A. I. Archakov

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 0.1xa0M 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) ofxa0-299xa0±xa016xa0mV andxa0-297.5xa0±xa021xa0mV (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.

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.

Interaction of 17α-hydroxylase, 17(20)-lyase (CYP17A1) inhibitors – abiraterone and galeterone – with human sterol 14α-demethylase (CYP51A1)

Abiraterone and galeterone induce type I differential spectral changes in human sterol 14α-demethylase (cytochrome P450 51A1, CYP51A1) with the sigmoidal shape of the binding curve. After approximation of the data by Hill model, the half-saturation concentrations (K0.5) were estimated as 22u202f±u202f1u202fμM and 16u202f±u202f1u202fμM and the Hill coefficients as 2.4u202f±u202f0.2 and 1.97u202f±u202f0.23 for abiraterone and galeterone, respectively. We analyzed the catalytic activity of CYP51A1 towards abiraterone and galeterone using an electrochemical system based on recombinant CYP51A1 immobilized on the screen-printed graphite electrode (SPE) modified by didodecyldimethylammonium bromide (DDAB) film. The study revealed the amperometric response of CYP51A1 upon addition of abiraterone, which may indicate the substrate properties of abiraterone towards CYP51A1. Galeterone caused negligible amperometric response of CYP51A1. Mass-spectrometric analysis of the products of CYP51A1-dependent electrocatalytic reaction at a controlled potential towards abiraterone and galeterone revealed products with m/z of 366.3 and 405.2, respectively, indicating monohydroxylation of abiraterone and galeterone. We have observed the sigmoidal character of the dependence of the catalytic current on abiraterone concentration. Analysis of molecular docking data demonstrated the ability of abiraterone and galeterone to bind to the active site of CYP51A1, but abiraterone occupies the position closer to the heme.

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.

Electrochemical methods in biomedical studies

Own experimental studies on the development of highly sensitive methods of electrochemical analysis applicable for biochemical research in the postgenomic era as well as electrochemical sensor systems for analysis of various biological objects have been summarized. Electroanalysis of catalytic activity of cytochrome P450 resulted in the development of a system for screening potential substrates and/or inhibitors of this class hemoproteins, as well as biologically active compounds modulating the catalytic function of this protein. The study of kinetics of bioaffinity troponin I/anti-troponin I (antibody to TnI) interactions in human plasma resulted in the development of a highly sensitive piezoelectric immunosensor, performing direct registration of biochemical interactions based on the difference of the kinetic parameters of specific and nonspecific bioaffinity interactions without additional administration of labels and without chemical modifications. The developed methods of direct registration of the electrochemical activity of bacterial cells Escherichia coli JM109 are applicable for real time evaluation of antibacterial activity of drug substances; this requires minimal volumes of cells (106 CFU/electrode). Special attention is paid to experimental data on preparation of polymers with molecular imprints (molecularly imprinted polymers, MIP) as analogues of antibodies and biorecognizing elements, carrying selective complementary analytes binding based on the “lock and key” principle.

Analysis of l -tyrosine based on electrocatalytic oxidative reactions via screen-printed electrodes modified with multi-walled carbon nanotubes and nanosized titanium oxide (TiO 2 )

Method for electrochemical determination of l-tyrosine with screen-printed electrodes (SPE) modified with multi-walled CNT or CNT/TiO2 as sensing elements was used for the electroanalysis of l-tyrosine (Tyr). It was demonstrated that SPE/CNT and SPE/CNT/TiO2 exhibited high electrocatalytic activity and good analytical performance towards oxidation of l-tyrosine. The linear range of Tyr in human serum was 0.025 ÷ 1xa0mM with the correlation coefficient R2u2009=u20090.97. Direct electrochemistry (without any mediator) of co-factor-free bovine serum albumin (BSA) and human serum albumin (HSA) was investigated by use of modified electrodes. Protein–ligand interactions based on the electrocatalytic oxidation of l-tyrosine during HSA interaction with hemin were analyzed by the change of peak height and oxidation peak area, corresponding to tyrosine oxidation accessibility.

Molecularly imprinted polymers (MIP) in electroanalysis of proteins

The review summarizes current knowledge on the main approaches used for creation of high affinity polymer analogs of antibodies (known as molecularly imprinted polymers, MIP) applicable for electroanalysis of functionally important proteins such as myoglobin, troponin T, albumin, ferritin, lysozyme, calmodulin. The main types of monomers for MIP preparation as well as methods convenient for analysis of MIP/protein interactions, such as surface plasmon resonance (SPR), nanogravimetry with the use of a quartz crystal resonator (QCM), spectral and electrochemical methods have been considered. Special attention is paid to experimental data on electrochemical registration of myoglobin by means of o-phenylenediaminebased MIP electrodes. It was shown that the imprinting factor calculated as a ratio of the myoglobin signal obtained after myoglobin insertion in MIP to the myoglobin signal obtained after myoglobin insertion in the polymer lacking the molecular template (NIP) is 2–4.

Determination of the “Amino Acid Conflicts” and amino acid substitutions in primary structures of 41 human proteins by the proteomic technologies

Proteomic studies of some human tissues and organs (skeletal muscles, myometrium, motor zone of the brain, prostate), and also cultivated myoblasts revealed 41 proteins, in which the presence of certain variants of amino acids (“conflicts”) was recognized at several “conflict” positions. Among the 93 registered “amino acid conflicts”, seven cases represented the results of the protein polymorphisms caused by corresponding substitution of individual amino acid. Proteomic analysis of prostate proteins revealed two isoforms of a prostate-specific antigen, formed due to alternative splicing. Thus, our results have shown that employment of the proteomic technologies may characterize various types of biochemical polymorphism in many human proteins.