Glutathione reductase (glutathione: NADP⁺ oxidoreductase, EC 1.8.1.7; GR) maintains the cellular level of reduced glutathione (GSH) by catalyzing the reduction of oxidized glutathione (GSSG) through a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reaction. Therefore, GR plays a crucial role in the cellular antioxidant defense system. In the present study, GR enzyme was purified from quail liver tissue in two steps using salting-out (ammonium sulfate precipitation) followed by affinity chromatography on 2',5'-ADP Sepharose-4B. The results indicated that the enzyme was purified 142.18-fold with a yield of 13.6% and a specific activity of 22.75 EU/mg protein. Enzyme activity was determined spectrophotometrically. The molecular mass of the GR enzyme subunits in its native state was estimated to be approximately 59 kDa by SDS–PAGE analysis. Characterization studies revealed that the optimum pH of the enzyme was 8.0 in K-phosphate buffer, while the optimum ionic strength was determined as 600 mM in the same buffer system. The optimum temperature for enzyme activity was found to be 85°C, and the enzyme exhibited maximum stability at pH 8.0 in Tris–HCl buffer. Kinetic parameters of the enzyme were also determined. The Km and Vmax values for NADPH, one of the physiological substrates of GR, were calculated as 0.109 mM and 0.603 EU/mL, respectively. For GSSG, the KM and Vmax values were determined to be 0.044 mM and 0.272 EU/mL, respectively.
TAHER et al. (Mon,) studied this question.