Abstract
Abstract Cytochrome P-450 (P450) catalyzes a wide variety of chemical reactions; however, its use for in vitro assays has several limitations, the most striking one is the use of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) coenzyme. In this work, the P450 activity using NADP+ reduced by an anionic organosiloxane, commonly named silica hydride, was evaluated. The results showed that the reduction of NADP+with silica hydride was concentration- and time-dependent. P-450 activity was maintained when NADP+ and silica hydride were added during the reaction; however, it was lower than when commercial NADPH was employed. This is due to the ability of silica hydride to reduce P450 iron atom as corroborated by the electronic paramagnetic resonance (EPR). Furthermore, this compound possibly chelates FeII because, in its presence, the P450 affinity for aniline diminishes. However, the P450 activity was the best when NADP+ was reduced by silica hydride before the former was added to the reaction. Therefore, this system could be apt for studying biotransformation reactions.
Highlights
Cytochrome P450 (P450) enzymes belong to a family of monooxygenase proteins, which catalyze a wide variety of oxidative reactions such as hydroxylation of aliphatic and aromatic carbons, N- and O-dealkylation, S-oxidation and epoxidation of olefins [1, 2]
Si-OH protons appear as a multiplet centered at 2.41 ppm, which integrates for 16.5 H-atoms; a broad signal for one H-atom centered at 1.6 ppm was assigned to –SiH2– moiety, and a quarter with a coupling constant of 81 Hz and centered at –0.31 ppm was assigned to six hydrides (Fig. 1), in agreement with the reported values [12]
Reduction of NADP+ by silica hydride The results showed that NADP+ was reduced by silica hydride, supporting the earlier reports in which this compound was able to reduce NAD+ to NADH [15, 16]
Summary
Cytochrome P450 (P450) enzymes belong to a family of monooxygenase proteins, which catalyze a wide variety of oxidative reactions such as hydroxylation of aliphatic and aromatic carbons, N- and O-dealkylation, S-oxidation and epoxidation of olefins [1, 2]. P450 showed catalytic activity when NADP+ and silica hydride were added to the reaction system; its activity was lower than when commercial NADPH was used (Fig. 4).
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