Electroanalysis in Pharmacogenomic Studies: Mechanisms of Drug Interaction with DNA.

The review discusses electrochemical methods for analysis of drug interactions with DNA. The electroanalysis method is based on the registration of interaction-induced changes in the electrochemical oxidation potential of heterocyclic nitrogenous bases in the DNA molecule and in the maximum oxidation current amplitude. The mechanisms of DNA-drug interactions can be identified based on the shift in the electrooxidation potential of heterocyclic nitrogenous bases toward more negative (cathodic) or positive (anodic) values. Drug intercalation into DNA shifts the electrochemical oxidation potential to positive values, indicating thermodynamically unfavorable process that hinders oxidation of nitrogenous bases in DNA. The potential shift toward the negative values indicates electrostatic interactions, e.g., drug binding in the DNA minor groove, since this process does not interfere with the electrochemical oxidation of bases. The concentration-dependent decrease in the intensity of electrochemical oxidation of DNA bases allows to quantify the type of interaction and calculate the binding constants.

Pharmacogenomic Studies of Antiviral Drug Favipiravir.

In this work, we conducted a study of the interaction between DNA and favipiravir (FAV). This chemotherapeutic compound is an antiviral drug for the treatment of COVID-19 and other infections caused by RNA viruses. This paper examines the electroanalytical characteristics of FAV. The determined concentrations correspond to therapeutically significant ones in the range of 50-500 µM (R2 = 0.943). We have shown that FAV can be electro-oxidized around the potential of +0.96 V ÷ +0.98 V (vs. Ag/AgCl). A mechanism for electrochemical oxidation of FAV was proposed. The effect of the drug on DNA was recorded as changes in the intensity of electrochemical oxidation of heterocyclic nucleobases (guanine, adenine and thymine) using screen-printed graphite electrodes modified with single-walled carbon nanotubes and titanium oxide nanoparticles. In this work, the binding constants (Kb) of FAV/dsDNA complexes for guanine, adenine and thymine were calculated. The values of the DNA-mediated electrochemical decline coefficient were calculated as the ratio of the intensity of signals for the electrochemical oxidation of guanine, adenine and thymine in the presence of FAV to the intensity of signals for the electro-oxidation of these bases without drug (S, %). Based on the analysis of electrochemical parameters, values of binding constants and spectral data, intercalation was proposed as the principal mechanism of the antiviral drug FAV interaction with DNA. The interaction with calf thymus DNA also confirmed the intercalation mechanism. However, an additional mode of interaction, such as a damage effect together with electrostatic interactions, was revealed in a prolonged exposure of DNA to FAV.

Catalytic and Electrocatalytic Mechanisms of Cytochromes P450 in the Development of Biosensors and Bioreactors.

Cytochromes P450 are a unique family of enzymes found in all Kingdoms of living organisms (animals, bacteria, plants, fungi, and archaea), whose main function is biotransformation of exogenous and endogenous compounds. The review discusses approaches to enhancing the efficiency of electrocatalysis by cytochromes P450 for their use in biotechnology and design of biosensors and describes main methods in the development of reconstituted and electrochemical catalytic systems based on the biochemical mechanism of cytochromes P450, as well as and modern trends for their practical application.

Effect of an NGR Peptide on the Efficacy of the Doxorubicin Phospholipid Delivery System.

