Electrophoretically mediated microanalysis of rhodanese
| Autoři | |
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| Rok publikování | 2002 |
| Druh | Článek ve sborníku |
| Konference | Proceeding of the 13th International Symposium on Capillary Electroseparation Techniques ITP 2002 |
| Fakulta / Pracoviště MU | |
| Citace | |
| Obor | Biochemie |
| Klíčová slova | EMMA; rhodanese |
| Popis | Ten years ago a new application for the evaluation of enzymatic reactions in capillary electrophoresis was proposed and developed, electrophoretically mediated microanalysis (EMMA). In this method, substrate(s) and enzyme are introduced into the capillary as distinct plugs, the first analyte injected being the one with the lower electrophoretic mobility. Upon the application of an electric field, the two zones interpenetrate due to the differences in their electrophoretic mobilities. Enzymatic reaction takes place and the resultant reaction product(s) and the unreacted substrate(s) are electrophoretically transported towards detector, where they are individually detected. Since its discovery by Bao and Regnier the EMMA methodology has been utilized in a number of biochemical systems - for assays of enzyme activities, determinations of substrates, Michaelis constants, etc. In this work, the EMMA method was applied to determine the kinetic parameters of rhodanese. Rhodanese (thiosulfate: cyanide sulfur transferase, EC 2.8.1.1) is responsible for the transfer of the sulfane sulfur of thiosulfate to an acceptor, which is likely to be cyanide under some physiological conditions: S2O32- + CN- = SCN- + SO32- Its physiological role has been debated for many years with proposals ranging from the detoxication of cyanide to suggestion that rhodanese is important in the bioenergetic oxidation of thiosulfate, in the generation of iron-sulfur protein complexes, in the lipoate metabolism and in the reactivation of nitrogenase. Several kinetic parameters of rhodanese was determined by means of EMMA - the Michaelis constant (Km) for thiosulfate and cyanide, Ki values of 2-oxoglutaric acid and the temperature optimum of the enzymatic reaction. Moreover, the kinetic mechanism of enzymatic reaction and types of inhibition of inhibitor were also elucidated. By best of our knowledge it is the first application of EMMA method for this purpose. |
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