[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.

[The influence of vitamin B group on monooxygenase activity of cytochrome P450 3A4: pharmacokinetics and electro analysis of catalytic properties].

It was shown that vitamin B group permit to shorten the longitude of diclofenak therapy and to reduce the daytime dose of this drug. All three schemes of diclofenac treatment - only diclofenac, diclofenac plus 2 tablets of Gitagamp (mixture of vitamin B group), and diclofenac plus 4 tablets of Gitagamp--gave maximum value of diclofenal in blood through 1 hour after treatment. In the case of diclofenak treatment without vitamins Cmax corresponds to 1137.2 +/- 82.4 ng/ml, with 2 tablets of Gitagamp--Cmax 1326.7 +/- 122.5 ng/ml, and with 4 tablets--Cmax 2200.4 +/- 111.3 ng/ml. Positive influence of vitamin B group on the decrease of pain syndrome was shown. Pharmacodynamics and pharmacokinetics data were confirmed in electrochemical experiments with cytochrome P450 3A4. For enzyme immobilization screen printed graphite electrodes modified with gold nanoparticles and synthetic membrane-like compound didodecyldimethylammonium bromide (DDAB/Au) were used. Electrochemical analysis reviled the influence of vitamin B group on metabolism of non steroid anti inflammation drug diclofenac catalyzed by cytochrome P450 3A4. Riboflavin was the most effective inhibitor of diclofenac hydroxylation by cytochrome P450 3A4 as was compared at 300 M concentration of vitamin B group (B1, B2, B6). These data confirmed the opportunity of pharmacokinetic parameters regulation and the level of pharmacodynamic effects by the influence of vitamin B group on the catalytic activity of cytochrome P450 3A4.

[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.

[Nanoelectrochemistry of cytochrome P450s: direct electron transfer and electrocatalysis].

The present study demonstrates the direct electron transfer between cytochrome P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14alpha-demethylase (CYP51MT) and screen printed graphite electrodes, modified with gold nanoparticles and didodecyldimethylammonium bromide (DDAB). Electrodetection of heme proteins is 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, lanosterol) and inhibitor ketoconazole were analyzed using cyclic voltammetry (CV), square wave (SWV) or differential pulse (DPV) voltammetry, amperometry.

[Electrochemical reduction of cytochrome P450s based on electrodes with immobilized substrates].

A new approach for the electrochemical reduction of cytochromes P450 (P450s, CYPs) with electrodes chemically modified with appropriate substrates of P450s ("reverse" electrodes) has been proposed. The method is based on the analysis of cyclic voltammograms, square wave voltammograms, amperograms and determination of such electrochemical characteristics as catalytic current and redox potential. The sensitivity of the proposed method is 0.2-1 nmol P450/electrode. The differences of maximal current and potentials in square wave voltammograms and catalytic current in amperometric measurements are more sensitive and reliable. Planar regime of screen-printed electrodes permits to use 20-60 microl of electrolyte volume. We investigated P450 2B4--benzphetamine or P450scc--cholesterol enzyme - substrate pairs. Electrochemical parameters of electrodes with nonspecific P450 substrate were differed from electrodes with appropriate substrates.

[Electrochemical reduction of cytochrome P450 as a way for construction of biosensors and bioreactors]. / Elektrokhimicheskoe vosstanovlenie tsitokhromov P450--put' k sozdaniiu biosensorov i bioreaktorov.

The present review describes the data on electrochemical reduction of cytochrome P450. Three generations of enzyme biosensors have been considered. The concept and potentialities of enzyme electrodes--transducers--as the main element on construction of electrochemical biosensors are described. Different types of electrodes for bioelectrochemistry are presented. New experimental approaches for immobilisation of cytochrome P450 based on nanotechnology are reported. Nanobiotechnology in electrochemistry has potential application for production of biosensors and bioreactors for medicine

[Study of interaction of cytochrome P450 2B4 with riboflavin by fluorescence spectrometry]. / Issledovanie vzaimodeistviia tsitokhroma P450 2B4 s riboflavinom metodami fluorestsentnoi spektroskopii.

Fluorescence quenching of riboflavin by cytochrome P450 2B4 was used to probe the ligand--enzyme binding interaction ((lambda ex = 385 nm, lambda em = 520 nm). Riboflavin is a component of a flavoprotein NADPH dependent cytochrome P450 reductase, an essential electron carrier during cytochrome P450 catalysis. Fluorescence titration measurements revealed that cytochrome P450 2B4 and riboflavin formed a complex with an apparent Kd = 8.8 +/- 1 microM. The fluorescence intensity of riboflavin decreased upon the addition of cytochrome P450 2B4, which may be caused by the resonance excitation energy transfer from the fluorescent donor riboflavin to the cytochrome P450 2B4 heme acceptor. These data suggest that there may exist specific sites of binding of riboflavin with the protein globule of cytochrome P450 2B4.

[Photoreduction of flavocytochrome P450 2B4]. / Fotovosstanovlenie flavotsitokhorma P450 2B4.

