The effect of antioxidants on electrocatalytic activity of cytochrome P450 3A4

The electrochemical analysis of cytochrome P450 3A4 catalytic activity has shown that vitamins C, A and E influence reduction of cytochrome P450 3A4. These data suggest a possibility of cross effects and interference of vitamins-antioxidants with drugs metabolised by cytochrome P450 3A4, during complex therapy of patients. These vitamins demonstrate antioxidant properties that lead to the increase of the cathodic current corresponding to heme reduction of this functionally significant hemoprotein. Ascorbic acid (0.028–0.56 mM) stimulated the cathodic peak (an electrochemical signal) of cytochrome P450 3A4. In the presence of diclofenac (Voltaren), a typical substrate of cytochrome P450 3A4, the increase in the catalytic current suggesting electrocatalysis and stimulating action of ascorbic acid was observed. In the presence of vitamins A and E the dose-dependent increase in the catalytic current of cytochrome P450 3A4 was observed in the range of vitamin concentrations from 10 to 100 μM. The maximal increase of 229 ± 20 and 162 ± 10% was observed at 100 μM vitamin A and vitamin E, respectively. In contrast to vitamin A, vitamin E in the presence of the cytochrome P450 inhibitor itraconazole did not increase the catalytic current. The latter implies existence of some substrate properties in vitamin E. The electrochemical approach for the analysis of catalytic activity of cytochrome P450 3A4 and studies of the effect of biologically active compounds on electrocatalysis is the sensitive and effective sensor approach, allowing to use low concentration of protein on an electrode (up to 10–15 mol/electrode), to carry out the analysis without involvement of protein redox partners, and to reveal drug-drug or drug-vitamins interaction in pre-clinical experiments.     

The role of proteins of the outer membrane of <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1 in the formation and stabilization of silver sulfide nanoparticles

Silver sulfide nanoparticles stable in aqueous solutions were obtained in presence of the cells of the bacterium Shewanella oneidensis MR-1 in aqueous solution containing an equimolar mixture of AgNO₃ and Na₂S₂O₃. Proteins absorbed on the surface of Ag₂S nanoparticles were identified for the first time by MALDITOF/TOF. Among these proteins, multiheme cytochromes MtrC and OmcA, as well as the MtrB membrane porin, which forms a complex on the outer cell membrane, were detected. It was shown that an insoluble precipitate consisting of agglomerated Ag₂S nanoparticles with a wide size distribution was formed in the absence of the cells. The role of the detected proteins in the mechanism of the formation and stabilization of the Ag₂S nanoparticles in the studied system is discussed.     

Nanoelectrochemistry of cytochrome P450s: Direct electron transfer and electrocatalysis

Direct electron transfer has been demonstrated between cytochrome P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14α-demethylase (CYP51MT) and screen printed graphite electrodes, modified by gold nanoparticles and didodecyldimethyl ammonium bromide (DDAB). The proposed method for preparation of enzymatic nanostructured electrodes may be used for electrodetection of this hemoprotein provided that 2–200 pmol P450 per electrode has been adsorbed. Electron transfer, direct electrochemical reduction and interaction with P450 substrates (oxygen, benzphetamine, lanosterol) and inhibitor ketoconazole were analyzed using cyclic voltammetry (CV), square wave (SWV) or differential pulse (DPV) voltammetry, and amperometry.     

Electrochemical reduction of flavocytochromes 2B4 and 1A2 and their catalytic activity

The present study shows that cytochromes P450 2B4 and 1A2 with a covalently attached riboflavin (semisynthetic flavocytochromes RfP450 2B4 and RfP450 1A2) can be reduced electrochemically on rhodium-graphite electrodes at a potential of -500 mV (vs Ag/AgCl). In the presence of substrates such as aminopyrine, aniline, 7-ethoxyresorufin, and 7-pentoxyresorufin, N-demethylation, p-hydroxylation, and O-dealkylation reactions proceeded, as was confirmed by product analysis. Rates of electrocatalytically driven reactions are comparable to those obtained using NAD(P)H as the source of reducing equivalents. These results suggest the practicality of developing flavocytochrome P450s as catalysts for oxidation reactions with different classes of organic substrates.     

