Electrophoretically mediated microanalysis with partial filling technique and indirect or direct detection as a tool for inhibition studies of enzymatic reaction
| Authors | |
|---|---|
| Year of publication | 2004 |
| Type | Article in Proceedings |
| Conference | Proceeding of 17th International Symposium on Microscale Separations and Capillary Electrophoresis |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | capillary electrophoresis; microscale separations |
| Description | The study of enzymes remains of great importance to scientific community and to society in general. They are utilised in many industrial applications. The traditional roles of enzymes in food and beverage manufacturing are still in use today. In modern times, the role of enzymes in consumer products and in chemical manufacturing has expanded greatly. Perhaps one of the most exciting fields of modern enzymology is the application of enzyme inhibitors. Many of the drugs function by inhibiting specific enzymes those are associated with the disease process. Enzyme inhibitors are developed as pesticides to protect crops. Even lots of chemical warfare agents act as enzyme inhibitors. For that reason new methodological approaches are looked out for the inhibition studies of enzymatic reaction. The inhibition study of the model enzyme - haloalkane dehalogenase [EC 3.8.1.5] 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 best 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 CTAB (pH 9.2) in combination with indirect detection and 0.1 M beta-alanine HCl (pH 3.5) in combination with direct detection. For the first time the chromate background electrolyte containing tetraalkylammonium salt as an EOF modifier in combination indirect detection was used for this purpose. The method was used not only to estimate KI but also for the determination of the inhibition type. Furthermore it can be applied for the kinetic studies of the other enzymes dealing with of the high mobile inorganic or organic anions as substrates or products. |
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