PURIFICATION OF HUMAN RECOMBINANT SUPEROXIDE-DISMUTASE BY ISOELECTRIC-FOCUSING IN A MULTICOMPARTMENT ELECTROLYZER WITH ZWITTERIONIC MEMBRANES

Human recombinant superoxide dismutase (SOD), purified to homogeneity, is resolved by both conventional isoelectric focusing and immobilized pH gradients into three bands, with isoelectric points (pIs) in the p H range 4.8 to 5.1, the pI 4.80 form representing the minor component. Due to the fact that this enzyme is expressed in E. coli, N-terminal acetylation or glycosylation should be ruled out. When purified by sma ll-scale preparative isoelectric focusing in immobilized pH gradient g els, it was found that, upon subsequent analysis, the pI 5.07 form wou ld band in the same position, but the intermediate pI 4.92 band would split into the upper (pI 5.07) and the lower (pI 4.80) species, in nea rly the same amounts, whereas the lowest pi component would always gen erate both the intermediate and upper forms. Enzymatic essays pointed out that these three isoforms had nearly the same specific activity, s lightly higher than that of the starting material. Metal analysis indi cated that all three forms contained the same metal/protein ratio, app roaching the value Cu2Zn2- SOD, as reported in the literature. Circula r dichroism spectra of the pI 4.80 and 5.07 forms showed the same prof ile in the 190-240 nm range, but marked differences in the 250--350 nm region. Treatment with EDTA produces 1-2 additional, slightly higher pI isoforms, whereas treatment with KCN generates a number of higher p I components, reaching pI values as high as pH 7, with nearly complete disappearance of the three major SOD isoforms. It is concluded that t hese three isoforms could represent interconvertible species, the high est pI component representing the most stable conformer.