AMINOPHENOLS AND PHENYLENEDIAMINES 591 



tatively.* iV",iV,A'',iV'-Tetramethyl-29-phenylenediamine is often a rather 

 potent enzyme inhibitor. In addition to the results above, it has been found 

 to inhibit several enzymes at concentrations around 0.01 mM, e. g., the 

 nitrate reductase of the halophilic Vibrio costicolvs (Robinson, 1954). It 

 can also serve as an electron donor and has been used to study terminal 

 phosphorylations in the respiratory sequence (Rowland, 1963 a). P:0 

 ratios around 1.0-1.3 were obtained with rat liver mitochondria. On the 

 other hand, it had been shown by Park et al. (1957) that liver mitochondria 

 oxidize this substance to a deep purple free radical, which at 0.2 mM un- 

 couples phosphorylation associated with the oxidation of /?-hydroxybutyr- 

 ate, dropping the P:0 ratio from 2.4 to 0.5. The uncoupling is antagonized 

 by Mg++, EDTA, and glutathione, and in each case there is a visible re- 

 duction in the purple color. There is little or no evidence to suggest that 

 the actions of tetramethyl-p-phenylenediamine are in any way related to 

 reactions with SH groups. 



There has been a good deal of work and argument relative to the effects 

 of p-aminophenol on xanthine oxidase, since Bernheim et al. (1937) claimed 

 that no other dehydrogenases in liver suspensions are inhibited at con- 

 centrations of p-aminophenol which inhibit almost completely. Neither 

 j)-benzoquinone nor o-aminophenol was found to possess this selectivity of 

 action. Using oxidized p-aminophenol (i. e., solutions allowed to stand for 

 a short while), Berheim and Berheim (1938) found 50% inhibition at a 

 concentration of 0.013 mM (this is the total concentration and we do not 

 know the concentration of p-quinoneimine). They emphasized that this is 

 a selective and somewhat unique type of dehydrogenase inhibition. Con- 

 sistent results could not be obtained with milk xanthine oxidase and it 

 was shown that milk contains some substance which inactivates the qui- 

 noneimine. Ball (1939) found that it requires 7 mM p-aminophenol to in- 

 hibit milk xanthine oxidase 77%. Richert et al. (1949) in general confirmed 

 the results of the Bernheims and also found that the inhibition does not 

 occur in the presence of substrate, indicating that p-quinoneimine (or 

 some other oxidation product) reacts readily at the substrate site. However, 

 Doisy et al. (1955), using a more purified preparation of rat liver xanthine 

 oxidase and chicken liver xanthine dehydrogenase, observed very little 

 inhibition by p-quinoneimine at 0.2 mM; although this was not explained, 

 it might mean that the actual inhibitor is a complex of jj-quinoneimine 

 with some liver component, or some component altered by the p-quinone- 



* It would seem reasonable in experiments with reactive and tightly bound inhi- 

 bitors acting on enzymes in homogenates to pay more attention to the kinetics of 

 the inhibition. Likewise in situations in which the active inhibitor is being formed 

 from a precursor it would be advisable to study the development of the inhibition 

 with time. More significant results would also be obtained if some effort toward purify- 

 ing enzyme preparations was made. 



