Capillary electrophoretic assay of acyl-CoA hydrolase activity.
Acyl-CoA hydrolases catalyze the hydrolysis of fatty acyl CoA thioesters to free fatty acids and coenzyme A. These enzymes play an important role in the maintenance of cellular acyl-CoA and free CoASH pools and in the detoxification of nonphysiological metabolites. The assays most commonly used for acyl-CoA hydrolase quantitation are spectrophotometric and radioisotopic methods, both of which have limitations. In this study, capillary electrophoresis was used as an effective analytical technique to characterize rat liver peroxisomal acyl-CoA hydrolase reactivity using octanoyl-CoA as a substrate at different reaction conditions. The substrate and product of acyl-CoA hydrolase were identified by their migration times and quantitated using the peak areas. The enzyme activity exhibited a typical Michaelis-Menten pattern with increasing octanoyl-CoA concentration. The apparent Km and Vmax of octanoyl-CoA hydrolysis were determined using the enzyme activity at varying substrate concentrations. The rate of hydrolysis of octanoyl-CoA with increasing enzyme concentration appeared as a hyperbolic plot. The enzyme activity became elevated with increasing incubation time, showing the highest activity at 20 min, after which it started to decrease as the incubation time increased. Thus, capillary electrophoresis has been shown to be an effective, rapid, and reproducible method for the characterization of acyl-CoA hydrolase.
Panuganti
Sree Divya
SD
Department of Chemistry, Oakland University, Rochester, MI 48309-4477, USA.
Moore
Kathleen Healy
KH
D000214:Acyl Coenzyme A / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D004796:Clinical Enzyme Tests; D019075:Electrophoresis, Capillary / Q000379:methods*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010170:Palmitoyl-CoA Hydrolase / Q000032:analysis / Q000378:metabolism*; D051381:Rats
D000214:Acyl Coenzyme A; C034681:octanoyl-coenzyme A; D010170:Palmitoyl-CoA Hydrolase