Self-Association of Band 3-Protein from Human Erythrocyte Membranes in Aqueous Solutions

Abstract

Band 3, the main integral protein of the human erythrocyte membrane, was solubilized and purified in high concentrations of acetic acid. After removal of the organic solvent by dialysis, the self-association of the protein in aqueous solutions was studied by analytical ultracentrifugation. Sedimentation velocity and sedimentation equilibrium experiments clearly demonstrated that, under appropriate conditions of protein preparation, at protein concentrations c < 200 .mu.g/ml, ionic strengths I < 10 mM and pH values remote from the isoelectric pH of the protein, band 3 showed a monomer/dimer/tetramer-association equilibrium. With some preparations and at higher values of c or I, hexamers and octamers contributed to the association equilibrium. The time needed for relaxation towards association equilibrium depended on the blood donor from whom the membranes were derived and varied between less than 1 min and more than several hours. The results of analytical ultracentrifugation, together with previously published data on the incorporation of band 3 into planar lipid bilayers, from chemical crosslinking and from EM suggested that band 3 will also show a monomer/dimer/tetramer-association equilibrium in the human erythrocyte membrane. This hypothesis contrasted with the widely-held assumption that, in the membrane, band 3 is a stable dimer; it is consistent with nearly all known data on band 3 self-association.