The oxidation of 4-aminobiphenyl by horseradish peroxidase

The oxidation of the carcinogen 4-aminobiphenyl (4-ABP) catalyzed by the model peroxidase enzyme horseradish peroxidase (HRP) was investigated. 4-ABP served as a reducing cosubstrate for HRP during the enzyme-catalyzed reduction of the synthetic hydroperoxide, 5-phenyl-4-penten-1-yl hydroperoxide, to its corresponding alcohol. Spectral analysis during the incubation of HRP, 4-ABP, and H2O2 showed an increase in absorbance at 230 and 325 nm and decrease at 270 nm, suggesting metabolite formation. Oxygen consumption was not detected in incubations of HRP, 4-ABP, and H2O2. However, oxygen uptake was observed after the addition of glutathione, which indicated that a free radical metabolite of 4-ABP was formed by the peroxidase. The 4-ABP free radical reacted with glutathione forming a glutathionyl radical which, in turn, reacted with and consumed oxygen. HPLC analysis of organic extracts of incubations with HRP, [3H]-4-ABP, and H2O2 showed the formation of one major peak identified by mass spectroscopy as 4,4'-azobis(biphenyl). The addition of glutathione to the incubations decreased the formation of 4-ABP metabolites, suggesting a reduction of the 4-ABP free radical and/or the formation of glutathione conjugates. Subsequent HPLC analysis of incubations including [35S]glutathione indicated formation of several unidentified 4-ABP-glutathione conjugates as well as recovery of parent compound. These studies suggest that HRP metabolizes 4-ABP by a one-electron oxidation mechanism, resulting in formation of a free radical. This radical can either react with a second radical to form azobis(biphenyl), be reduced by glutathione back to parent, or react with glutathione to form glutathione conjugates.