Cross-linking of hemoglobin to the cytoplasmic surface of human erythrocyte membranes. Identification of band 3 as a site for hemoglobin binding in Cu2+-o-phenanthroline catalyzed cross-linking.

The addition of hemoglobin to isolated membrane ghosts of human erythrocytes followed by catalytic oxidation with Cu2+-o-phenanthroline results in the covalent attachment of hemoglobin to the membrane. A decrease in the mobility of Band 3 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to an increase in molecular weight of approximately 16,000 suggests that Band 3 is a site for the covalent attachment of hemoglobin to the membrane under these conditions. This conclusion was confirmed using two-dimensional gels in which the covalent linkage was cleaved with 2-mercaptoethanol in the second dimension. 14C-labeled hemoglobin was used to show that the new band was composed of one globin monomer per Band 3 monomer. Additionally, it was demonstrated that only the beta chain of hemoglobin is covalently bound to the Band 3 protein.