title: On-line coupling of immobilized cytochrome P450 microreactor and capillary electrophoresis: A promising tool for drug development.
abstract: In this work, the combination of an immobilized enzyme microreactor (IMER) based on the clinically important isoform cytochrome P450 2C9 (CYP2C9) with capillary electrophoresis (CE) is presented. The CYP2C9 was attached to magnetic SiMAG-carboxyl microparticles using the carbodiimide method. The formation of an IMER in the inlet part of the separation capillary was ensured by two permanent magnets fixed in a cassette from the CE apparatus in the repulsive arrangement. The resulting on-line system provides an integration of enzyme reaction mixing and incubation, reaction products separation, detection and quantification into a single fully automated procedure with the possibility of repetitive use of the enzyme and minuscule amounts of reactant consumption. The on-line kinetic and inhibition studies of CYP2C9's reaction with diclofenac as a model substrate and sulfaphenazole as a model inhibitor were conducted in order to demonstrate its practical applicability. Values of the apparent Michalis-Menten constant, apparent maximum reaction velocity, Hill coefficient, apparent inhibition constant and half-maximal inhibition concentration were determined on the basis of the calculation of the effective substrate and inhibitor concentrations inside the capillary IMER using a model described by the Hagen-Poisseulle law and a novel enhanced model that reflects the influence of the reactants' diffusion during the injection process.
authors.affiliation: Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Schejbal
authors.affiliation: Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Roman
authors.initials: R
authors.lastname: Řemínek
authors.affiliation: Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Lukáš
authors.initials: L
authors.lastname: Zeman
authors.affiliation: Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Aleš
authors.initials: A
authors.lastname: Mádr
authors.affiliation: Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic. Electronic address: glatz@chemi.muni.cz.
authors.forename: Zdeněk
authors.initials: Z
authors.lastname: Glatz
mesh_terms: D019149:Bioreactors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D004008:Diclofenac / Q000737:chemistry; D004058:Diffusion; D055808:Drug Discovery / Q000295:instrumentation* / Q000379:methods*; D019075:Electrophoresis, Capillary; D004800:Enzymes, Immobilized / Q000378:metabolism; D007700:Kinetics
chemical_list: D004800:Enzymes, Immobilized; D004008:Diclofenac; D003577:Cytochrome P-450 Enzyme System
keywords: Capillary electrophoresis; Cytochrome P450; Drug metabolism; Immobilized enzyme reactor