[Adrenodoxins and their role in the cytochrome P450 systems]. / Adrenodoksiny i ikh rol' v sistemakh tsitokhroma R450.

The role of partner proteins in the formation of functional complexes in cytochrome P450 systems was investigated by means of optical biosensor technique. Kinetic constants and equilibrium dissociation constants of complexes of cytochrome CYP11A1 (P450scc) with wild-type adrenodoxin (Adx WT) and mutant forms of adrenodoxin R106D and D109R were determined using an optical biosensor. Wild-type adrenodoxin (Kd = (1.23±0.09)⋅10⁻6 M) and mutant D109R (Kd = (2.37±0.09)⋅10⁻8 M) formed complexes with cytochrome P450scc. For the R106D mutant, no complex formation was detected. To investigate the possibility of the participation of adrenodoxins and their mutant variants in the process of electron transfer as electron donors in mitochondrial cytochrome P450 systems, the electrochemical properties of these iron-sulfur proteins Adx WT and mutant forms of adrenodoxins were studied. Adx WT, mutant forms R106D and D109R have redox potentials E1/2 significantly more negative than cytochromes P450 (-579±10 mV, -590±15 mV, and -528±10 mV, respectively). These results suggest that Adx WT and mutant forms may be electron donors in the cytochrome P450 systems.

[Modeling of drug-drug interactions between omeprazole and erythromycin with cytochrome P450 3A4 in vitro assay]. / Modelirovanie mezhlekarstvennogo vzaimodeistviia omeprazola i éritromitsina s ispol'zovaniem tsitokhrom R450-zavisimoi sistemy in vitro.

In the present study the electrochemical system based on recombinant cytochrome P450 3A4 (CYP3A4) was used for the investigation of potential drug-drug interaction between medicinal preparations employed for Helicobacter pylori eradication therapy. Drug interactions were demonstrated in association of omeprazole as a proton pump inhibitor (PPI) and macrolide antibiotic erythromycin during cytochrome P450 3A4-mediated metabolism. It was shown that in the presence of omeprazole the rate of N-demethylase activity of CYP3A4 to erythromycin measured by means of product (formaldehyde) formation decreased. Mass-spectrometry analysis of omeprazole sulfone as a CYP3A4-mediated metabolite demonstrated the absence of erythromycin influence on CYP3A4-dependent omeprazole metabolism. This phenomenon may be explained by lower spectral dissociation constant of CYP3A4-omeprazole complex (Kd = 18±2 µM) than that of CYP3A4-erythromycin complex (Kd = 52 µM). Using the electrochemical model of electrochemically-driven drug metabolism it is possible to register CYP3A4-mediated catalytic conversion of certain drugs. In vitro experiments of potential CYP3A4-mediated drug-drug interactions are in accordance with in silico modeling with program PASS and PoSMNA descriptors in the case of omeprazole/erythromycin combinations.

[Electroanalytical and electrocatalytical characteristics of cytochrome P450 3A4 using electrodes modified with nanocomposite carbon nanomaterials]. / Tsitokhrom R450 3A4 kak ferment biotransformatsii lekarstv: rol' modifikatsii sensornykh sistem v élektrokatalize i élektroanalize.

The electroanalytical characteristics of recombinant cytochrome P450 3A4 (P450 3A4) immobilized on the surface of screen-printed graphite electrodes modified with multi-walled carbon nanotubes have been studied. The role and the influence of graphite working electrode modification with carbon nanotubes on electroanalytical characteristics of cytochrome P450 3A4 have been demonstrated. The conditions for the immobilization of cytochrome P450 3A4 on the obtained screen-printed graphite electrodes modified with carbon multi-walled nanotubes have been optimized. The electrochemical parameters of the oxidation and reduction of the heme iron of the enzyme have been estimated. The midpoint potential E0' was -0.35±0.01 V vs Ag/AgCl; the calculated heterogeneous electron transfer rate constant ks, was 0.57±0.04 s-1; the amount of electroactive cytochrome P450 3A4 on the electrode Г0, was determined as (2.6±0.6)⋅10-10 mol/cm2. The functioning mechanism of P450 3A4-based electrochemical sensor followed the "protein film voltammetry". In order to develop electrochemical analysis of drugs being substrates of that hemoprotein and respective medical biosensors the voltammetric study of catalytic activity of immobilized cytochrome P450 3A4 was carried out. Electrocatalytic properties of cytochrome P450 3A4, immobilized on modified screen-printed graphite electrodes, has been investigated using erythromycin (macrolide antibiotics). It has been shown that the modification of electrodes plays a decisive role for the study of the properties of cytochromes P450 in electrochemical investigations. Smart electrodes can serve as sensors for analytical purposes, as well as electrocatalysts for the study of biotransformation processes 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 (e.g. didodecyldimethylammonium bromide) are effective in enzyme-dependent electrocatalysis.

