The aim of the project is to improve and enlarge the applicability of capillary electrophoresis especially its modification EMMA (Electrophoretically Mediated MicroAnalysis) method for the assay and study of membrane bound enzymes. Important group of detoxication enzymes – cytochromes P450 will be studied with respect to enzyme-drug interactions. Special attention will be paid on the combination of the EMMA methodology with a partial filling technique. In this EMMA modification introduced by Van Dyck et all. the part of capillary is filled with the buffer suitable for enzyme reaction whereas the rest of the capillary is filled with the background electrolyte optimal for separation of substrate(s) and product(s). The basic limitation of the EMMA method – the necessity to have the electrophoretic conditions compatible with both the separation of substrate(s) and product(s) of the enzymatic reaction and the enzymatic reaction itself – is thus eliminated. The methodology will be used for the assays and studies of different cytochrome P450 isoenzymes. Various modes of capillary electrophoresis – capillary zone electrophoresis, micellar electrokinetic capillary chromatography, even chiral capillary electrophoresis will be used depending on the nature of studied compounds and their metabolites. Detection will be performed by means of UV-VIS spectrometry; off-line MS will be used for the metabolite identification. Different sources of cytochromes P450 will be used – microsomal fraction, if available their recombinant forms. Main advantage of the method is low sample consumption what is especially important in the case of enzymes, substrates and inhibitors. Moreover the automatization of all assay steps – sampling, mixing, separation and quantitation not only minimised the possible errors but also saved the experimental time.

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