title: The effect of mild diamide oxidation on the structure and function of human erythrocyte spectrin.
abstract: Oxidants can alter erythrocyte membrane properties and cause ultimate hemolysis, but the mechanisms responsible for these changes are not understood. A protein skeleton preserves the normal integrity of the erythrocyte membrane. In this study, we investigated the effects of limited chemical oxidation on the structure and function of the major skeletal protein, spectrin. After mild treatment of spectrin with 2.5 microM diamide, with formation of an average of only one disulfide bond, we observed a 50% reduction in the ability of protein 4.1 to amplify spectrin-actin binding. The oxidized spectrin specifically lacked the ability to bind protein 4.1, whereas all other spectrin functions remained intact. However, oxidation also produced a structural change in spectrin. A rapidly migrating species appeared on non-denaturing gels in a dose-dependent manner with increasing diamide concentrations. By electron microscopy, the oxidized spectrin appeared as single-stranded signet rings with irregular knob-like protrusions. Fifty per cent of spectrin was converted to the ring form after the formation of an average of two disulfide bonds. Both the structural and functional defects were reversed by chemical reduction. The loss of spectrin function or the structural transformation in spectrin may contribute to erythrocyte membrane failure in the oxidative environment.
mesh_terms: D001391:Azo Compounds / Q000494:pharmacology*; D001798:Blood Proteins / Q000378:metabolism; D003598:Cytoskeletal Proteins; D003958:Diamide / Q000494:pharmacology*; D004220:Disulfides / Q000032:analysis; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008854:Microscopy, Electron; D009479:Neuropeptides; D011485:Protein Binding; D013049:Spectrin / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
chemical_list: D001391:Azo Compounds; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D004220:Disulfides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009479:Neuropeptides; D013439:Sulfhydryl Reagents; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D003958:Diamide; D013049:Spectrin; D004229:Dithiothreitol