Determination of Haloalkane Dehalogenase Activity by Capillary Zone Electrophoresis
Glatz, Z., Marini, M.V., Wimmerova, M., Damborsky, J., Nagata, Y.
JOURNAL OF CHROMATOGRAPHY A 895: 219-225 (2000)
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 monito red by sequential capillary zone electrophoresis runs. The determinations were performed in a 75 mm fused-silica capillary using 5 mM chromate, 0.5 mM 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 b-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 bot cases 25oC. The method is rapid, can be automated, and requires only small amount of enzyme preparation and substrate.