On-line coupling of immobilized cytochrome P450 microreactor and capillary electrophoresis: A promising tool for drug development
| Autor: | Roman Řemínek, Jan Schejbal, Lukáš Zeman, Aleš Mádr, Zdeněk Glatz |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Diclofenac
Immobilized enzyme Capillary action Diffusion 010402 general chemistry Sulfaphenazole 01 natural sciences Biochemistry Analytical Chemistry chemistry.chemical_compound Capillary electrophoresis Bioreactors Cytochrome P-450 Enzyme System Drug Discovery medicine Carbodiimide Chromatography Chemistry 010401 analytical chemistry Organic Chemistry Substrate (chemistry) Electrophoresis Capillary General Medicine Enzymes Immobilized 0104 chemical sciences Kinetics Microreactor medicine.drug |
| Zdroj: | Journal of chromatography. A. 1437 |
| ISSN: | 1873-3778 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect