Purification of glutathione reductase from chicken liver and investigation of kinetic properties.
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.
Erat
Mustafa
M
Biotechnology Application and Research Center, Atatürk University, 25240 Erzurum, Turkey.
Demir
Hülya
H
Sakiroğlu
Halis
H
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
D002021:Buffers; D009249:NADP; D005980:Glutathione Reductase; D000645:Ammonium Sulfate; D019803:Glutathione Disulfide