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.     

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 14α-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. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 4, pp. 542–549.     

Construction and characterization of bioelectrocatalytic sensors based on cytochromes P450.

Semisynthetic flavocytochromes RfP450 1A2, RfP450 2B4 and RfP450scc--molecular conjugates of protein with riboflavin--could be reduced on rhodium-graphite screen-printed thick film electrodes as was confirmed by cyclic voltammograms of immobilized enzymes. Amperometric enzyme electrodes for direct measurement of organic pollutants were developed. The efficiency of controlled potential electrolysis for the reduction of flavocytochromes P450 was comparable with traditional reduction by pyridine nucleotides. The rate constants for substrates conversion obtained by electrochemical methods were close to those obtained using NAD(P)H as an electron source.     

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.     

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

The electrochemical redox reactions of the recombinant form of human cytochrome P450 17A1 (CYP17A1) were investigated. The hemoprotein was immobilized on the electrode modified with a biocompatible nanocomposite material based on the membrane-like synthetic surfactant didodecyldimethylammonium bromide (DDAB) and gold nanoparticles. Analytical characteristics of DDAB/Au/CYP17A1 electrodes were investigated using cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Analysis of electrochemical behavior of cytochrome P450 17A1 was performed in the presence of a natural substrate, pregnenolone (1), known inhibitor, ketoconazole (2), and in the presence of synthetic derivatives of pregnenolone: acetyl pregnenolone (3), cyclopregnenolone (4), and tetrabromo-pregnenolone (5). Ketoconazole, the azole inhibitor of cytochromes P450, blocked catalytic current in the presence of the substrate, pregnenolone (1). Compounds 3–5 did not demonstrate substrate properties towards the electrode/CYP17A1 system. Compound 3 did not influence catalytic activity with pregnenolone, whereas compounds 4 and 5 demonstrated some inhibitory activity. Thus, electrochemical redox reactions of CYP17A1 may serve as an adequate substitution of the reconstituted system, which requires additional redox partners for manifestation of catalytic activity of hemoproteins of the cytochrome P450 superfamily.     

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.     

Can peroxygenase and microperoxidase substitute cytochrome P450 in biosensors

Aromatic peroxygenase (APO) from the basidiomycetous mushroom Agrocybe aegerita (AaeAPO) and microperoxidases (MPs) obtained from cytochrome c exhibit a broad substrate spectrum including hydroxylation of selected aromatic substrates, demethylation and epoxidation by means of hydrogen peroxide. It overlaps with that of cytochrome P450 (P450), making MPs and APOs to alternate recognition elements in biosensors for the detection of typical P450 substrates. Here, we discuss recently developed approaches using microperoxidases and peroxygenases in view of their potential to supplement P450 enzymes as recognition elements in biosensors for aromatic compounds. Starting as early as the 1970s, the direct electron transfer between electrodes and the heme group of heme peptides called microperoxidases has been used as a model of oxidoreductases. These MP-modified electrodes are used as hydrogen peroxide detectors based on the catalytic current generated by electrically contacted microperoxidase molecules. A similar catalytic reaction has been obtained for the electrode-immobilised heme protein AaeAPO. However, up to now, no MP-based sensors for substrates have been described. In this review, we present biosensors which indicate 4-nitrophenol, aniline, naphthalene and p-aminophenol based on the peroxide-dependent substrate conversion by electrode-immobilised MP and AaeAPO. In these enzyme electrodes, the signal is generated by the conversion of all substrates, thus representing in complex media an overall parameter. The performance of these sensors and their further development are discussed in comparison with P450-based electrodes.     

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 measurement of intraprotein and interprotein electron transfer

Intramolecular and intermolecular direct (unmediated) electron transfer was studied by electrochemical techniques in a flavohemoprotein cytochrome P450 BM3 (CYP102A1 from Bacillius megaterium) and between cytochromes b ₅ and c. P450 BM3 was immobilized on a screen printed graphite electrode modified with a biocompatible nanocomposite material based on didodecyldimethylammonium bromide (DDAB) and gold nanoparticles. Analytical characteristics of SPG/DDAB/Au/P450 BM3 electrodes were studied with cyclic voltammetry and square wave voltammetry. The electron transport chain in P450 BM3 immobilized on the nanostructured electrode is: electrode → FAD → FMN → heme; i.e., electron transfer takes place inside the cytochrome, in evidence of functional interaction between its diflavin and heme domains. The effects of substrate (lauric acid) or inhibitor (metyrapone or imidazole) binding on the electro-chemical parameters of P450 BM3 were assessed. Electrochemical analysis has also demonstrated intermolecular electron transfer between electrode-immobilized and soluble cytochromes properly differing in redox potentials.     

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.     

Molecular Lego: design of molecular assemblies of P450 enzymes for nanobiotechnology.