It was shown that noncovalent complexes of riboflavins and cytochrome P450 2B4 (flavocytochrome P450 2B4) can be used for photoinduced intramolecular electron transfer between the isoalloxazine cycle of flavins and the cytochrome P450 2B4 heme. The measurement of the photocurrent generated by photoreduction of noncovalent flavocytochrome P450 2B4 was carried out. It was found that, in the presence of typical substrates for cytochromes P450, the cathode photocurrent generated by both riboflavin and a mixture of riboflavin with cytochrome P450 decreases. A comparison of photocurrents in the presence and absence of substrates enabled one to register xenobiotics in solutions and use the photosensitivity of artificial flavocytochrome P450 for the determination of xenobiotic concentration. It was demonstrated that artificial flavocytochromes may serve as molecular amplifiers of the photocurrent generated upon the reduction of flavins. The introduction of flavin residues into the cytochrome P450 molecule transformed this hemoprotein into a photoreceptor and a photodiod and, in addition, into a photoactivated enzyme.

[Peroxide-dependent oxidation of substrates of polysynthetic flavocytochrome 2B4]. / Peroksid-zavisimoe okislenie substratov polusinteticheskim flavotsitokhromom 2B4.

Semisynthetic flavocytochromes, obtained by covalent binding of riboflavins with cytochrome P450 2B4, were able to catalyse H2O2-supported aniline p-hydroxylation, amidopyrine N-demethylation and p-nitroanisole O-dealkylation. Rates of these reactions were considerably higher than the rates of corresponding NAD(P)H-dependent reactions and comparable with H2O2-dependent reactions, catalysed by cytochrome P450. Kinetic parameters (Km and kcat) of hydrogen peroxide-supported oxidation of aniline, amidopyrine and p-nitroanisole in the presence of flavocytochrome 2B4 were determined. Cyanide and SKF-525 A inhibited H2O2-dependent reactions of flavocytochrome 2B4. The intensity of luminol chemiluminescence in the presence of hydrogen peroxide was studied. Chemiluminescent response was shown to be influenced by cyanide ions and SKF 525 A. The difference in chemiluminescence intensity of cytochrome P450 2B4 and semisynthetic flavocytochrome 2B4 was found.

[The use of alternative sources of electrons in mono-oxygenase reactions catalyzed by cytochrome P450. Physico-chemical methods as factors acting on enzymatic activity of proteins]. / Ispol'zovanie al'ternativnykh istochnikov élektronov v monooksigenaznykh reaktsiiakh, kataliziruemykh tsitokhromami P450. Fiziko-khimicheskie metody kak faktory vozdeistviia na fermentativnuiu aktivnost' belkov.

Influence of physico-chemical methods on catalytic activities of enzymes, (cytochrome P450 in particular), has been reviewed. Authors discussed the properties of semisynthetic flavohemoproteins based on cytochrome P450. With emphasis on recovery of alternative electron sources for redox enzymes. As alternative electron sources we have discussed electrochemical methods which allow electrons to be used as a reagent for redox system and photoreduction of photosensitive molecules. The effects of the chemical modification, high pressure, temperature, organic solvents on enzyme activity have also been analysed.

[The interaction of organophosphorus amino acid analogs with cytochrome P-450 2B4]. / Vzaimodeistvie fosfororganicheskikh analogov aminokislot s tsitokhromom P450 2B4.

Interaction between phosphorus amino acids analogs, 1-aminoalkylphosphonous and 1-aminoalkylthiophosphonic acids, and microsomes from the liver of phenobarbital-induced rabbits was studied. The phosphorus amino acids analogs cause type I and reverse type I spectral changes, respectively. A new reaction in the microsomal monooxygenase system was revealed. In the presence of NADPH, 1-aminoalkylphosphonous acids can be transformed to the corresponding 1-aminoalkylphosphonic acids by the reaction P-H-->P-OH. The reaction was monitored by 1H-NMR spectroscopy. 1-Aminoalkylphosphonous acids also serve as substrates for the NADPH-dependent monooxygenase system. 31P-NMR has shown that the oxidative desulfuration produces 1-aminoalkylphosphonous acids according to the reaction P=S-->P=O. Neither 1-aminoalkylphosphonous nor 1-aminoalkylphosphonic acids are deaminated in the NADPH-dependent monooxygenase system as follows from 1H-NMR spectroscopy.

[Inhibition of the angiotensin-converting enzyme from bovine kidney by organophosphorus analogs of amino acids]. / Ingibirovanie angiotenzinprevrashchaiushchego fermenta iz pochek byka fosfororganicheskimo analogami aminokislot.

Reactions of amino acid phosphoorganic derivatives with angiotensin-converting enzyme (dipeptidyl carboxypeptidase) were studied. Substitution of the amino acid carboxyl group by HS- or P(O) (OH)2-groups did not cause the enzyme considerable inhibition. The enzymatic activity was inhibited by 20-50% in presence of 1 X 10(-3) M Asp, Met and Cys phosphoorganic derivatives. These amino acid derivatives may be used as potential antihypertensive drugs because of their metabolic stability and inhibitory action on the enzyme.