title: Purification of glutathione reductase from chicken liver and investigation of kinetic properties.
abstract: Glutathione reductase was purified from chicken liver and some characteristics of the enzyme were investigated. The purification procedure was composed of four steps: preparation of homogenate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Owing to the four consecutive procedures, the enzyme was purified 1714-fold, with a yield of 38%. Specific activity at the final step was 120 enzyme unit (EU)/mg of protein. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was found to be 100 kDa by Sephadex G-200 gel filtration chromatography, and the subunit molecular weight was found to be 43 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, and optimum temperature were 7.0, 7.4, 0.75 M Tris-HCl buffer including 1 mM EDTA, and 50 degrees C, respectively. KM and Vmax values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme.
authors.affiliation: Biotechnology Application and Research Center, Atatürk University, 25240 Erzurum, Turkey.
authors.forename: Mustafa
authors.initials: M
authors.lastname: Erat
authors.forename: Hülya
authors.initials: H
authors.lastname: Demir
authors.forename: Halis
authors.initials: H
authors.lastname: Sakiroğlu
mesh_terms: D000645:Ammonium Sulfate; D000818:Animals; D002021:Buffers; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005590:Fractional Precipitation; D019803:Glutathione Disulfide / Q000378:metabolism; D005980:Glutathione Reductase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D009994:Osmolar Concentration; D013696:Temperature
chemical_list: D002021:Buffers; D009249:NADP; D005980:Glutathione Reductase; D000645:Ammonium Sulfate; D019803:Glutathione Disulfide