This paper reports on the application of the molecular Lego approach to P450 enzymes. Protein domains are used as catalytic (P450 BM3 haem domain and human P450 2E1) or electron transfer (flavodoxin and P450 BM3 reductase) modules. The objectives are to build assemblies with improved electrochemical properties, to construct soluble human P450 enzymes, and to generate libraries of new P450 catalytic modules based on P450 BM3. A rationally designed, gene-fused assembly (BMP-FLD) was obtained from the soluble haem domain of cytochrome P450 BM3 from Bacillus megaterium (BMP) and flavodoxin from Desulfovibrio vulgaris (FLD). The assembly was expressed successfully and characterised in its active form, displaying improved electrochemical properties. Solubilisation of the human, membrane-bound P450 2E1 (2E1) was achieved by fusing key elements of the 2E1 enzyme with selected parts of P450 BM3. An assembly containing the first 54 residues of P450 BM3, the whole sequence of P450 2E1 from residue 81 and the reductase domain of P450 BM3 was constructed. The 2E1-BM3 assembly was successfully expressed in the cytosol of Escherichia coli. The soluble form of 2E1-BM3 was reduced in carbon monoxide atmosphere and displayed the typical absorption peak at 450 nm, characteristic of a folded and active P450 enzyme. Finally, the alkali method previously developed in this laboratory was used to screen for P450 activity within a library of random mutants of P450 BM3. A number of variants active towards non-physiological substrates, such as pesticides and polyaromatic hydrocarbons were identified, providing new P450 catalytic modules. The combination of these three areas of research provide interesting tools for exploitation in nanobiotechnology.     

Epoxidation of styrene by human cyt P450 1A2 by thin film electrolysis and peroxide activation compared to solution reactions

Films of human cytochrome P450 1A2 (cyt P450 1A2) and polystyrene sulfonate were constructed on carbon cloth electrodes using layer-by-layer alternate absorption and evaluated for electrochemical- and H(2)O(2)-driven enzyme-catalyzed oxidation of styrene to styrene oxide. At -0.6 V vs. saturated calomel reference electrode in an electrochemical cell, epoxidation of styrene was mediated by initial catalytic reduction of dioxygen to H(2)O(2) which activates the enzyme for the catalytic oxidation. Slightly larger turnover rates for cyt P450 1A2 were found for the electrolytic and H(2)O(2) (10 mM) driven reactions compared to conventional enzymatic reactions using cyt P450s, reductases, and electron donors for cytochromes P450 1A2. Cyt P450(cam) gave comparable turnover rates in film electrolysis and solution reactions. Results demonstrate that cyt P450 1A2 catalyzes styrene epoxidation faster than cyt P450(cam), and suggests the usefulness of this thin-film electrolytic method for relative turnover rate studies of cyt P450s.     

Assembly of electroactive layer-by-layer films of hemoglobin and polycationic poly(diallyldimethylammonium)

Layer-by-layer (PDDA/Hb)(n) films were assembled by alternate adsorption of positively charged poly(diallyldimethylammonium) (PDDA) and negatively charged hemoglobin (Hb) at pH 9.2 from their aqueous solutions on pyrolytic graphite electrodes and other substrates. The assembly process was monitored and confirmed by quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV). CVs of (PDDA/Hb)(n) films showed a pair of well-defined, nearly reversible peaks at about -0.34 V vs SCE at pH 7.0, characteristic of Hb heme Fe(III)/Fe(II) redox couple. Positions of Soret absorption band and infrared amide II band of Hb in (PDDA/Hb)(8) films suggest that Hb in the films keeps its secondary structure similar to its native state. The electrochemical parameters of (PDDA/Hb)(8) films were estimated by square wave voltammetry, and the thickness of the PDDA/Hb bilayer was estimated by QCM and scanning electron microscopy. Trichloroacetic acid and nitrite (NO(2)(-)) were catalytically reduced at (PDDA/Hb)(8) film electrodes. The electrochemical catalytic reactions of O(2) and H(2)O(2) on (PDDA/Hb)(8) films were also studied.     

Electrochemical reduction of sterol-14α-demethylase from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> (CYP51b1)

The electrochemical reduction of the heme protein sterol-14α-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. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 6, pp. 805–811.     

Spectroelectrochemistry of cytochrome P450cam

The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described. The electrodes were modified with either 4,4(')-dithiodipyridin or sodium dithionite. Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV-visible absorption spectra were recorded. Reversible oxidation and reduction could be observed with both 4,4(')-dithiodipyridin and dithionite modified electrodes. A formal potential (E(0')) of -373mV vs Ag/AgCl 1M KCl was determined. The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis.     

