Application of a new ion exchanger TSK-GEL DEAE-5PW, to the purification of Cu,Zn-superoxide dismutase of bovine erythrocytes

• Published in: Journal of Chromatography A 1985-01, Vol.327, p.301-311
• Main Authors: Inouye, Kuniyo, Nakamura, Koji, Mitoma, Yasutami, Matsumoto, Masako, Igarashi, Tatsuo
• Format: Article
• Language: English
• Subjects: cattle; erythrocytes; ion-exchange chromatography; superoxide dismutase
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(01)81659-9

We have developed an effective method for the purification of Cu,Zn-superoxide 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 × 21.5 mm I.D.) were established. The acetone-precipitated crude enzyme was dissolved in 20 m M Tris—HCl 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.