Enzyme Assay for Glutathione Reductase Improved

Glutathione reductase assay GR activity was determined following the procedures of Halliwell and Foyer (1978) with slight modifications, by measuring the decrease in absorbance at 340 nm. The reaction mixture with a total volume of  1 ml contains 100 µl of    0.5 M  potassium phosphate containing 1 mM EDTA (pH 7.8), 810 µl of distilled water, 50 µl of crude extract, 20 µl of 10 mM NADPH in 50 mM potassium phosphate containing 1 mM EDTA (pH 7.8) and 20 µl of 10 mM oxidized glutathione(GSSG) in 10 mM potassium phosphate. The components were added in the 1-mL quartz cuvet   in the same order with GSSG as the last component. Since the enzyme extract was crude, 2 minutes was allotted for the nonspecific oxidation of NADPH before adding the GSSG. The reaction mixture was carefully mixed and read immediately at 340 nm  at an interval time  of 1.5 seconds for a  total time of  300 seconds using the DU 800 Spectrophotometer All measurements were made in triplicates. The activity of the enzyme was calculated using molar absorptivity constant of NADPH (6.2 mM^-1cm^-1). The activity of glutathione reductase was expressed in terms of µmol NADPH oxidized per mg protein per minute.


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