Determination of haloalkane dehalogenase activity by capillary zone electrophoresis

Abstract

A new sensitive method has been developed for the determination of haloalkane dehalogenase activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of product — bromide or chloride ions — was monitored by sequential capillary zone electrophoresis runs. The determinations were performed in a 75 μm fused-silica capillary using 5 m M chromate, 0.5 m M tetradecyltrimethylammonium bromide (pH 8.4) as a background electrolyte, separation voltage 15 kV (negative polarity) and indirect detection at sample wavelength 315 nm, reference wavelength 375 nm for brominated and chlorinated substrates, respectively 0.1 M β-alanine–HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity) and direct detection at 200 nm for brominated substrates. The temperature of capillary was in both cases 25°C. The method is rapid, can be automated, and requires only small amount of enzyme preparation and substrate.