Purification of glutathione reductase from chicken liver and investigation of kinetic properties |
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Authors: | Mustafa Erat Hülya Demir Halis ?akiroglu |
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Institution: | (1) Biotechnology Application and Research Center, Atatürk University, 25240 Erzurum, Turkey;(2) Arts and Science Faculty, Department of Chemistry, Atatürk University, 25240 Erzurum, Turkey |
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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°C, respectively. K
M and V
max values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme. |
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Keywords: | Glutathione reductase purification chicken liver enzyme gel filtration chromatography affinity chromatography |
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