Electroanalysis of myoglobin based on electropolymerized molecularly imprinted polymer poly-o-phenylenediamine and carbon nanotubes/screen printed electrode.

Electroanalysis of myoglobin as a marker of acute myocardial infarction by means of screenprinted electrodes modified with multiwalled carbon nanotubes and polymeric artificial antibodies is developed. Plastic antibodies to myoglobin (molecularly imprinted polymers, MIPs) based on o-phenylenediamine were produced by electropolymerization. Molecular imprinting technology in biosensor analysis was used as alternative to natural receptors (namely, antibodies) and demonstrated high sensitivity (1.5 × 10(-2) A/nmol of myoglobin) and selectivity.

Electrosynthesis and binding properties of molecularly imprinted poly-o-phenylenediamine as artificial antibodies for electroanalysis of myoglobin.

Molecularly imprinted poly-o-phenylenediamine with template myoglobin molecules (i.e., polymeric antibodies to myoglobin, molecularly imprinted polymer, MIP) was synthesized via electropolymerization. Electropolymerization, washing, and the interaction of the polymeric antibodies with myoglobin was examined by square wave voltammetry and microgravimetry. The analysis of myoglobin was carried out through direct electrochemical detection of the reduction peak of Fe(3+) of the hemeprotein on screen-printed graphite electrodes modified by the MIP. According to the electrochemical analysis, MIP surfaces demonstrated remarkably higher ability to bind the protein compared to that of surfaces prepared by the same route under the same conditions but in the absence of myoglobin (surfaces of the non-imprinted polymer, NIP). The imprinting factor I max(MIP)/I max(NIP) was found to be 2-4. The equilibrium dissociation constant K d of the interaction of myoglobin with MIP electrodes was evaluated as (2.4 ± 0.5) × 10(-8) M. The lower detection limit of myoglobin by a MIP sensor was determined as 0.5 × 10(-9) M, the range of detectable concentrations being 10(-9)-10(-5) M.

[Functionalization of screen printed electrodes with organic-inorganic hybrid nano-composites for bio-sensing applications].

New types of organic-inorganic hybrid nanocomposites based on nanosized Titanium (IV) oxide TiO2 (<100 nm particle size) and carbon nanotubes (CNT, outer diameter 10-15 nm, inner diamentre 2-6 nm, length 0.1-10 µm) and phosphatidilcholine were elaborated for improvement of analytical characteristics of screen printed electrodes. These nanomaterials were employed as an interface for the immobilization of skeletal myoglobin. Electrochemical behavior of myoglobin on such interfaces was characterized with cyclic voltammetry (CV) and square wave voltammetry (SWV). Direct unmediated electron transfer between myoglobin and electrodes modified with organic-inorganic hybrid nanocomposites was registered. TiO2 film and CNT film are biocompartible nanomaterials for myoglobin as was demonstrated with UV-Vis spectra. The midpoint potential of Fe3+/Fe2+ pair of myoglobin corresponded to Е1/2=-0,263 V for CNT film, and Е1/2=-0,468 V for TiO2 nanocomposite (vs. Ag/AgCl reference electrode).

[Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes b5].

Molecular interactions between proteins redox partners (cytochromes Р450 3А4, 3А5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes Р450 3А4 and 3А5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435  -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 in the presence of its substrate testosterone.

[Molecularly imprinted polymers in electro analysis of proteins].

In the review the main approaches to creation of recognition materials capable of competing with biological specific receptors, (polymeric analogs of antibodies or molecularly imprinted polymers, MIP) for the electro analysis of functionally significant proteins such as a myoglobin, troponin T, albumin, human ferritin, calmodulin are considered. The main types of monomers for MIP fabrication, and methods for MIP/protein interactions, such as a surface plasmon resonance (SPR), nanogravimetry with use of the quartz crystal resonator (QCM), spectral and electrochemical methods are discussed. Experimental data on electrochemical registration of a myoglobin using MIP/electrode are presented. For a development of electrochemical sensor systems based on MIPs, o-phenylenediamine (1,2-diaminobenzene was used as a monomer. It was shown that the imprinting factor Imax(MIP)/Imax(NIP), calculated as a myoglobin signal ratio when embedding in MIP to a myoglobin signal when embedding in the polymer received without molecular template (NIP) corresponds 2-4.

