A NOVEL APPROACH IN CYTOCHROME P450 INHIBITORS’ SCREENING: IMMOBILIZED ENZYME MICROREACTOR FOR ON-LINE STUDIES USING CAPILLARY ELECTROPHORESIS
| Název česky | Nový přístup při screeningu inhibitorů cytochromů P450: Imobilizovaný enzymatický reactor pro on-line studie využívající kapilární elektroforézu |
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| Autoři | |
| Rok publikování | 2014 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | In this work we present an IMER integrated into capillary based on CYP 2C9 isoform, representing approximately 20 % of all CYP in human liver which is responsible for metabolizing more than 10 % of commonly used drugs. The CYP 2C9 was immobilized on magnetic microparticles Si-MAG carboxyl using the carbodiimide method and the formation of IMER was established by two permanent magnets placed in cassette for capillary electrophoresis. The enzymatic reaction was instantly followed by capillary zone electrophoresis separation so that the product could be detected and quantified with UV-DAD. Unfortunately CYP enzymes are membrane bound, which makes their immobilization rather cumbersome and displayed features are usually inferior in comparison to soluble enzymes, further optimization of incubation and separation was therefore necessary. All challenges were in the end overcome and optimized method was used to determine Michalis Menten constant for diclofenac and to characterise the inhibition of previous reaction by sulphophenazole. All acquired data were in good correlation with literature, proving this method suitable for inhibition studies. Furthermore the consumption of enzyme and reactants is significantly lower compared to generally used methods like fluorescence microassays. |
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