Rapid method for the determination of glutathione transferase isoenzymes in crude extracts.
The analysis of glutathione transferase (GST) isoenzyme patterns is of interest in many fields as hepatic glutathione transferase activity is increased by exposure to a variety of xenobiotics and its isoenzymatic forms are induced differentially. A high-performance liquid chromatography method has been developed for the rapid determination of individual isoenzyme levels in crude extracts using an anion-exchange column connected to an on-line system to automatically detect GST activity with 1-chloro-2,4-dinitrobenzene as the substrate. When 50-200 microliters of a cytosolic fraction of fish liver containing up to 15 mg/ml of protein and less than 2 units of GST were injected, a high resolution and highly reproducible chromatogram was obtained. The activity profile determined automatically showed eight to twelve peaks (depending on the sample) that were quantified and could be classified into three groups. Starting from intact tissue, a complete isoenzyme pattern could be obtained in less than 3 h. The method has been applied to ecotoxicological studies with fish samples.
Martínez-Lara
E
E
Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Córdoba, Spain.
Pascual
P
P
Toribio
F
F
López-Barea
J
J
Bárcena
J A
JA
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004137:Dinitrochlorobenzene / Q000378:metabolism; D005399:Fishes; D005982:Glutathione Transferase / Q000032:analysis*; D007202:Indicators and Reagents; D007527:Isoenzymes / Q000032:analysis*; D008099:Liver / Q000201:enzymology*
D004137:Dinitrochlorobenzene; D007202:Indicators and Reagents; D007527:Isoenzymes; D005982:Glutathione Transferase