This study is a continuation of an investigation into the effect of a targeted component, a peptide with an NGR, on the properties of the previously developed doxorubicin phospholipid delivery system. The NGR peptide has an affinity for aminopeptidase N (known as the CD13 marker on the membrane surface of tumor cells) and has been extensively used to target drug delivery systems. This article presents the results of a study investigating the physical properties of the phospholipid composition with and without the peptide chain: particle size, zeta potential, stability in fluids, and dependence of doxorubicin release from nanoparticles at different pH levels (5.0, 6.5, 7.4). The cytotoxic effect of the compositions has also been shown to depend on the dose of the drug used for incubation, the presence of the targeted component in the composition, and the time of incubation time of the substances. There was a significant difference in the cytotoxic effect on HT-1080 (CD13-positive) and MCF-7 (CD13-negative) cells. Cell death pathway analysis has shown that death occurred mainly by apoptosis. We also present data on the effect of doxorubicin embedded in phospholipid nanoparticles with the targeted peptide on DNA assessed by differential pulse voltammetry, the mechanism of action being electrostatic interactions. The interactions of native dsDNA with doxorubicin encapsulated in phospholipid nanoparticles with the targeted peptide were studied electrochemically by differential pulse voltammetry. Here, we have highlighted that the targeted peptide in the doxorubicin composition moved specific interaction of the drug with dsDNA from intercalative mode to electrostatic interactions.

Interaction of Doxorubicin Embedded into Phospholipid Nanoparticles and Targeted Peptide-Modified Phospholipid Nanoparticles with DNA.

The interactions of dsDNA with new targeted drug delivery derivatives of doxorubicin (DOX), such as DOX embedded into phospholipid nanoparticles (NPhs) and DOX with the NGR targeted peptide-modified NPhs were studied electrochemically by differential pulse voltammetry technique. Screen-printed electrodes (SPEs), modified with stable fine dispersions of carbon nanotubes (CNTs), were used for quantitative electrochemical investigations of direct electrochemical oxidation of guanine, adenine, and thymine heterocyclic bases of dsDNA, and their changes in the presence of DOX nanoderivatives. Analysing the shifts of peak potentials of nucleobases in the presence of drug, we have shown that the doxorubicin with NGR targeted peptide changed the mode of interaction in DNA-drug complexes from intercalative to electrostatic. Binding constants (Kb) of DNA-drug complexes were calculated in accordance with adenine, guanine, and thymine oxidation signals. Based on our experiments, we have proven that the surface modification of a drug delivery system with NGR targeted peptide dramatically changed the mechanism of interaction of drug with genetic material. DNA-mediated drug toxicity was calculated based on the concentration-dependent "response" of heterocyclic nucleobases on drug influence. DOX, DOX-loaded phospholipid nanoparticles (NPhs), and DOX with NGR addressed peptide-modified NPhs were moderately toxic in the concentration range of 0.5-290 µM.

Approaches for increasing the electrocatalitic efficiency of cytochrome P450 3A4.

The electrochemically driven cytochrome P450 reactions have great promise as drug sensing device, new drug searching tool and bioreactor with broad synthetic application. In the present work, we proposed approaches for the increasing the efficiency of cytochrome P450 3A4 electrocatalysis, based on fine regulation and reproduction of nature hemeprotein catalytic cycle and electron transfer pathways on electrode. To analyze the comparative electrochemical and electrocatalytic activity, cytochrome P450 3A4 was immobilized on electrodes modified with a membrane-like synthetic surfactant, didodecyldimethylammonium bromide (DDAB). We used riboflavin, FMN and FAD as low molecular models of NADPH-dependent cytochrome P450 reductase for the improving and enhancement properties of catalytically responsible cytochrome P450 3A4-electrode. The efficiencies of electrocatalysis of erythromycin N-demethylation as well-known cytochrome P450 3A4 substrate in the case of riboflavin, FAD and FMN as electron transfer mediators were 135 ± 6, 171 ± 15 and 203 ± 10 %, respectively (in comparison with 100 ± 18 % erythromycin N-demethylation in the case of cytochrome P450 3A4-electrode as catalyst). Molecular modeling of cytochrome P450 3A4 complexes with riboflavin, FMN and FAD confirms possibility of binding isoalloxazine ring of riboflavin to the protein on the proximal side of hemeprotein, which is the place for binding of redox partners of the cytochrome P450.

Predicting drug-drug interactions by electrochemically driven cytochrome P450 3A4 reactions.

