6533b853fe1ef96bd12accbc

RESEARCH PRODUCT

On the spectral intermediate at 440 nm formed during mixed function substrate oxidation.

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Abstract The spectral shoulder formed at 440 nm in microsomes oxidising hexobarbital and other drugs has been investigated and some of its properties characterised. Hexobarbital, pentobarbital, ethylmorphine and barbital produce this shoulder, while acetanilide, aniline, desmethylimipramine, imipramine, metyrapone and SKF 525-A do not. The formation of the 440 nm shoulder depends on the presence of NADPH and oxygen and is reduced in size when NADH is also present. At saturating substrate concentrations the size of the 440 nm shoulder is correlated to the cytochrome P-450 content. The hexobarbital induced shoulder can be inhibited by drug metabolism inhibitors such as metyrapone, imipramine and desmethylimipramine, whereas acetanilide, aniline and SKF 525-A are indifferent. The size of the absorption at 440 nm is proportional to substrate induced oxygen consumption up to a maximum, after which the oxygen consumption alone increases with increasing substrate concentration. This may indicate a more rapid cycling within the reaction sequence caused by oversaturating substrate concentrations. The substrate induced oxygen consumption is more sensitive to low inhibitor concentrations than the peak formation, suggesting a more complex relationship between the two. The reported results lend further support for the hypothesis that the absorption at 440 nm represents an intermediary complex in the catalytic cycle of cytochrome P-450 mediated mixed function oxidation.