Purification and characterization of glutathione reductase enzyme from sheep heart tissue

Glutathione reductase (GR; E.C.1.8.1.7) is an enzyme that catalyzes the reduction of GSSG to GSH and is a member of the flavin coenzyme (FAD) and pyridine-nucleotide (NADPH) dependent disulfide oxidoreductase family. In this study, glutathione reductase enzyme was purified from sheep heart tissue. In this purification process, 30-70% ammonium sulfate precipitation and 2', 5'-ADP Sepharose 4B affinity chromatography methods were applied. GR enzyme, which has a protein specific activity of 5.35 EU/mg, was purified 191-fold with a yield of 72.82%. The purity of the enzyme was checked by SDS-PAGE method and the molecular mass was found to be approximately 70 kDa under denaturing conditions. In the characterization studies, the optimum pH, stable pH, optimum ionic strength and optimum temperature values of the enzyme were found to be 6.0 (in 0.1 M KH2PO4 buffer), 8.0 (in 0.1 M KH2PO4 buffer), 240 mM (0.1 M KH2PO4) and 70°C, respectively. In addition, the KM and Vmax values for NADPH and GSSG substrates were determined to be 7.776 mM, 0.1678 EÜ/mL and 0.593 mM, 0.0306 EÜ/mL, respectively.