Substrate and position specificity of hematin-activated monooxygenation reactions

Rates of hydroxylation of benzo[a]pyrene (BaP), benzo[e]pyrene (BeP), chrysene, acetanilide (AC), 7,12-dimethylbenz[a]anthracene (DMBA), and 17β-estradiol (E₂) in vitro could be increased by as much as 70-fold by additions of micromolar quantities of hematin. Such increases were observed primarily when extrahepatic tissues were utilized as the enzyme source; the greatest increases occurred with rabbit brain. Mixed-function oxygenations of aniline, ethylmorphine, benzphetamine, N-2-fluorenylacetamide (FAA), dibenz[a, h] anthracene(DBA), and benz[a] anthracene (BA), were affected minimally or not at all by hematin additions. Analyses of metabolites of BaP, AC and DMBA with high-pressure liquid chromatography revealed a high degree of position specificity for the hematinmediated reactions. This specificity was dependent upon the enzyme source, e.g. with rabbit kidney as enzyme source and AC as substrate, hematin additions resulted in only minor increases (30-40 per cent) in quantities of 3- and 4-hydroxylated metabolites and decreases (approximately 40-50 per cent; possibly a result of further degradation) in amounts of 2-hydroxylated AC. With hematin additions to rabbit brain homogenates, quantities of the measured 2-hydroxylated AC increased by 10 to 12-fold and of the 3-hydroxylated product by 3 to 4-fold, but no detectable changes in 4-hydroxylated AC were observed. With BaP as substrate, hematin elicited the formation of large quantities of an unidentified and hitherto undetected metabolite. Results of the study were consistent with the concept that hematin additions result in the reconstitution of a number of functionally distinct, tissue-specific cytochrome P-450 apoproteins.