State of Association of Band 3 Protein from Bovine Erythrocyte Membrane in Nonionic Detergent

Abstract

The state of association of bovine Band 3, which is a major intrinsic protein of erythrocyte membranes, was examined in nonaethyleneglycol dodecyl ether (C₁₂E₉) solution by ultracen-trifugation, gel filtration, gel electrophoresis and cross-linking studies. The molecular weight of bovine Band 3 was 107, 000±5% as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. When Band 3 was purified in C₁₂E₉ solution with the aid of 2, 3-dimethylmaleic anhydride (DMMA), which is a known dissociating reagent for peripheral proteins from erythrocyte membranes and for Band 3-Band 4.2 complex, the protein was present as a monomer and was not oxidatively cross-linked. On the other hand, DMMA-untreated Band 3 was present as oligomeric forms composed mainly of the dimer and tetramer, and the oligomer in C₁₂E₉ was as susceptible to oxidative cross-linking as Band 3 in ghosts. The oligomeric form apparently retained a more ordered conformational state than the monomeric form. These results indicate that the bovine Band 3 oligomer is a stable form in C₁₂E₉, but the present result showing the coexistence of dimer and tetramer in C₁₂E₉ contrasts with the reported observation that human Band 3 is present as a stable dimer in a nonionic detergent, Triton X-100.
It was shown that polyacrylamide gradient gel electrophoresis in the presence of C₁₂E₉ gave better resolution of the associated species of Band 3 than ultracentrifugation or gel filtration, and this method made it possible to determine the Stokes radii of Band 3-C₁₂E₉ complexes. This result suggests the usefulness of electrophoretic methods in the presence of nonionic detergent for studies of the state of association of other membrane proteins.