[Electrochemical methods for biomedical investigations].

In the review, authors discussed recently published experimental data concerning highly sensitive electrochemical methods and technologies for biomedical investigations in the postgenomic era. Developments in electrochemical biosensors systems for the analysis of various bio objects are also considered: cytochrome P450s, cardiac markers, bacterial cells, the analysis of proteins based on electro oxidized amino acids as a tool for analysis of conformational events. The electroanalysis of catalytic activity of cytochromes P450 allowed developing system for screening of potential substrates, inhibitors or modulators of catalytic functions of this class of hemoproteins. The highly sensitive quartz crystal microbalance (QCM) immunosensor has been developed for analysis of bio affinity interactions of antibodies with troponin I in plasma. The QCM technique allowed real-time monitoring of the kinetic differences in specific interactions and nonspecific sorption, with out multiple labeling procedures and separation steps. The affinity binding process was characterized by the association (ka) and the dissociation (kd) kinetic constants and the equilibrium association (K) constant, calculated using experimental data. Based on the electroactivity of bacterial cells, the electrochemical system for determination of sensitivity of the microbial cells to antibiotics cefepime, ampicillin, amikacin, and erythromycin was proposed. It was shown that the minimally detectable cell number corresponds to 106 CFU per electrode. The electrochemical method allows estimating the degree of E.coli JM109 cells resistance to antibiotics within 2-5 h. Electrosynthesis of polymeric analogs of antibodies for myoglobin (molecularly imprinted polymer, MIP) on the surface of graphite screen-printed electrodes as sensor elements with o- phenylenediamine as the functional monomer was developed. Molecularly imprinted polymers demonstrate selective complementary binding of a template protein molecule (myoglobin) by the "key-lock" principle.

Taurine modulates catalytic activity of cytochrome P450 3A4.

The influence of the biologically active compound taurine on the stability and catalytic properties of the hemoprotein cytochrome P450 3A4 has been investigated. The catalytic properties were analyzed by electrochemical methods (cyclic and square-wave voltammetry) using cytochrome P450 3A4 immobilized on the electrode. Taurine at concentrations in the range 10-70 µM stimulated the electrochemical reduction of cytochrome P450 3A4, and the reduction was the highest (115 ± 3%) in the presence of 50 µM taurine. Taurine pronouncedly attenuated the itraconazol-caused inhibition of the P450 isoenzyme P450 3A4. Taurine protected cytochrome P450 3A4 due to stabilizing it during electrolysis at controlled voltage in the presence of erythromycin as a substrate. This protection was manifested by an increase in the amount of the "residual" reduced form of the hemoprotein (52 ± 5 and 71 ± 8%, respectively).

[Role of antioxidants in electro catalytic activity of cytochrome P450 3A4].

The electrochemical analysis of cytochrome Р450 3А4 catalytic activity has shown that vitamins C, A and Е influence on electron transfer and Fe3+/Fe2+ reduction process of cytochrome Р450 3А4. These data allow to assume possibility of cross effects and interference of vitamins-antioxidants with drugs metabolised by cytochrome Р450 3А4, at carrying out of complex therapy. This class of vitamins shows antioxidant properties that lead to increase of the cathodic current corresponding to heme reduction of this functionally significant haemoprotein. Ascorbic acid of 0.028-0.56 mM concentration stimulates cathodic peak (an electrochemical signal) of cytochrome Р450 3А4. At the presence of diclofenac (Voltaren) - a typical substrate of cytochrome Р450 3А4 - the increase growth of a catalytic current testifying to an electrocatalysis and stimulating action of ascorbic acid is observed. In the presence of vitamins A and Е also is registered dose-dependent (in a range of 10-100 M) increase in a catalytic current of cytochrome Р450 3А4: the maximum increase corresponds to 229 ± 20% for 100 M of vitamin A, and 162±10% for 100 M of vitamin E. Vitamin E in the presence of P450's inhibitor itraconazole doesn't give essential increase in a reductive current, unlike retinol (vitamin A). This effect can manifest substrate properties of tocopherol (vitamin E). The electrochemical approach for the analysis of catalytic activity of cytochrome Р450 3А4 and studies of influence of biologically active compounds on an electrocatalysis is the sensitive and effective sensor approach, allowing to use low concentration of protein on an electrode (till 10-15 mol/electrode), to carry out the analysis without participation of protein redox partners, and to reveal drug-drug or drug-vitamins interaction in pre-clinical experiments.

[Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes b5].

Molecular interactions between proteins redox partners (cytochromes P450 3A4, 3A5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 - -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 to testosterone.

[Electrochemical measurement of intraprorein and interprotein electron transfer].

Intramolecular and intermolecular direct (unmediated) electron transfer was studied by means of electrochemical techniques in flavohemoprotein cytochrome P450 BM3 (CYP102A1 from Bacillius megaterium) and between cytochrome b5 and cytochrome c. Flavohemoprotein cytochrome P450 BM3 was immobilized on a screen printed graphite electrode, modified with a biocompatible nanocomposite material based on the didodecyldimethylammonium bromide DDAB and gold nanoparticles. Analytical characterictics of DDAB/Au/P450 BM3 electrodes were studied with cyclic voltammetry and square wave voltammetry. It was shown that intramolecular electron transfer was realized between diflavin (FAD/FMN) and heme domain of CYP102A1. An electron transport chain of flavohemoprotein P450 BM3 immobilized at nanostructued electrode is realized as: electrode --> FAD --> FMN --> heme. Electron transfer occurs inside the protein, and it is an evidence of functional interaction between diflavin and heme domains. The effect of a substrate (lauric acid) or inhibitors (metyrapone or imidazole) binding on the electrochemical parameters of flavohemoprotein P450 BM3 was also studied. Interprotein electron transfer was analyzed between cytochrome b5 and cytochrome c. Electrochemical analysis revealed that electron transfer takes place in protein-protein complexes with participants possessing different redox potentials.

[Electrochemical sensor systems based on one dimensional (1D) nanostructures for analysis of bioaffinity interactions].

It was shown that modification of screen printed graphite electrodes with gold nanoparticles (AuNPs) decorated Pb nanowires (PbNWs) demonstrates the enhancement of sensor's analytical characteristics such as effective surface area, electro catalytic properties and heterogeneous electron transfer kinetics. The reason for such improvement may be the synergistic effect ofAuNPs and PbNWs. Nanowires ensembles on electrode surface were employed for the detection of hemeproteins cytochrome P450 2B4, cytochrome c, and cardiac myoglobin in human plasma. Composite materials based on nanoparticles with different dimentions (3D three dimensional gold nanoparticles and 1D one dimensional Pb nanowires make it possible to construct biosensors with low detection limit of proteins.

[Screening of potential substrates or inhibitors of cytochrome P450 17A1 (CYP17A1) by electrochemical methods].

The electrochemical reduction of the recombinant form of human cytochrome P450 17A1 (CYP17A1) was investigated. Hemeprotein was immobilized on electrode modified with biocompatable nanocomposite material based on the membrane-like synthetic surfactant didodecyldimethylammonium bromide (DDAB) and gold nanoparticles. Analytical characteristics of DDAB/Au/CYP17A1 electrodes were investigated with cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Analysis of electrochemical behaviour of cytochrome P450 17A1 was conducted in the presence of substrate pregnenolone (1), inhibitor ketoconazole (2), and in the presence of synthetic derivatives of pregnenolone: acetylpregnenolone (3), cyclopregnenolone (4), and tetrabrompregnenolone (5). Ketoconazole, azole inhibitor of cytochromes P450, blocked catalytic current in the presence of substrate pregnenolone (1). Compounds 3-5 did not demonstrate substrate properties towards electrode/CYP17A1 system. Compound 3 did not block catalytic activity towards pregnenolone, but compounds 4 and 5 inhibited such activity. Electrochemical reduction of CYP17A1 may serve as an adequate substitution of the reconstituted system which requires additional redox partners - for the exhibition of catalytic activity of hemoproteins of the cytochrome P450 superfamily.

[Electrochemical immunoanalysis of cardiac myoglobin].

