464 5. QUINONES 



where the + charges are the binding loci for succinate. Substances which 

 are not reactive with SH groups (e. g. 9,10-anthraquinone) or not capable 

 of being oxidized to a quinone structure (e. g. resorcinol) are not inhibitory. 

 The inhibition progresses slowly and is not complete in 60 min when quinone 

 concentrations of 0.01-0.1 mM are used. The presence of succinate delays 

 the inhibition and malonate protects the enzyme against quinones, indicat- 

 ing that the SH group attacked lies in the region binding succinate and mal- 

 onate. Since malonate does not protect urease against the quinones, direct 

 reaction of the quinones with malonate is disposed of. Quinonoid substances 

 such as p-aminophenol and jj-phenylenediamine are equally inhibitory. 

 However, actual proof of such a combination as depicted was not obtained, 

 and other mechanisms can be imagined. The role of oxidation cannot 

 be eliminated since oxidants such as iodine, methylene blue, and ferricyanide 

 are inhibitory, and the oxidation need not involve only the SH groups. Cer- 

 tain amino groups may be particularly reactive on an enzyme surface, and 

 Anderson (1961) has shown that inhibition of alkaline phosphatase by qui- 

 nones is accompanied by the liberation of ammonia. Some enzymes, e. g. 

 catalase, which are usually not thought of as typical SH enzymes, may be 

 potently inhibited by quinones (Ogura et al., 1950). In the case of catalase 

 no changes in the absorption spectrum occur during inhibition, so the pro- 

 tein was considered to be the site of the reaction. Typical SH enzymes, 

 e. g. 3-phosphoglyceraldehyde dehydrogenase, are quite sensitive to qui- 

 nonoid substances (Holzer, 1956; Holzer et al., 1956), but the ethylenimino- 

 benzoquinones generally used could react with the enzyme SH groups in 

 a different manner. Furthermore, no direct titration of enzyme SH groups 

 has been carried out with the quinones. One should thus at this time ac- 

 cept the 1,4 addition of enzyme SH groups to quinones with caution, 

 although there is certainly no reason, in view of the rapid reaction of qui- 

 nones with many thiols, to believe that this may not be an important mech- 

 anism. 



Reactive Form of the Quinones 



A good deal of attention has been paid to determining if the quinone, 

 hydroquinone, or semiquinone is the inhibitory species in particular cases, 

 and this is quite important because of the bearing on the possible mech- 

 anisms of the inhibitions. We have pointed out that it is virtually im- 

 possible to assure the presence of only one species in most cases, even 

 though very pure quinone or hydroquinone is added to the enzyme pre- 

 paration, and even though oxygen is carefully excluded in the latter case, 

 since oxidation-reduction reactions with the enzyme or other components 

 of the mixture may occur. However, it is usually possible to obtain reason- 

 ably good evidence for the importance of one or the other species. Fisch- 

 gold (1934) found that p-benzohydroquinone, treated with hydrogen and 

 palladized asbestos to reduce any quinone present, produced no inhibition 



