Physicochemical Properties of Charge Isomers of Recombinant Human Superoxide Dismutase

Abstract

Recombinant human Cu₂Zn₂SOD expressed in Escherichia coli consisted of mainly three isomers with isoelectric points of 5.14 (A), 5.06 (B), and 4.99 (C). Each isomer was isolated by DEAE-Toyopearl chromatography and the physicochemical properties were investigat-ed. No significant differences in chemical and spectrophotometric properties, such as specific activity, metal contents, amino acid composition, and UV and ESR spectra, were found. The result of labeling of free cysteine residues with ABD-F showed the disulfide bond to be formed between ⁵⁷Cys and ¹⁴⁶Cys in every isomer. A few differences were found in the CD spectrum around 260 nm and in the elution patterns on reverse-phase HPLC. The isoelectric points of the three isomers became the same after treatment by reduction and carboxymethylation and even after reduction only, pI of isomers tended to be at the value of component (A). These results suggest that the three isomers are identical in primary structure but slightly different in secondary or tertiary structure. These differences are probably derived from structural alterations around ¹¹¹Cys.