Purification and partial characterization of superoxide dismutase from the thermophilic bacteria Thermothrix sp.

Abstract

Superoxide dismutase (SOD: EC 1.15.1.1.), a high molecular weight component of the antioxidant defense system, provided promising results in the treatment of oxidative damage. Thermothrix sp., isolated from thermal spa water in Serbia, showed high superoxide dismutase activity. The SOD, from cell free extract, was purified to homogenity by ammonium sulfate precipitation. Sephadex G 75 gel filtration chromatography and QAE Sephadex ion exchange chromatography. The specific activity of the purified enzyme was 9191 U/mg. The purified enzyme was analyzed and partially characterized. SOD was localized in polyacrylamide gel by activity staining, based on the reduction of nitroblue tetrazolium (NBT) by superoxide. The enzyme molecular weight determined by gel chromatography is 37 kD. According to SDS PAGE it is composed of two subunits of equal size, joined by noncovalent interactions. The isoelectric point, assessed by isoelectric focusing is 5.3. The optimum pH for enzyme activity was in the range of 8 to 10. The optimum temperature for SOD activity was 60°C. After one hour of incubation at 40, 50 and 60°C the SOD activity increases, but at 80°C, the SOD is denaturated. After 24 hours of incubation at 25°C SOD activity only slightly decreases.