Study of Enzymes by means of capillary electrophoresis (Lecture)
| Authors | |
|---|---|
| Year of publication | 2005 |
| Type | Article in Proceedings |
| Conference | Sborník konference "Chiranal 2005" |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | capillary electrophoresis; enzymes |
| Description | 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. Measurements of enzyme activity by CE, which have been under active investigation in recent years, include the enzyme reaction prior CE analysis and the enzyme reaction in the separation capillaries during CE 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. The applications of this new methodology on other enzyme systems will be also shortly given. |
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