Hughes M F, Smith B J, Eling T E
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Chem Res Toxicol. 1992 May-Jun;5(3):340-5. doi: 10.1021/tx00027a004.
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.
研究了辣根过氧化物酶（HRP）这种典型过氧化物酶对致癌物4-氨基联苯（4-ABP）的氧化作用。在酶催化合成氢过氧化物5-苯基-4-戊烯-1-基氢过氧化物还原为相应醇的过程中，4-ABP作为HRP的还原性共底物。HRP、4-ABP和H2O2孵育期间的光谱分析表明，230和325nm处吸光度增加，270nm处吸光度降低，提示有代谢产物生成。在HRP、4-ABP和H2O2的孵育体系中未检测到耗氧量。然而，添加谷胱甘肽后观察到了氧气摄取，这表明过氧化物酶形成了4-ABP的自由基代谢产物。4-ABP自由基与谷胱甘肽反应形成谷胱甘肽自由基，后者进而与氧气反应并消耗氧气。对含有HRP、[3H]-4-ABP和H2O2的孵育体系的有机提取物进行HPLC分析，结果显示形成了一个主要峰，通过质谱鉴定为4,4'-偶氮双（联苯）。向孵育体系中添加谷胱甘肽可减少4-ABP代谢产物的形成，提示4-ABP自由基的还原和/或谷胱甘肽结合物的形成。随后对含有[35S]谷胱甘肽的孵育体系进行HPLC分析，结果表明形成了几种未鉴定的4-ABP-谷胱甘肽结合物，同时也回收了母体化合物。这些研究表明，HRP通过单电子氧化机制代谢4-ABP，导致自由基的形成。该自由基既可以与另一个自由基反应形成偶氮双（联苯），被谷胱甘肽还原回母体，也可以与谷胱甘肽反应形成谷胱甘肽结合物。
