Application of Capillary Zone Electrophoresis for the Study of Enzymes
| 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 |
| Popis | Enzymes are biological catalysts that play an important role in biochemical reactions necessary for normal growth, maturation and reproduction through whole live world. Due to their low concentrations in the samples containing large amount of other proteins, direct measurements of enzymes by masses are impossible. However enzymes can be measured more easily by their catalytic activities, which are the most relevant properties of enzymes in the biochemical context. The accurate measurement of enzymatic activity in biological samples is important in many fields of biochemistry, not only in routine biochemistry and in fundamental research but also in clinical and pharmacological research and diagnosis. The enzyme assay is also important procedure in elucidation of enzyme properties and function. Determination of kinetic parameters is usually undertaken to characterize an enzyme, to provide a quantitative evaluation of substrate specificity and to study kinetic mechanisms. In the last decade capillary electromigration separation techniques have become powerful techniques, which can provide highly efficient separations and large peak capacities. Today the availability of sophisticated automated equipment for CZE makes the technique suitable to be implement in routine laboratories for separation of a variety of molecules including metal ions, organic and inorganic ions, drugs, peptides, proteins and DNA fragments. All these parameters predict CZE as one of the method of choice also for determination of enzyme activities. Measurements of enzyme activity by CZE, which have been under active investigation in recent years, include the enzyme reaction prior CZE analysis and the enzyme reaction during CZE also known as electrophoretically mediated microanalysis (EMMA). Both these approaches were applied in the complex study on rhodanese, important enzyme catalysing detoxication of cyanide to less toxic thiocyanate after reaction with a sulfur donor, such as thiosulfate. The enzyme activity of rhodanese and the effects of temperature and pH on enzymatic reaction were evaluated by the off-capillary approach, whereas the kinetic parameters such as the Michaelis and inhibition constants were determined by means of the EMMA methodology. |
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