Study of recombinant cytochrome P450 2C9 activity with diclofenac by MEKC
| Autor: | Zdenek Glatz, Josef Tomandl, Jan Jurica, Jiri Konecný |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Diclofenac
Metabolite Clinical Biochemistry Kinetics Sensitivity and Specificity 030226 pharmacology & pharmacy 01 natural sciences Biochemistry Michaelis–Menten kinetics Analytical Chemistry 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Humans Chromatography Micellar Electrokinetic Capillary Cytochrome P-450 CYP2C9 chemistry.chemical_classification Chromatography biology Chemistry 010401 analytical chemistry Reproducibility of Results Substrate (chemistry) Cytochrome P450 Metabolism Recombinant Proteins 3. Good health 0104 chemical sciences Enzyme biology.protein Aryl Hydrocarbon Hydroxylases Drug metabolism |
| Zdroj: | ELECTROPHORESIS. 28:1229-1234 |
| ISSN: | 1522-2683 0173-0835 |
| Popis: | Cytochrome P450 2C9 (CYP2C9) is one of the most important isoforms in human liver involved in the metabolism of a large number of therapeutic agents. The aim of this paper is to demonstrate the applicability of CE for the determination of the enzymatic activity of CYP2C9 with diclofenac as a probe substrate. MEKC with SDS as a pseudostationary phase was used for this purpose. Compared to other assays, the MEKC-based method is rapid, can be automated and requires only a small quantity of enzymes and substrate. Moreover, the enzymatic reaction can be monitored with high sensitivity and repeatability even when the reaction mixture is used for the analysis without any pretreatment. The kinetic study on the given enzymatic reaction was also performed since the basic characterization of drug biotransformation generally begins with the enzyme kinetic analysis of metabolite formation. As a result, the Michaelis constant and maximum reaction velocity were evaluated, the values 3.44 +/- 0.45 microM and 19.78 +/- 0.76 nmol min(-1) nmol(-1), respectively, were in agreement with the literature data. On the other hand, a slight deviation from typical Michaelis-Menten kinetics with a weak positive cooperativity was found at diclofenac concentrations below 2 microM. The same atypical kinetic behavior of CYP2C9 was also observed by other authors. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text