OBJECTIVES: Human cytochrome P450 3A4 is the most abundant hepatic and intestinal Phase I enzyme that metabolizes approximately 60% marketed drugs. Simultaneous administration of several drugs may result in appearance of drug-drug interaction. Due to the great interest in the combination therapy, the exploration of the role of drug as "perpetrator" or "victim" is important task in pharmacology. In this work the model systems based on electrochemically driven cytochrome P450 3A4 for the analysis of drug combinations was used. We have shown that the analysis of electrochemical parameters of cytochrome P450 3A4 and especially, potential of the start of catalysis, Eonset, possess predictive properties in the determination of the leading ("perpetrator") properties of drug. Based on these experimental data, we concluded, that the more positive potential of the start of catalysis, Eonset, the more pronounced the role of drug as leading medication. METHODS: Electrochemically driven cytochrome P450 3A4 was used as probe and measuring tool for the estimation of the role of interacting drugs. RESULTS: It is shown that the electrochemical non-invasive model systems for monitoring the catalytic activity of cytochrome P450 3A4 can be used as prognostic devise in assessment of drug/drug interacting medications. CONCLUSIONS: Cytochrome P450 3A4 activity was studied in electrochemically driven system. Method was implemented to monitor drug/drug interactions. Based on the obtained experimental data, we can conclude that electrochemical parameter such as potential of onset of catalysis, Eonset, has predictive efficiency in assessment of drug/drug interacting medications in the case of the co-administration.

Electroanalysis of Biomolecules: Rational Selection of Sensor Construction.

Methods of electrochemical analysis of biological objects based on the reaction of electro-oxidation/electro-reduction of molecules are presented. Polymer nanocomposite materials that modify electrodes to increase sensitivity of electrochemical events on the surface of electrodes are described. Examples of applications electrochemical biosensors constructed with nanocomposite material for detection of biological molecules are presented, advantages and drawbacks of different applications are discussed.

Electrochemical studies of the interaction of rifampicin and nanosome/rifampicin with dsDNA.

The interactions of dsDNA with rifampicin (RF) or with rifampicin after encapsulation in phospholipid micelles (nanosome/rifampicin) (NRF) were studied electrochemically. Screen-printed electrodes (SPEs) modified by stable dispersions of multi-wolled carbon nanotubes (MWCNTs) in aqueous solution of poly(1,2-butadiene)-block-poly(2-(dimethylamino)ethyl methacrylate) (PB290-b-PDMAEMA240) diblock copolymer were used for quantitative electrochemical investigation of direct electrochemical oxidation of guanine at E = 0.591 V (vs. Ag/AgCl) and adenine at E = 0.874 V (vs. Ag/AgCl) of dsDNA and its change in the presence of RF or NRF. Due to RF or NRF interaction with dsDNA, the differential pulse voltammetry (DPV) peak currents of guanine and adenine decreased and the peak potentials shifted to more positive values with increasing drug concentration (RF or NRF). Binding constants (Kb) of complexes RF-dsDNA and NRF-dsDNA were calculated based on adenine and guanine oxidation signals. The Kb values for RF-dsDNA were 1.48 × 104 M-1/8.56 × 104 M-1, while for NRF-dsDNA were 2.51 × 104 M-1/1.78 × 103 M-1 (based on adenine or guanine oxidation signals, respectively). The values of Kb revealed intercalation mode of interaction with dsDNA for RF and mixed type of interaction (intercalation and electrostatic mode) for NRF. The estimated values of ΔG (Gibbs free energy) of the complex formation confirmed that drug-dsDNA interactions are spontaneous and favourable reactions.

No effect of lipoic acid on catalytic activity of cytochrome P450 3A4.

Objectives α-Lipoic acid is used as an antioxidant in multivitamin formulations to restore the normal level of intracellular glutathione after depletion caused by environmental pollutants or during physiological aging of the body, as a chelating agent, as a dietary supplement, in anti-aging compositions. Lipoic acid (LA) acts as a buffer in cancer therapy and in therapy of diseases associated with oxidative stress. The effect of LA on the catalytic functions of cytochrome P450 3A4 as the main enzyme of the biotransformation of drugs was studied. It was shown that LA in the concentration range of 50-200 µM affects the stage of electron transfer (stage of cytochrome P450 3A4 heme reduction), decreasing the cathodic reduction current by an average of 20 ± 5%. The kinetic parameters (k cat) of the N-demethylation reaction of erythromycin, the antibiotic of the macrolide group, used as a marker substrate for the comparative analysis of the catalytic activity of cytochrome P450 3A4, both in the presence of α-lipoic acid and in the cytochrome P450 3A4-erythromycin complex, amounted to comparable values of 3.5 ± 0.9 and 3.4 ± 0.9 min-1, respectively. Based on these experimental data, we can conclude that there is no significant effect of α-lipoic acid on the catalysis of cytochrome P450 3A4. These results can be projected on the possibility of using α-lipoic acid in complex therapy without negative impact on the enzymatic cytochrome P450 system. Methods The analysis was performed in electrochemical non-invasive model systems for recording the catalytic activity of cytochrome P450 3A4, using screen-printed electrodes, modified with membranous didodecyldimethylammonium bromide. Results It was shown that LA did not affect the N-demethylation of macrolide antibiotic erythromycin. Catalytic constant (k cat) of N-demethylation of erythromycin corresponds to 3.4 ± 0.9 min-1 and in the presence of LA corresponds to 3.5 ± 0.9 min-1. Conclusions Based on the obtained experimental data, we can conclude that there is no significant effect of α-lipoic acid on individual stages and processes of catalysis of cytochrome P450 3A4. LA can be recommended for inclusion in complex therapy as an antioxidant, antitoxic and chelating compound without negative impact on the enzymatic cytochrome P450 3A4 activity of the human body.

Rational Design of Amphiphilic Diblock Copolymer/MWCNT Surface Modifiers and Their Application for Direct Electrochemical Sensing of DNA.

We demonstrate the application of amphiphilic ionic poly(n-butylmethacrylate)-block- poly(2-(dimethylamino)ethyl methacrylate) diblock copolymers (PnBMA40-b-PDMAEMA40, PnBMA40-b-PDMAEMA120, PnBMA70-b-PDMAEMA120) for dispersing multiwalled carbon nanotubes (MWCNTs) in aqueous media, a subsequent efficient surface modification of screen-printed electrodes (SPEs), and the application of the modified SPEs for DNA electrochemistry. Stable and fine aqueous dispersions of MWCNTs were obtained with PnBMAx-b-PDMAEMAy diblock copolymers, regardless of the structure of the copolymer and the amount of MWCNTs in the dispersions. The effect of the diblock copolymer structure was important when the dispersions of MWCNTs were deposited as modifying layers on surfaces of SPEs, resulting in considerable increases of the electroactive surface areas and great acceleration of the electron transfer rate. The SPE/(PnBMAx-b-PDMAEMAy + MWCNT) constructs were further exploited for direct electrochemical oxidation of the guanine (G) and adenine (A) residues in a model salmon sperm double-stranded DNA (dsDNA). Two well-defined irreversible oxidation peaks were observed at about +600 and +900 mV, corresponding to the electrochemical oxidation of G and A residues, respectively. A multi-parametric optimization of dsDNA electrochemistry enables one to get the limits of detection (LOD) as low as 5 µg/mL (0.25 µM) and 1 µg/mL (0.05 µM) for G and A residues, respectively. The achieved sensitivity of DNA assay enables quantification of the A and G residues of dsDNA in the presence of human serum and DNA in isolated human leukocytes.

The influence of taurine and L-carnitine on 6 ß-hydroxycortisol/cortisol ratio in human urine of healthy volunteers.

