Purification, characterization of glutathion reductase enzyme from sheep splen tissue and investigation of the effects of some chemical substances on enzyme activity

In this study, the enzyme Glutathione reductase (Glutathione: NADP+ oxidoreductase, EC 1.8.1.7; GR), which is the key enzyme of antioxidant metabolism, was purified from sheep spleen tissue. Purification was carried out using 20-70% ammonium sulfate precipitation and 2', 5'-ADP Sepharose 4B affinity chromatography. As a result, the enzyme was purified in 40.61% yield and 1564.8 fold. The purity level of was checked by SDS-PAGE and characterizated. In characterization studies; optimum pH: 8.0 (KH2PO4 buffer), optimum ionic strength: 100 mM (KH2PO4 buffer), stable pH: 7.0 (KH2PO4 buffer), optimum temperature: 400C, subunit molecular mass: 91 kDa. In addition, KM constant and Vmax values of the substrates were determined in order to determine the affinity of the enzyme to GSSG and NADPH substrates. For NADPH; KM constant: 0.0061 mM, Vmax value was 0.259 EU/mL, for GSSG, KM constant: 0.351 mM, Vmax value was 0.604 EU/mL. According to these results; It was determined that the affinity of the GR enzyme to the NADPH substrate was higher than its affinity to the GSSG substrate. In addition, the effects of some drugs on sheep spleen GR enzyme activity were investigated. It was determined that ampicillin, streptomycin sulfate, gentamicin, cefoperazone sodium and precort-lyo showed inhibitory effects. IC50 values of these drugs; precort-lyo: 1.27 mM, ampicillin: 3.22 mM, streptomycin sulfate: 7.95 mM, cefoperazone sodium: 16.97 mM, gentamicin: 17.20 mM, Ki constants; ampicillin : 1.057 ± 0.110, gentamicin : 20.770 ± 8.169, streptomycin sulfate : 3.386 ± 1.305, cefoperazone sodium : 4.910 ± 0.960 and precort-lio : 0.466 ± 0.387, inhibition types; Ampicillin and gentamicin were determined as noncompetitive, streptomycin sulfate, cefoperazone sodium and precort-lyo were determined competitively.