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.
Issledovany élektroanaliticheskie kharakteristiki rekombinantnogo tsitokhroma R450 3A4 (R450 3A4), immobilizovannogo na poverkhnosti pechatnykh grafitovykh élektrodov, modifitsirovannykh mnogostenochnymi uglerodnymi nanotrubkami. Pokazana rol' i ustanovleno vliianie modifikatsii grafitovogo rabochego élektroda uglerodnymi nanotrubkami na élektroanaliticheskie parametry étogo tsitokhroma. Optimizirovany usloviia ego immobilizatsii na poluchennykh pechatnykh grafitovykh élektrodakh, modifitsirovannykh mnogostenochnymi uglerodnymi nanotrubkami. Opredeleny élektrokhimicheskie parametry protsessa okisleniia i vosstanovleniia iona zheleza gema v sostave fermenta (polupotentsial (E0′) -0,35±0,01 V (otnositel'no khloridserebrianogo élektroda sravneniia, Ag/AgCl); élektrokhimicheskaia konstanta skorosti geterogennogo perenosa élektrona (ks) 0,57±0,04 s-1; kolichestvo élektroaktivnogo R450 3A4 na modifitsirovannom élektrode G0, mol'/sm2 (2,6±0,6)⋅10-10. Ustanovlen mekhanizm funktsionirovaniia étogo élektrokhimicheskogo sensora po printsipu “protein film voltammetry” (vol'tamperometrii, protekaiushcheĭ v plenke belka na poverkhnosti élektroda). Provedeno vol'tamperometricheskoe issledovanie kataliticheskoĭ aktivnosti immobilizovannogo R450 3A4 s tsel'iu razrabotki élektroanaliza lekarstvennykh preparatov, predstavliaiushchikh soboĭ substraty dannogo gemoproteina, dlia razrabotki biosensorov meditsinskogo naznacheniia. Issledovany élektrokataliticheskie svoĭstva rekombinantnogo P450 3A4, immobilizovannogo na modifitsirovannykh pechatnykh grafitovykh élektrodakh, po otnosheniiu k substratu – antibiotiku gruppy makrolidov éritromitsinu. Pokazano, chto modifikatsiia élektrodov igraet reshaiushchuiu rol' dlia issledovaniĭ svoĭstv tsitokhromov R450 v élektrokhimicheskikh issledovaniiakh. Smart-élektrody mogut vypolniat' funktsii kak élektroanalizatorov dlia analiticheskikh tseleĭ, tak i élektrokatalizatorov dlia issledovaniia protsessov biotransformatsii i metabolicheskikh protsessov. Élektrody, modifitsirovannye uglerodnymi nanomaterialami, primenimy dlia analiticheskikh tseleĭ pri registratsii gemoproteinov. Élektrody, modifitsirovannye sinteticheskimi membranopodobnymi soedineniiami (naprimer, didodetsildimetilammoniia bromidom), éffektivny pri provedenii ferment-zavisimogo élektrokataliza.
Keywords: carbon nanotubes; cytochrome P450; electroanalytical parameters; electrocatalysis; erythromycin.