Method targeting the direct monitoring of myoglobin based on analysis of electrochemical parameters of modified electrodes were proposed. Method of direct detection is based on interaction of myoglobin with anti-myoglobin with subsequent electrochemical registration of hemeprotein. Myocardial infarction biomarker myoglobin was quantified at biological level using screen printed electrodes modified with gold nanoparticles stabilized with didodecyldimethylammonium bromide (DDAB) and antibodies. Proposed method did not require signal enhancement and amplification and also labeled secondary antibodies. Electro analysis has high specificity and sensitivity. Myoglobin -antibodies interaction was studied also with electrochemical impedance spectroscopy. Sensor has low detection limit and broad diapason of working concentrations (17.8 ng/ml-1780 ng/ml; 1 nM-10 nM). Method based on gold nanoparticles detection on the surface of electrodes was treated for myoglobin identification. AuNP worked as an electrochemical sensing platform: the oxidation of gold surface (resulted in gold oxide formation) upon polarization served as a basis for analytical response. The difference of cathodic peak area and peak high of gold oxide reduction in the case of electrodes with antibodies and electrodes with antibodies-myoglobin complex, was registered.

[Sensor systems for medical application based on hemoproteins and nanocomposite materials].

Recent advances in nanotechnologies stimulate the development of sensor systems based on nanocomposite materials. This review discusses the prospects and challenges of sensors coupled with functionally important for medicine hemoproteins and nanoscale materials. Authors summarized their own experimental results and literature data on hemoprotein-based sensor systems. Mechanisms and the main function principles of electrochemical nanosensors are also discussed.

Electrochemistry of cytochromes p450: analysis of current-voltage characteristics of electrodes with immobilized cytochromes p450 for the screening of substrates and inhibitors.

In the current study, an approach to elucidating the substrate specificity of cytochromes P450 based on the analysis of current-voltage characteristics of voltammograms and amperograms is proposed. Data on the electrochemical behavior of bioelectrodes with immobilized cytochromes P450 2B4, 1A2, 3A4, 11A1 (P450scc), and 51b1 (Mycobacterium tuberculosis sterol 14alpha-demethylase or CYP51 MT) in the presence of typical substrates and inhibitors for these hemoprotein forms are reported. Immobilization of the enzymes was accomplished by using graphite screen-printed electrodes modified with gold nanoparticles and with the synthetic membrane-like compound didodecyldimethylammonium bromide. The method of electro-analysis can be applied to the search of potential substrates and inhibitors of cytochromes P450 and to creation of multichannel electrochemical plates (chips, panels) with immobilized cytochromes P450.

Electrochemical reduction of sterol-14alpha-demethylase from Mycobacterium tuberculosis (CYP51b1).

The electrochemical reduction of the heme protein sterol-14alpha-demethylase from Mycobacterium tuberculosis (CYP51b1, or further CYP51) was investigated. Direct electron transfer was demonstrated between CYP51 and graphite screen-printed electrodes modified with gold nanoparticles and with the membrane-like synthetic surfactant didodecyl dimethylammonium bromide. The formal potential of the Fe3+/Fe2+ pair, E(1/2), is equal to -273 mV (vs. Ag/AgCl). The cathodic current corresponding to the reduction of oxygen by immobilized heme protein was registered in the presence of oxygen. Addition of lanosterol, one of the substrates of the CYP51 family, to the oxygenated solution caused a concentration-dependent increase in the reduction current in voltammetric and amperometric experiments. Ketoconazole, an inhibitor of CYP51, inhibited the catalytic cathodic current in the presence of lanosterol. Electrochemical reduction of CYP51 may serve as an adequate alternative to the reconstituted system, which requires additional redox partners for the exhibition of catalytic activity of heme proteins of the cytochrome P450 superfamily.

Electrochemical properties of cytochroms P450 using nanostructured electrodes: direct electron transfer and electro catalysis.

The present study demonstrates direct electron transfer between cytochromes P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14alpha-demethylase (CYP51b1) on the one hand and screen-printed graphite electrodes, modified with gold nanoparticles and didodecyldimethylammonium bromide (DDAB) on the other. Electro detection of heme proteins was possible when 2-200 pmol P450/electrode were adsorbed on the surface of nanostructured electrochemical interfaces. Electron transfer, direct electrochemical reduction and interaction with P450 substrates (oxygen, benzphetamine, and lanosterol) and with P450 inhibitor (ketoconazole) were analyzed using cyclic voltammetry (CV), square wave voltammetry (SWV) differential pulse voltammetry (DPV), and amperometry.