title: The oxidation of 4-aminobiphenyl by horseradish peroxidase.
abstract: 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.
authors.affiliation: Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
authors.forename: M F
authors.initials: MF
authors.lastname: Hughes
authors.forename: B J
authors.initials: BJ
authors.lastname: Smith
authors.forename: T E
authors.initials: TE
authors.lastname: Eling
mesh_terms: D000611:Aminobiphenyl Compounds / Q000737:chemistry*; D005609:Free Radicals; D005978:Glutathione / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000611:Aminobiphenyl Compounds; D005609:Free Radicals; C006757:4-biphenylamine; D006735:Horseradish Peroxidase; D005978:Glutathione