PURIFICATION AND PROPERTIES OF CYTOSOLIC COPPER, ZINC SUPEROXIDE-DISMUTASE FROM WATERMELON (CITRULLUS-VULGARIS SCHRAD) COTYLEDONS

Cytosolic copperzinc-superoxide dismutase (CuZn- SOD I; EC 1.15.1.1) w as purified to homogeneity from watermelon (Citrullus vulgaris Schrad. ) cotyledons. The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fa st Protein Liquid Chromatography, gel filtration column chromatography , and affinity chromatography on concanavalin A-Sepharose. CuZn-SOD I was purified 310-fold with a yield of 12.6 micrograms enzyme per gram cotyledons, and had a specific activity of 3,540 units per milligram p rotein. The relative molecular mass for cytosolic CuZn-SOD was 34000, and it was composed by two equal subunits of 16.3 kDa. CuZn-SOD I did not contain neutral carbohydrates in its molecule, and its ultraviolet and visible absorption spectra showed two absorption maxima at 254 nm and 580 nm. Metal analysis showed that the enzyme contained 1 gram-at om Cu and 1 gram-atom Zn per mole dimer. Cytosolic CuZn-SOD was recogn ized by the antibody against peroxisomal CuZn-SOD from watermelon coty ledons, and its enzymatic activity was inhibited by this antibody. By IEF (pH 4.2-4.9), using a new method for vertical slab gels set up in our laboratory, purified cytosolic CuZn-SOD was resolved into two equa l isoforms with isoelectric points of 4.63 and 4.66.