Cytochrome P450 3A4 as a Drug Metabolizing Enzyme: the Role of Sensor System Modifications in Electocatalysis and Electroanalysis

Abstract

The electroanalytical characteristics of the recombinant cytochrome P450 3A4 (CYP3A4) immobilized on the surface of screen-printed graphite electrodes modified by multi-walled carbon nanotubes (MWCNTs) have been investigated. The role and the influence of the modification of a graphite working electrode by carbon nanotubes on the electroanalytical parameters of this cytochrome have been demonstrated. The conditions for its immobilization on the obtained screen-printed graphite electrodes modified by MWCNTs have been optimized. The electrochemical parameters of the oxidation and reduction of the enzyme heme iron ion were determined: these included midpoint-potential (E0') of –0.35 ± 0.01 V (relative to the silver/silver chloride reference electrode, Ag/AgCl); the electrochemical rate constant of heterogeneous electron transfer (ks) of 0.57 ± 0.04 s–1, the amount of electroactive CYP3A4 on the modified electrode (Γ0) of (2.6 ± 0.6) × 10–10 mol/cm2. The functioning mechanism of this electrochemical sensor followed the principle of “protein film voltammetry” (i.e. voltammetry of a protein film on the electrode surface). In order to develop medical biosensors using immobilized CYP3A4 for electroanalysis of drug substances, the substrates of this hemoprotein, we performed a voltammetric study of the catalytic activity of immobilized CYP3A4. We investigated electrocatalytic properties of the recombinant CYP3A4 immobilized on modified screen-printed graphite electrode using macrolide antibiotic erythromycin as a substrate. The modification of electrodes has been shown to play a decisive role for the study of the properties of cytochromes P450 in electrochemical studies. Smart electrodes can serve as electroanalyzers for analytical purposes, as well as electrocatalysts for the study of biotransformation and metabolic processes. Electrodes modified with carbon nanomaterials are applicable for analytical purposes in the registration of hemoproteins. Electrodes modified with synthetic membrane-like compounds (for example, didodecyldimethylammonium bromide) are effective in enzyme-dependent electrocatalysis.