A new format of electrodes for the electrochemical reduction of cytochromes P450

New approach to the electrochemical reduction of cytochromes P450 (P450s, CYPs) at electrodes chemically modified with appropriate substrates for P450s ("reverse" electrodes) was proposed. The method is based on the analysis of cyclic voltammograms, square-wave voltammograms and amperograms with subsequent determination of electrochemical characteristics such as catalytic current and redox potential. The sensitivity of proposed method is 0.2-1 nmol P450/electrode. The changes of maximal current and of redox potentials in square-wave voltammograms as well as the changes of catalytic current in amperometric experiments proved to be informative and reliable. Planar regime of screen-printed electrodes (strip-type sensors) enabled to utilise 20-60 microl of electrolyte volume. The enzyme-substrate pairs P450 2B4/benzphetamine and P450scc/cholesterol were investigated. Electrochemical parameters of electrodes with unspecific P450 substrates differed considerably from electrodes with appropriate substrates.     

Au-nanoparticles as an electrochemical sensing platform for aptamer-thrombin interaction

A novel electrochemical method for the detection of bioaffinity interactions based on a gold-nanoparticles sensing platform and on the usage of stripping voltammetry technique was developed. The oxidation of gold surface (resulted in gold oxide formation) upon polarization served as a basis for analytical response. As a model, thrombin-thrombin binding aptamer couple was chosen. The aptamer was immobilized on a screen-printed electrode modified with gold-nanoparticles by avidin-biotin technology. Cathodic peak area was found proportional to thrombin quantity specifically adsorbed onto electrode surface. Sigmoid calibration curve as is typical for immunoassay was obtained, with thrombin detection limit of 10(-9)M. Linear range corresponds from 10(-8) to 10(-5)M thrombin concentration or 2 x 10(-14) to 2 x 10(-11)mol/electrode (R=0.996). Binding of thrombin to an aptamer has also been detected using the ferricyanide/ferrocyanide redox couple as electrochemical indicator.     

A New Method for Quantitative Determination of Steroid Metabolites of Cytochrome P450-Dependent Reactions Using Fluorescent Spectroscopy

A method for determination of hydroxylase activity of cytochrome P450 3A4 (CYP3A4) towards its substrate hydrocortisone using fluorescent analysis of the product was developed. 6β-hydroxycortisol, formed during CYP3A4-dependent electrocatalysis, has a characteristic fluorescent peak at λ = 427 ± 2 nm after treating with the sulfuric acid : ethanol (3 : 1) mixture and excitation at λ = 365 nm, which is different from the substrate (hydrocortisone) fluorescence (λ = 525 ± 2 nm). The limit of detection of 6β-hydroxycortisol was 0.32 μM. The developed analytical approach was used to determine the kinetic parameters of CYP3A4-dependent hydrocortisone hydroxylation.     

Scrutiny of electrochemically-driven electrocatalysis of C-19 steroid 1α-hydroxylase (CYP260A1) from Sorangium cellulosum So ce56

Direct electrochemistry and bioelectrocatalysis of a newly discovered C-19 steroid 1α-hydroxylase (CYP260A1) from the myxobacterium Sorangium cellulosum So ce56 were investigated. CYP260A1 was immobilized on screen-printed graphite electrodes (SPE) modified with gold nanoparticles, stabilized by didodecyldimethylammonium bromide (SPE/DDAB/Au). Cyclic voltammograms in argon-saturated substrate free 0.1 M potassium phosphate buffer, pH 7.4, and in enzyme-substrate complex with androstenedione demonstrated a redox processes with a single redox couple of E^0′ of −299 ± 16 mV and −297.5 ± 21 mV (vs. Ag/AgCl), respectively. CYP260A1 exhibited an electrocatalytic activity detected by an increase of the reduction current in the presence of dissolved oxygen and upon addition of the substrate (androstenedione) in the air-saturated buffer. The catalytic current of the enzyme correlated with substrate concentration in the electrochemical system and this dependence can be described by electrochemical Michaelis-Menten model. The products of CYP260A1-depended electrolysis at controlled working electrode potential of androstenedione were analyzed by mass-spectrometry. MS analysis revealed a mono-hydroxylated product of CYP260A1-dependent electrocatalytic reaction towards androstenedione.     

Prediction of the three-dimensional structure of aflatoxin of Aspergillus flavus by homology modelling

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server. The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the 3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of inhibition of aflatoxin.