Purification and characterization of superoxide dismutase from garlic

Abstract

An efficient and easily scaled up method to isolate superoxide dismutase from garlic is proposed. The separation and purification procedure consists of phosphate buffer extraction, heat treatment and a two-stage ultrafiltration process. The enzyme was purified 139-fold with a specific activity of 2867 U/mg protein and a yield of 91%. The native molecular mass of superoxide dismutase estimated by fast protein liquid chromatography on a Superose 6 column was 28 kDa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed a single band near 14 kDa, suggesting that native enzyme was homo-dimeric. The optimal pH for enzyme activity was found to be 7.0, and at this pH the enzyme exhibited maximum activity at 50 °C in 50 mM sodium phosphate buffer. Among various metal ions examined, Cu 2+ and Zn 2+ exerted a positive effect on superoxide dismutase activity, whereas Hg 2+ was found to be a strong inhibitor. The final purified enzyme had an isoelectric point of 5.1–5.4 and a sheet content of 46%, consistent with the literature values. This shows that the purified SOD folded with a reasonable secondary structure.