Background Cytochrome P450s (CYPs, EC 1.14.14.1) are the main enzymes of drug metabolism. The functional significance of CYPs also includes the metabolism of foreign chemicals and endogenic biologically active compounds. The CYP3A4 isoform contributes to the metabolism of about half of all marketed medicinal preparations. The aim of this study was to investigate the effects of two biologically active compounds: 2-aminoethane-sulfonic acid (taurine) and 3-hydroxy-4-trimethylaminobutyrate (L-carnitine) on urinary 6ß-hydroxycortisol/cortisol (6ß-OHC/cortisol) metabolic ratio as a biomarker of the CYP3A4 activity of healthy volunteers. Taurine is used for the treatment of chronic heart failure and liver disease. Cardiologists, nephrologists, neurologists, gerontologists in addition to the main etiopathogenetic therapies, use L-carnitine. The quantification of the 6ß-OHC/cortisol metabolic ratio as a biomarker of CYP3A4 activity in human urine was used for the assessment of CYP3A4 catalytic activity as a non-invasive test. Methods The study included 18 healthy male volunteers (aged from 18 to 35 years old). The volunteers took taurine in a dose of 500 mg twice a day or L-carnitine in a dose of 2.5 mL 3 times a day for 14 consecutive days. The test drug was given 20 min before meals. The collection of urine samples was performed before and after 3, 7, 10, and 14 days after taurine intake. The metabolic ratio of 6ß-OHC/cortisol in morning spot urine samples was studied by the liquid chromatography/mass spectroscopy (LC/MS) method. Results The ratio of 6-6ß-OHC/cortisol was used as a biomarker to study the taurine and L-carnitine influence on CYP3A4 metabolism of cortisol. The ratio of urinary 6ß-OCH/cortisol in the morning urine samples of volunteers before the beginning of taurine therapy (baseline ratio) was 2.71 ± 0.2. Seven days after the administration of taurine in a dose of 500 mg twice a day, the 6ß-OCH/cortisol ratio was 3.3 ± 0.2, which indicated the increased catalytic activity of CYP3A4 towards cortisol. As for the L-carnitine supplementation, analysis of the 6ß-OCH/cortisol ratio in the urine for 14 days did not show any significant changes in this baseline ratio, indicating the lack of L-carnitine influence on the catalytic activity of CYP3A4 to cortisol. Conclusions The results obtained demonstrated the influence of taurine on 6ß-OCH/cortisol metabolic ratio as a biomarker of CYP3A4 catalytic activity to cortisol. L-carnitine did not affect the activity of CYP3A4. The lack of a clinically meaningful effect of L-carnitine was established.

From electrochemistry to enzyme kinetics of cytochrome P450.

This review is an attempt to describe advancements in the electrochemistry of cytochrome P450 enzymes (EC 1.14.14.1) and to study molecular aspects and catalytic behavior of enzymatic electrocatalysis. Electroanalysis of cytochrome P450 demonstrates how to translate theoretical laws and equations of classical electrochemistry for the calculation of the kinetic parameters of enzymatic reactions and then translation of kinetic parameters to interpretation of drug-drug interactions. The functional significance of cytochrome P450s (CYPs) includes the metabolism of drugs, foreign chemicals, and endogenic compounds. The pharmaceutical industry needs sensitive and cost-effective systems for screening new drugs and investigation of drug-drug interactions. The development of different types of CYP-based biosensors is now in great demand. This review also highlights the characteristics of electrode processes and electrode properties for optimization of the cytochrome P450 electroanalysis. Electrochemical cytochrome P450-biosensors are the most studied. In this review, we analyzed electrode/cytochrome P450 systems in terms of the mechanisms underlying P450-catalyzed reactions. Screening of potential substrates or inhibitors of cytochromes P450 by means of electrodes were described.

Molecular imprinting coupled with electrochemical analysis for plasma samples classification in acute myocardial infarction diagnostic.

