High-performance liquid chromatographic resolution of NADP+ after induction of fluorescence and its application to assay for an NADPH-dependent enzyme: application to the determination of glutathione reductase activity in plant leaf extracts.
A method is described for the determination of glutathione reductase activity (GR; EC 1.6.4.2) in plant extracts utilizing HPLC quantitation of NADP+ following the reduction of glutathione disulfides. After protein incubation, fluorescence of NADP+ was induced under strongly basic conditions, and the product was directly resolved from the reaction medium by isocratic reversed-phase elution on a silica-coated alumina support which took 2 min. The mobile phase was acetonitrile-water (50:50) delivered at a flow rate of 1.5 ml/min. The adduct (stable for at least 7 days) was detected fluorometrically and quantitated by direct integration of peak area.
Norman
H A
HA
Weed Science Laboratory, U.S. Department of Agriculture, Beltsville, Maryland, 20705, USA.
Pillai
P
P
D002851:Chromatography, High Pressure Liquid; D005453:Fluorescence; D005980:Glutathione Reductase / Q000378:metabolism*; D009249:NADP / Q000032:analysis*; D010936:Plant Extracts / Q000737:chemistry*
D010936:Plant Extracts; D009249:NADP; D005980:Glutathione Reductase