Direct voltammetry and electrocatalytic properties of catalase incorporated in polyacrylamide hydrogel films

The direct voltammetry and electrocatalytic properties of catalase (Cat) in polyacrylamide (PAM) hydrogel films cast on pyrolytic graphite (PG) electrodes were investigated. Cat-PAM film electrodes showed a pair of well-defined and nearly reversible cyclic voltammetry peaks for Cat Fe(III)/Fe(II) redox couples at approximately -0.46 V vs. SCE in pH 7.0 buffers. The electron transfer between catalase and PG electrodes was greatly facilitated in the microenvironment of PAM films. The apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E degrees ') were estimated by fitting square wave voltammograms with non-linear regression analysis. The formal potential of Cat Fe(III)/Fe(II) couples in PAM films had a linear relationship with pH between pH 4.0 and 9.0 with a slope of -56 mV pH(-1), suggesting that one proton is coupled with single-electron transfer for each heme group of catalase in the electrode reaction. UV-Vis absorption spectroscopy demonstrated that catalase retained a near native conformation in PAM films at medium pH. The embedded catalase in PAM films showed the electrocatalytic activity toward dioxygen and hydrogen peroxide. Possible mechanism of catalytic reduction of H(2)O(2) at Cat-PAM film electrodes was proposed.     

Direct electrochemistry of hemoglobin in the hyaluronic acid films

Hemoglobin (Hb) in the hyaluronic acid (HA) was cast at pyrolytic graphite (PG) electrodes for researching its electrochemical and electrocatalytic properties. The formal potential and electron transfer rate constant of Hb on HA films were determined, and the stability of the films, the pH effect, and the influence of supporting electrolyte concentrations upon Hb electrochemistry on the films were investigated by cyclic voltammetry and square wave voltammetry. UV-Vis absorption and reflectance absorption infrared (RAIR) spectra showed that the protein on HA film retained near-native secondary structure. The stable Hb-HA/PG gave analytically useful electrochemical catalytic responses to hydrogen peroxide. Thus, the property of the HA film for sorption and retention of water maybe utilized to develop some new biosensors.     

Direct electrochemistry of hemoglobin in the hyaluronic acid films

Hemoglobin (Hb) in the hyaluronic acid (HA) was cast at pyrolytic graphite (PG) electrodes for researching its electrochemical and electrocatalytic properties. The formal potential and electron transfer rate constant of Hb on HA films were determined, and the stability of the films, the pH effect, and the influence of supporting electrolyte concentrations upon Hb electrochemistry on the films were investigated by cyclic voltammetry and square wave voltammetry. UV–Vis absorption and reflectance absorption infrared (RAIR) spectra showed that the protein on HA film retained near-native secondary structure. The stable Hb–HA/PG gave analytically useful electrochemical catalytic responses to hydrogen peroxide. Thus, the property of the HA film for sorption and retention of water maybe utilized to develop some new biosensors.     

Evaluation of alpha-cyano ethers as fluorescent substrates for assay of cytochrome P450 enzyme activity

We have previously reported the synthesis of four alpha-cyano-containing ethers based on 2-naphthaldehyde (2-NA) as cytochrome P450 (P450) fluorescent substrates. Activity detection was based on the formation of fluorescent 2-NA following substrate hydrolysis. A major limitation of these substrates was the need to remove NADPH, a required cofactor for P450 oxidation, before measuring 2-NA fluorescence. In this article, we report the synthesis of a new series of novel P450 substrates using 6-dimethylamino-2-naphthaldehyde (6-DMANA), which has a green fluorescent emission that is well separated from the NADPH spectrum. A major advantage of the 6-DMANA substrates is that NADPH removal is not required before fluorescence detection. We used eight alpha-cyano ether-based substrates to determine the O-dealkylation activity of human, mouse, and rat liver microsomes. In addition, substrate activities were compared with the commercial substrate 7-ethoxyresorufin (7-ER). The catalytic turnover rates of both the 6-DMANA- and 2-NA-based substrates were in some cases threefold faster than the catalytic turnover rate of 7-ER. The 2-NA-based substrates had greater turnover than did the 6-DMANA-based substrates. Murine and rat liver microsomes prepared from animals that had been treated with various P450 inducers were used to examine for isozyme-selective turnover of the substrates. The vastly improved optical properties and synthetic flexibility of the alpha-cyano ether compounds suggest that they are possibly good general P450 substrates.     

Study on an amperometric immunosensor based on Nafion-cysteine composite membrane for detection of carcinoembryonic antigen

In this work, protonated l-cysteine was entrapped in Nafion (Nf) membrane by cation exchange function, forming Nf-Cys (cysteine) composite membrane, which was more stable, compact, biocompatible, and favorable for mass and electron transfer compared with Nf film solely. Then gold (Au) nanoparticles were adsorbed onto the electrode surface by thiol groups on the composite membrane. After that, nano-Au monolayer was formed, onto which carcinoembryonic antibody was loaded to prepare carcinoembryonic antigen (CEA) immunosensor. The results indicated that the immunosensor had good current response for CEA using potassium ferricyanide as the redox probe. A linear concentration range of 0.01 to 100 ng/ml with a detection limit of 3.3 pg/ml (signal/noise = 3) was observed. Moreover, the morphology of the modified Au substrates was investigated with atomic force microscopy, and the electrochemical properties and performance of modified electrodes were investigated by cyclic voltammograms and electrochemical impendence spectroscopy. The results exhibited that the immunosensor has advantages of simple preparation, high sensitivity, good stability, and long life expectancy. Thus, the method can be used for CEA analysis.