Electroanalysis of myoglobin (Mb) in 10 plasma samples of healthy donors (HDs) and 14 plasma samples of patients with acute myocardial infarction (AMI) was carried out with screen-printed electrodes modified first with multi-walled carbon nanotubes (MWCNT) and then with a molecularly imprinted polymer film (MIP), viz., myoglobin-imprinted electropolymerized poly(o-phenylenediamine). The differential pulse voltammetry (DPV) parameters, such as a maximum amplitude of reduction peak current (A, nA), a reduction peak area (S, nA × V), and a peak potential (P, V), were measured for the MWCNT/MIP-sensors after their incubation with non-diluted plasma. The relevance of the multi-parameter electrochemical data for accurate discrimination between HDs and patients with AMI was assessed on the basis of electrochemical threshold values (this requires the reference standard method (RAMP® immunoassay)) or alternatively on the basis of the computational cluster assay (this does not require any reference standard method). The multi-parameter electrochemical analysis of biosamples combined with computational cluster assay was found to provide better accuracy in classification of plasma samples to the groups of HDs or AMI patients.

Electrosynthesis and binding properties of molecularly imprinted poly-o-phenylenediamine for selective recognition and direct electrochemical detection of myoglobin.

Electrosynthesis of molecularly imprinted polymer (MIP) templated with myoglobin (Mb) and the reference non-imprinted polymer (NIP) was examined with o-phenylenediamine (o-PD) as a monomer. Mass-sensitive quartz crystal microbalance with dissipation monitoring supplied by an electrochemical module (EQCM-D) was applied to characterize and optimize MIP/NIP electrosynthesis. Mb rebinding was detected by direct electrocatalytic reduction of Mb by square wave voltammetry (SWV) or differential pulse voltammetry (DPV). The results obtained showed high specificity of polymeric antibodies to template Mb, with an imprinting factor determined as a ratio Imax(MIP)/Imax(NIP) of 2-4. The prepared MIP sensor is characterized by an apparent dissociation constant of (3.3±0.5)×10(-9)M and has a broad range of working concentrations of 1nM-1µÐœ, with the detection limit of 0.5nM (9ng/ml). Mb rebinding was examined in Mb-free diluted human serum spiked with Mb as well as in plasma samples of patients with acute myocardial infarction (AMI) and in control plasma of healthy donors in order to demonstrate the potential medical application of developed MIP sensors.

Facilitated biosensing via direct electron transfer of myoglobin integrated into diblock copolymer/multi-walled carbon nanotube nanocomposites.

Nanocomposite materials were prepared by sequential drop-casting of multi-walled carbon nanotube (MWCNT) suspensions and amphiphilic polybutadiene-block-poly(2-(N,N-dimethylamino)ethyl methacrylate) (PB290-b-PDMAEMA240) diblock copolymer micellar solution on screen-printed electrodes (SPEs). These nanocomposite materials were found to be very favorable for integration of myoglobin (Mb) and facilitate direct electron transfer from the electrodes to heme proteins. In that respect, PB290-b-PDMAEMA240 was demonstrated to be a well-suited binding agent. In aqueous solutions, it forms core-corona micelles (shown by cryogenic transmission electron microscopy, cryo-TEM, and nanoparticle tracking analysis, NTA), which at pH 7 in phosphate buffer exhibit good adhesion to carbon materials (shown by atomic force microscopy, AFM, scanning electron microscopy, SEM, and scanning transmission electron microscopy, STEM) and build up uniform thin films on a hydrophobic graphite-based substrate. As demonstrated by a quartz crystal microbalance with dissipation monitoring, QCM-D, attractive interactions between Mb and PB290-b-PDMAEMA240 take place when both components are subsequently deposited onto a solid substrate. Spectroscopic studies confirmed that the absorption maximum of Mb remains unaltered, suggesting that at least some protein globules retain their tertiary structure. Cyclic voltammetry, CV, and square wave voltammetry, SWV, show a remarkable (ca. 180-fold) increase of the reductive current of Mb after its incorporation into the SPE/MWCNT/PB290-b-PDMAEMA240 matrix. The herein developed analytical approach was used for the detection of cardiac myoglobin as a very early marker of acute myocardial infarction (AMI) both in plasma of healthy donors and patients with AMI.

