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  • Title: Application of a new ion exchanger TSK-GEL DEAE-5PW, to the purification of Cu,Zn-superoxide dismutase of bovine erythrocytes.
    Author: Inouye K, Nakamura K, Mitoma Y, Matsumoto M, Igarashi T.
    Journal: J Chromatogr; 1985 Jun 26; 327():301-11. PubMed ID: 4030961.
    Abstract:
    We have developed an effective method for the purification of Cu,Zn-super-oxide dismutase [E.C. 1.15.1.1] from bovine erythrocytes. This enzyme functions as a scavenger of superoxide radical, and it seems to be a key enzyme in the metabolism of active oxygen species. Application of this enzyme as a drug has been considered, and for this purpose a highly purified preparation is necessary. The first purification of this enzyme was reported by McCord and Fridovich [J. Biol. Chem., 244 (1969) 6049]. The limiting step in their method is the removal of the small amount of contaminating protein from the acetone-precipitated crude preparation (3000 units/mg protein). We found that a new, high-performance ion exchanger, TSK-GEL DEAE-5PW, improves this step and makes possible the large-scale preparation of pure protein. Optimal conditions for a TSK-GEL DEAE-5PW preparative column (150 X 21.5 mm I.D.) were established. The acetone-precipitated crude enzyme was dissolved in 20 mM Tris-HC1 buffer (pH 7.5), protein concentration 11.0 mg/ml, applied to the column, and eluted with a linear gradient of sodium chloride from 0 to 0.3 M. The flow-rate and sample volume were set to 4.0 ml/min and 8.0 ml, respectively. Under these conditions, the fractionated Cu,Zn-superoxide dismutase showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ion exchange high-performance liquid chromatography, and a specific activity of 3800 units/mg protein. The recovery of activity was 75%, and the cycle time was 120 min. A yield of 80-85 mg of purified enzyme was obtained per cycle. The preparation thus isolated has the highest purity and activity so far reported. We conclude that TSK-GEL DEAE-5PW is the practical choice for large-scale purification of Cu,Zn-superoxide dismutase.
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