Study of Enzyme Kinetics of Phenol Sulfotransferase using EMMA
| Autoři | |
|---|---|
| Rok publikování | 2003 |
| Druh | Článek ve sborníku |
| Konference | Proceeding of 27th Symposium on HPLC and Related Techniques |
| Fakulta / Pracoviště MU | |
| Citace | |
| Obor | Biochemie |
| Klíčová slova | CZE; Enzymes; Phenol Sulfotransferase |
| Popis | Sulfonation represents a major pathway for the biotransformation/detoxication of drugs and xenobiotics, as well as endogenous compounds such as cholesterol, catecholamines, steroid and thyroid hormones, and bile acid. The responsible enzymes are called the cytosolic sulfotransferases. This family of sulfotransferases (SULT) belongs to the Phase II group of drug metabolizing enzymes and catalyzes the sulfonation reaction involving the transfer of an electrophilic sulfonate group from the co-substrate adenosine 3-phosphate 5-phosphosulfate (PAPS) to a nucleophilic acceptor substrate. Humans have at least 11 different SULT enzymes, forming three subfamilies based on their amino acid sequence identity and substrate specifity: SULT1 (phenol sulfotransferase family), SULT2 (hydroxysteroid sulfotransferase family), SULT4 (brain sulfotransferase). In this work we used an isoenzyme from the SULT1A subfamily that is responsible for the sulfonation of small phenolic substrates such as p-nitrophenol, 2-naphtol and dopamine. The purpose of this study was to determine whether it is possible to assay SULT1A (EC 2.8.2.-) activity with p-nitrophenol and dopamine as different substrates by capillary electrophoresis. Capillary electrophoretic systems have been successfully applied for on-line enzyme-catalyzed reactions by a methodology known as electrophoretically mediated microanalysis (EMMA), firstly described by Bao and Regnier. EMMA utilizes the different electrophoretic mobilities of enzyme, substrate, and product to initiate reaction inside the capillary and to separate the components from each other for final in-capillary quantitation. Compared to spectrophotometric and other discontinuous assays, the method is rapid, can be automated, does not need expensive radiolabeled substrates and requires only small amounts of reagents, which is especially important in the case of enzymes (injection volumes of analytes are in the order of nl). In consequence the EMMA methodology could serve as a progressive tool of modern enzymology in the context of metabolic research. |
| Související projekty: |