A novel application of cyclic voltammetry for direct investigation of metabolic intermediates in microsomal azo reduction

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Abstract

We have established that reduction of azo dyes structurally related to 4-(dimethylamino)-azobenzene (DAB) by rat liver microsomal cytochrome P-450 requires a polar electron-donating substituent on one ring. Reduction of azo dyes containing only electron-donating substituents is insensitive to both oxygen and CO (I substrates). However, reduction of azo dyes containing electron-withdrawing substituents as well is sensitive to both oxygen and CO (S substrates). Positive, irreversible potentials were observed by cyclic voltammetry (CV) in anhydrous solutions for both I and S substrates but not for the nonreducible azo dyes. This positive potential permits electron transfer to dyes from NADPH-cytochrome P-450 reductase and from cytochrome P-450, both of which have negative potentials. Reduction products retaining electron-donating groups (amino, phenolic) also exhibited positive potentials, implying that these groups contribute the positive potential in the dye molecule. All substrates also exhibited two negative potentials with a clear distinction between I and S substrates. The latter exhibited, on average, potentials that were less negative than the former by about 0.6 V. This is consistent with the more rapid reduction of S substrates by liver microsomes [Zbaida and Levine (1990) Biochem. Pharmacol. 40, 2415-2423]. Admitting air to the system quenched the first potential for S but not for I substrates, which is consistent with the oxygen sensitivities of their reduction. Addition of water significantly shifted the second negative potential to a more positive value, ultimately leading to single negative potential. The water permits rapid protonation of the two-electron-reduced intermediate, facilitating further reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

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