Purification and characterization of iron superoxide dismutase and copper-zinc superoxide dismutase from Acanthamoeba castellanii

Two superoxide dismutases (SOD I and SOD II) were purified from Acanthamoeb a castellanii and characterized for several biochemical properties. Analysi s of the primary structure and inhibition studies revealed that SOD I is ir on SOD (Fe-SOD), with a molecular mass of 50 kDa, and SOD II is copper-zinc SOD (Cu,Zn-SOD), with a molecular mass of 38 kDa. Both enzymes have a homo dimeric structure consisting of 2 identical subunits, each with a molecular mass of 26 and 19 kDa for SOD I and SOD II, respectively. The isoelectric points of SOD I and SOD II were 6.4 and 3.5, respectively, and there were n o isoenzyme forms detected. Both enzymes show a broad optimal pH of 7.0-11. 0. Because no differences were observed in the apparent molecular weight of SOD I after addition of the reducing agent 2-mercaptoethanol, the subunits do not appear to be linked covalently by disulfide bonds. However, the sub units of SOD II were covalently linked by intra- and interdisulfide bonds. Western blot analyses showed that the 2 enzymes have different antigenicity . Both enzymes occur as cytoplasmic and detergent-extractable fractions. Th ese enzymes may be potential virulence factors of A. castellanii by acting both as antioxidants and antiinflammatory agents. These enzymes may be attr active targets for chemotherapy and immunodiagnosis of acanthamoebiasis.