Irreversible Inliihition of Catalase 243 



theories (see Lemberg and Legge, 1949). Keilin and Hartree (1945) on the 

 basis of their finding that in the presence of a continuous supply of hydrogen 

 peroxide catalase could oxidize various low molecular weight alcohols, 

 proposed that the function of catalase was that of a peroxidase. The results 

 presented above indicate that erythrocytes contain a substance or substances 

 that can act as catalase donors, thus supporting the 'peroxidase theory' of 

 catalase action in red blood cells. Indeed, it was shown that the relative lack 

 of inhibition of catalase by AT in rat erythrocyte haemolysates (Margoliash 

 and Novogrodsky, 1958) is due to the presence of a dialysable catalase donor 

 in erythrocytes. This donor is probably a sulfhydryl substance since N-ethyl- 

 maleimide completely blocks its activity with respect to catalase. It should, 

 however, be noted that the fact that AT does cause the irreversible inhibition 

 of catalase in the liver and kidney of laboratory animals (Heim et al., 1956) 

 while the injection of a catalase donor such as ethanol prevents these inhibi- 

 tions (Nelson, 1958) shows that both liver and kidney do not contain a 

 significant concentration of catalase donors. Catalase therefore, cannot be 

 acting to any large extent as a peroxidase in these tissues. 



Heim et al., (1956) first observed that AT injected into rats and mice 

 causes a large and rapid decrease of the catalase activity of liver and kidney 

 suspensions, but has no effect on blood catalase activity, even on continued 

 administration. Feinstein (1958) and Feinstein and Dainko (1959) have found 

 that the concentration of AT in erythrocytes following the injection of the 

 drug is only about one-eighth that in the liver or kidney. This result might 

 possibly be due to rapid equilibration of the drug present inside the red 

 blood cells with the fluid outside the cells. In such a case washing the red 

 cell suspension could decrease considerably the apparent AT content before 

 it was determined, as compared to that of unwashed liver or kidney homo- 

 genates. Moreover, since the reaction of AT with catalase in the presence 

 of hydrogen peroxide is irreversible (Margoliash and Novogrodsky, 1958), 

 one would expect a decrease of the catalatic activity of the blood in vivo 

 even though at lower concentrations of the drug it occurred more slowly 

 than in the liver or kidney. The normal occurrence of a catalase donor in 

 erythrocytes shown by the present experiments, affords another explanation 

 of the lack of effect of AT on blood catalase activity in vivo. 



The kinetic equation developed for the irreversible inhibition of catalase 

 by the AT group of inhibitors (Margoliash et al., 1960) in the presence of 

 hydrogen peroxide and a catalase donor, indicates that the rate of inhibition 

 depends on the ratio of the donor concentration to the hydrogen peroxide 

 concentration. The results of the experiments in which the dilution of the 

 haemolysates and the concentration of the hydrogen peroxide source were 

 varied qualitatively verify this conclusion. A quantitative verification which 

 would require an immediate accurate estimation of the hydrogen peroxide 

 concentration has so far not been undertaken. It should be noted that 



