Impermeant maleimides. Oriented probes of erythrocyte membrane proteins

Maleimides impermeant to human erythrocyte membranes have been synthesized and applied to studies of the sulfhydryl groups of the membrane. Reaction of radioactive dextran-maleimide and glutathione-maleimide with either intact erythrocytes or ghosts yields sulfhydryl titers for the outer (exofacial) and inner (endofacial) surfaces, respectively, of 1.5 to 1.7 and 27 to 28 amol/cell. Corresponding values for sulfhydryl groups within the membrane interior, as estimated with radioactive N-ethylmaleimide, are 16 to 22 amol/cell. After exofacial labeling of intact cells with [35S]glutathione-maleimide, autoradiography of sodium dodecyl sulfate-polyacrylamide gels demonstrates four bands (alpha, beta, gamma, and delta) containing, respectively, 13%, 63%, 11%, and 13% of the radioactivity. The major beta-band corresponds in position to polypeptides of molecular weight 40,000 to 70,000 and to Coomassie brilliant blue-stained Band 5. Selective extraction demonstrates that the major Band 5 protein is not identical with the labeled beta-band polypeptides. Following endofacial labeling of ghosts with [35S]glutathione-maleimide, autoradiography reveals radioactivity in all of the major Coomassie brilliant blue bands. The impermeant maleimides described are also applicable to studies of discrete functional proteins of the erythrocyte membrane, including the hexose transport mechanism and the major Rho antigenic site.