Direct assay of glutathione peroxidase activity using high-performance capillary electrophoresis.
A fast, sensitive and direct method has been developed for the determination of glutathione peroxidase activity (both selenium- and non-selenium-dependent) in cell-free preparations. The assay is based on the separation and quantitation of reduced and oxidized glutathione by capillary electrophoresis. The electrophoretic separation buffer was 100 mM sodium tetraborate (pH 8.2) containing 100 mM sodium dodecylsulphate. A micellar electrokinetic mechanism took place under these conditions, and a total mass recovery was observed for both peptides. The reproducibility of migration times was excellent (less than 3% variability). A linear detector response range was observed in the range 5-50 U/ml, and both the reproducibility and accuracy were satisfied. Samples out of this linear range could be analysed by either increasing the reaction time or diluting the enzyme preparation. The results obtained with the new direct capillary electrophoresis assay were compared with those derived from a reversed phase high-performance liquid chromatographic and spectrophotometric coupled assay. A very good agreement was found between the two direct assay methods in all samples. Capillary electrophoresis is a versatile technique that allows the automation of the glutathione peroxidase assay in a reproducible manner and within a relatively short time with sufficient accuracy and precision.
Pascual
P
P
Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Córdoba, Spain.
Martinez-Lara
E
E
Bárcena
J A
JA
López-Barea
J
J
Toribio
F
F
D000818:Animals; D002417:Cattle; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D004586:Electrophoresis / Q000379:methods*; D004912:Erythrocytes / Q000201:enzymology; D005399:Fishes; D005978:Glutathione / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000032:analysis*; D008099:Liver / Q000201:enzymology; D008297:Male; D010084:Oxidation-Reduction; D051381:Rats; D013056:Spectrophotometry, Ultraviolet
D005979:Glutathione Peroxidase; D005978:Glutathione