Purification and characterization of Band 3, the major intrinsic membrane protein of the bovine erythrocyte membrane.
Band 3 from bovine erythrocyte membranes was isolated in a state of high purity by the following steps in the presence of a nonionic detergent, nonaethyleneglycol n-dodecyl ether (C12E9): (1) selective removal of Band 2.6 from ghosts by solubilization with 2% C12E9 (2) extraction of Band 3-rich fraction with 4% C12E9 from 2% C12E9-treated membrane residues, and (3) purification of Band 3 by aminoethyl-conjugated Sepharose 4B column chromatography. Human Band 3 was also purified in good yield by aminoethyl-conjugated Sepharose 4B column chromatography of erythrocyte membrane proteins solubilized with 1% C12E9 and treated with 2,3-dimethymaleic anhydride. There were no significant differences in CD spectra in C12E9, amino acid compositions, and migration mobilities in sodium dodecyl sulfate-gel electrophoresis between bovine and human Band 3. Calculations of average hydrophobicity and discriminant function demonstrated that bovine Band 3 could be categorized as a typical integral membrane protein. Bovine Band 3 showed a tendency to form a dimer and higher aggregates in 0.1% C12E9; these were resistant to dissociation into monomers in sodium dodecyl sulfate solution and, further, the protein retained residual secondary structure in highly concentrated guanidine hydrochloride solution, indicating the possible presence of an extended sequence of hydrophobic amino acid residues.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins / Q000302:isolation & purification*; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis*; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate
D000596:Amino Acids; D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins; D008565:Membrane Proteins; D012967:Sodium Dodecyl Sulfate