title: 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.
abstract: 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.
authors.affiliation: Weed Science Laboratory, U.S. Department of Agriculture, Beltsville, Maryland, 20705, USA.
authors.forename: H A
authors.initials: HA
authors.lastname: Norman
authors.forename: P
authors.initials: P
authors.lastname: Pillai
mesh_terms: D002851:Chromatography, High Pressure Liquid; D005453:Fluorescence; D005980:Glutathione Reductase / Q000378:metabolism*; D009249:NADP / Q000032:analysis*; D010936:Plant Extracts / Q000737:chemistry*
chemical_list: D010936:Plant Extracts; D009249:NADP; D005980:Glutathione Reductase