NADPH Dependent Cytochrome P-450 Reactions: Mode of Inhibition by the N—Butanol Fraction of Desmodium Adscendens

Abstract

The n-butanol fraction (nBF) of Desmxhvw adscendens, a plant used for the management of asthma, is' an inhibitor of NADPH-dependent cytochrome P-450 (CYP) reactions. Its mechanism of action as an inhibitor is however net knewn. In this study, flavoprotein reductase activity, spectral changes associated with binding and spectral properties of reduced cytochrome c and CYP were used to investigate the mode of inhibition. nBF reduced cytochrome c but not CYP directly. In the presence of NADPH, the rate of formation of CYP2 (the reduced form of CYP) was enhanced by the addition of nBF. These effects were observed in reactions without substrate, indicating that in the absence of a substrate, nBF does not prevent the NADPH reduction of CYP, but rather enhances it. In the presence of a substrate, as exemplified by a spectrophotometric assay of the EROD reaction, nBF was found to change the spectrum of oxidized CYP. The results indicate that in the presence of nBF, the binding site of CYP is altered to prevent substrate binding. Therefore, the node of inhibition of nBF relates to substrate binding. nBF could be inhibiting the NADPH-dependent CYP reactions by binding to CYP and preventing substrate binding. nBF could also inhibit the flew of electrons from NADPH to substrate-bound CYP by interfering with the flavoprotein reductase activity when it is bound to CYP. On the other hand, nBF could have the same effect on the rate of reduction of CYP to CYP2 in the presence of a substrate as it did in the absence of a substrate, leading to the formation of the CYP-O2-substrate temary complex, in which case the effect of nBF would be to prevent the breaking up of the temary complex to form products, the build-up of which causes the substrate to remain unmetabolized.