A novel application of cyclic voltammetry for direct investigation of metabolic intermediates in microsomal azo reduction.
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)
Zbaida
S
S
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
Levine
W G
WG
D000818:Animals; D001391:Azo Compounds / Q000737:chemistry / Q000378:metabolism*; D004563:Electrochemistry / Q000379:methods; D008862:Microsomes, Liver / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009601:Nitroreductases; D010084:Oxidation-Reduction; D051381:Rats; D013379:Substrate Specificity; D004124:p-Dimethylaminoazobenzene / Q000737:chemistry / Q000378:metabolism
D001391:Azo Compounds; D004124:p-Dimethylaminoazobenzene; D009247:NADH, NADPH Oxidoreductases; D009601:Nitroreductases; C021106:azoreductase; C009850:azobenzene