Application of capillary zone electrophoresis for the study and assay of enzymes
| Authors | |
|---|---|
| Year of publication | 2002 |
| Type | Article in Proceedings |
| Conference | Book of abstracts of 6th Meeting of biochemists and molecular biologists |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | EMMA; enzymes |
| Description | 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 in the capillary as distinct plugs, the first analyte injected being the one with the lower electrophoretic mobility. Upon the application of an electric field, these two zones interpenetrate due 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 substrates, Michaelis constants, inhibitors and inhibition constants, etc. In this communication the combination of the EMMA methodology with a partial filling technique was applied to study the kinetic parameters of bi-substrate enzymatic reaction of rhodanese. In this set-up the part of the capillary is filled with the buffer best for the enzymatic reaction whereas the rest of the capillary with the background electrolyte optimal for separation of substrates and products. The basic limitation of EMMA methodology 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 is thus overcome. |
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