Electrochemical methods for detection of post-translational modifications of proteins.

Post-translational modifications of proteins play a key role in the regulation of various cellular processes. The analysis and identification of post-translational modifications are probably the most versatile and difficult, but also most frequently studied area of interest in proteomics research. This review focuses on the electroactivity of amino acids as a tool for analysis of post-translational modifications of proteins. The most attention is paid to the electrochemical detection of phosphorylation/dephosphorylation and glycosylation of proteins, to the best-studied and functionally-significant modifications, and, also, to the electrochemical analysis of activity of enzymes responsible for carrying out phosphorylation/dephosphorylation of proteins. Recent advances in electrochemistry with special references to proteomics are outlined and innovative technologies for protein detection are highlighted.

Electrochemical approach for acute myocardial infarction diagnosis based on direct antibodies-free analysis of human blood plasma.

A novel direct antibodies-free electrochemical approach for acute myocardial infarction (AMI) diagnosis has been developed. For this purpose, a combination of the electrochemical assay of plasma samples with chemometrics was proposed. Screen printed carbon electrodes modified with didodecyldimethylammonium bromide were used for plasma charactrerization by cyclic (CV) and square wave voltammetry and square wave (SWV) voltammetry. It was shown that the cathodic peak in voltammograms at about -250 mV vs. Ag/AgCl can be associated with AMI. In parallel tests, cardiac myoglobin and troponin I, the AMI biomarkers, were determined in each sample by RAMP immunoassay. The applicability of the electrochemical testing for AMI diagnostics was confirmed by statistical methods: generalized linear model (GLM), linear discriminant analysis (LDA) and quadratic discriminant analysis (QDA), artificial neural net (multi-layer perception, MLP), and support vector machine (SVM), all of which were created to obtain the "True-False" distribution prediction where "True" and "False" are, respectively, positive and negative decision about an illness event.

Electrochemical investigations of cytochrome P450.

In this paper we summarized our experimental data on the electrochemical reduction of cytochrome P450. Electrode/cytochrome P450 systems were analyzed in terms of the mechanisms underlying P450-catalyzed reactions. Bioelectrocatalysis-based screening of potential substrates or inhibitors of cytochrome P450, stoichiometry of the electrocatalytic cycle, redox thermodynamics and the peroxide shunt pathway were described. Characteristics, performance and potential application of cytochrome P450-electrodes are discussed.

Stoichiometry of electrocatalytic cycle of cytochrome P450 2B4.

Stoichiometry of the electrocatalytical cycle of cytochrome P450 2B4 was studied in kinetic mode according to bielectrode scheme. Graphite screen-printed electrodes with immobilized cytochrome P450 2B4 were used as the operating electrode (at the potential E(0')=-450 mV) and electrodes, modified with cytochrome c (E(0')=-50 mV) or Prussian Blue (E(0')=0), as measuring electrodes (for H(2)O(2)) and Clark-type electrode (for O(2)). Benzphetamine N-demethylation rate was 17+/-3 nmol/nmol of enzyme/min, peroxide production was 4.8+/-0.7 nmol/nmol of enzyme/min (substrate-free system), 3.3+/-0.6 nmol/nmol of enzyme/min (0.5 mM benzphetamine), the oxygen consumption rate by capital P450 2B4 was 19.4+/-0.6 nmol/nmol of enzyme/min (in the presence of benzphetamine), 4.8+/-0.4 nmol/nmol of enzyme/min (without substrate). Based on stoichiometry of P450 electrocatalysis adequacy of electrochemical reduction and P450-monooxygenase system was revealed.