polidocanol and 2-3-dimethylmaleic-anhydride

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 ultracentrifugation, 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 a 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.

Topics: Animals; Cattle; Chymotrypsin; Erythrocyte Membrane; Erythrocytes; Macromolecular Substances; Maleic Anhydrides; Membrane Proteins; Molecular Weight; Peptide Fragments; Polidocanol; Polyethylene Glycols; Sodium Dodecyl Sulfate; Solubility; Spectrometry, Fluorescence