Gel filtration chromatographic studies of the isolated membrane domain of band 3

Abstract

We have investigated the oligomeric state of the membrane domain of band 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 (polyox-yethylene-9-lauryl ether) buffer containing phosphatidylcholine and sodium chloride, which significantly slow a dilution-induced band 3 conformational change, and an associated aggregation process. Under these conditions native MDB3 eluted predominantly as a single Gaussian peak with a Stokes radius of 76 ± 14 Å, at all protein concentrations studied between 0 2 and 12 μM. This value agrees with the calculated Stokes radius (74Å) determined from the crystal structure of the MDB3 dimer. The Stokes radius of the MDB3 monomer was obtained experimentally by treating native MDB3 with 0·5% SDS, and exchanging the SDS for C₁₂E₉ on the Sepharose column. SDS-treated 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 ± 14Å, which is essentially the same as the native MDB3 dimer. The peak representing the smaller material had a Stokes radius of 36 ± 9Å, 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 micellar solutions composed of C₁₂E₉ and phosphatidylcholine, and that the dimer can be dissociated to monomers only by denaturation.

Key Words:

• band 3
• membrane proteins
• anion exchanger (AE1)
• human red blood cell