method. It has been shown by 

 Theorell and Agner (2780) that it cannot be used for the activity esti- 

 mation of pure catalase solutions, since the oxygen evolution lags behind the 

 disappearance of hydrogen peroxide as measured oxidimetrically {cf. also 

 Roughton. 23G1). 



The kinetics of the destruction of hydrogen peroxide by catalase are 



* The important papers of Chance {Jt25a, 42ob; cf. also ol^b) appeared too late 

 for inclusion in this discussion, but should be consulted. 



