Electrophoretically mediated microanalysis as a tool for the enzyme study
| Authors | |
|---|---|
| Year of publication | 2006 |
| Type | Article in Proceedings |
| Conference | CD of the abstracts of the 29th international symposium on capillary chromatography |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | cytochromes P450; diclofenac; MEKC |
| Description | Fourteen years ago a new application for the evaluation of enzymatic reactions in capillary electrophoresis (CE) was proposed and developed, electrophoretically mediated microanalysis (EMMA). In this methodology, the capillary is used not only as a separation medium but also as a reaction chamber. 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. In this communication the EMMA methodology was applied to complex kinetic study of haloalkane dehalogenase from Sphingomonas paucimobilis UT26. The Michaelis constants for different substrates, the inhibition constant for inhibitor 1,2-dichloroethane, substrate inhibition of 1,2-dibromoethane and the effect of temperature on enzymatic reaction were evaluated by means of the EMMA methodology with partial filling technique. EMMA combines all advantages of CE in the study of enzyme kinetics. The minimal sample and reagent requirements as well as high separation efficiency of CE are fully utilŹized. The absence of manual procedures and complete automation miniŹmize the risk of cross contamination and strongly reduce the assay cost. |
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