BOVINE ERYTHROCYTE SUPEROXIDE-DISMUTASE - DIAZO COUPLING, SUBUNIT INTERACTIONS, AND ELECTROPHORETIC VARIANTS

The copper and zinc-containing superoxide dismutase of bovine erythrocytes was modified and inactivated by several diazonium reagents. Treatment of native or of zinc-only enzymes, with diazonium lH-tetrazole, derivatized 1 tyrosine, 1 histidine, and 10 lysine residues per subunit. The partial inactivation, accompanying this treatment, appeared to be due to modification of the lysine residues, since acetylation caused a similar extent of lysine modification and of inactivation. Apoenzyme exhibited greater reactivity toward diazonium lH-tetrazole, reflecting the exposure of two histidine residues per subunit in the active site region. Diazo coupling with diazonium lH-tetrazole grossly increased the anodic mobility of the enzyme and yielded a derivative useful for studies of subunit interactions. Admixture of native enzyme with diazo-coupled, H2O2-inactivated enzyme, in the presence of 8.0 M urea, followed by removal of the urea, generated a new, enzymatically active, species. The new species exhibited an electrophoretic mobility nearly intermediate between that of the native and diazo-coupled proteins, but had the same molecular weight as the parental species. When isolated from polyacrylamide gels and incubated at pH 7.8 for 2 h at 25 °C, the new species gave rise to both parental species, demonstrating that it was an unstable hybrid. The specific activity of the hybrid was approximately half that of the native enzyme. A native subunit thus exhibited the same catalytic activity, whether paired with another native subunit or with a chemically modified and catalytically inactive subunit. Mutually inhibitory interactions between subunits, giving rise to half of the sites reactivity, thus seems unlikely. © 1979, American Chemical Society. All rights reserved.