GEL-FILTRATION CHROMATOGRAPHIC STUDIES OF THE ISOLATED MEMBRANE DOMAIN OF BAND-3

We have investigated the oligomeric state of the membrane domain of ba nd 3 (MDB3) in non-ionic detergent solution using Sepharose CL-4B gel filtration chromatography to study the hydrodynamic properties of the protein as a function of its concentration. The studies were performed in a C12E9 (polyoxyethylene-9-lauryl ether) buffer containing phospha tidylcholine and sodium chloride, which significantly slow a dilution- induced band 3 conformational change, and an associated aggregation pr ocess. Under these conditions native MDB3 eluted predominantly as sing le Gaussian peak with a Stokes radius of 76 +/- 14 Angstrom, at all pr otein concentrations studied between 0.2 and 12 mu M. This value agree s with the calculated Stokes radius (74 Angstrom) determined from the crystal structure of the MDB3 dimer. The Stokes radius of the MDB3 mon omer was obtained experimentally by treating native MDB3 with 0.5% SDS , and exchanging the SDS for C12E9 On the Sepharose column. SDS-treate d MDB3 showed two peaks whose ratio was strongly dependent on applied protein concentration. The peak representing the largest material had a Stokes radius of 69.7 +/- 14 Angstrom, which is essentially the same as the native MDB3 dimer. The peak representing the smaller material had a Stokes radius of 36 +/- 9 Angstrom, and was assigned as the MDB3 monomer in C12E9 Evidence is discussed which indicates that the C12E9 monomer specifically self-associates to form a functional MDB3 dimer. We conclude that native MDB3 exists as a stable dimer in mixed micell ar solutions composed of C12E9 and phosphatidylcholine, and that the d imer can be dissociated to monomers only by denaturation.