Effect of pH on the self-association of erythrocyte band 3 in situ

The human erythrocyte anion exchanger (band 3) contains a cytoplasmic domai n (cdb3) that exists in a reversible, pH-dependent structural equilibrium a mong three native conformations. To understand how this conformational equi librium might influence the association state of band 3, we have incubated stripped erythrocyte membranes in solutions ranging from pH 6.0 to pH 10.5 and have examined the oligomeric state of the protein by size exclusion hig h performance liquid chromatography. We demonstrate that incubation of memb ranes in slightly acidic conditions favors dimer formation, whereas extende d incubation at higher pHs (pH > 9) leads to irreversible formation of an o ligomeric species larger than the tetramer. Since the pH dependence of the conformational state of the cytoplasmic domain exhibits a similar pH profil e, we suggest that the conformation of the cytoplasmic domain can modulate the self-association of band 3. Importantly, this modulation would appear t o require the structural interactions present within the intact protein, si nce the isolated membrane-spanning domain does not display any pH dependenc e of association. The irreversible nature of the alkali-induced aggregation further suggests that a secondary reaction subsequent to band 3 associatio n is required to stabilize the high molecular weight aggregate. Although we were able to eliminate covalent bond formation in this irreversible aggreg ation process, the exact nature of the secondary reaction remains to be elu cidated. (C) 2001 Elsevier Science B.V. All rights reserved.