Electrophoretically mediated microanalysis with partial filling technique and indirect or direct detection as a tool for inhibition studies of enzymatic reactions
| Authors | |
|---|---|
| Year of publication | 2004 |
| Type | Article in Periodical |
| Magazine / Source | Electrophoresis |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | inhibition; EMMA; partial filling technique; direct or indirect detection |
| Description | The inhibition study of the model enzyme haloalkane dehalogenase from Sphingomonas paucimobilis was performed by a combination of the electrophoretically mediated microanalysis with a partial filling technique and indirect or direct detection. In this set-up, the part of capillary is filled with the buffer suitable for the enzymatic reaction 20 mM glycine buffer (pH 8.6) whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. Two different background electrolytes and corresponding detection approaches were used to show the versatility of the developed method. The inhibition effect of 1,2-dichloroethane on the dehalogenation of brominated substrate 1-bromobutane was studied by means of 10 mM chromate - 0.1 mM cetyltrimethylammonium bromide (pH 9.2) in combination with an indirect detection or 0.1 M beta-alanine - hydrochloric acid (pH 3.5) in combination with a direct detection. The method was used not only to estimate inhibition constant Ki 0.44 mM by means of indirect detection method and 0.63 mM by means of direct detection method but also to determine the inhibition type. Compared to spectrophotometric and other discontinuous assays, the method is rapid, can be automated, and requires only small amount of reagents that is especially important in the case of enzymes and inhibitors. Consequently it has a great potential for such determinations in other enzyme-inhibitor systems. |
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