Affinity chromatography of Band 3, the anion transport protein of erythrocyte membranes

• Published in: The Journal of biological chemistry Vol. 261; no. 21; pp. 9770 - 9778
• Main Authors: Pimplikar, S W, Reithmeier, R A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25-07-1986; American Society for Biochemistry and Molecular Biology
• Subjects: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid - analogs & derivatives; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid - metabolism; Anion Exchange Protein 1, Erythrocyte - isolation & purification; Biological and medical sciences; Cell physiology; Chromatography, Affinity - methods; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane - analysis; Fundamental and applied biological sciences. Psychology; Hormonal regulation; Humans; Molecular and cellular biology; Stilbenes - metabolism; Temperature; Human; Anions; Band 3 protein; Purification; Affinity chromatography; Membrane transport; Plasma membrane; Red blood cell; Ion transport
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)67581-7

Affinity chromatography of Band 3 was performed using a series of affinity matrices synthesized with various inhibitor ligands and spacer arms. Hydrophilic spacer arms greater than four atoms in length were essential for Band 3 binding. An affinity resin prepared by reacting 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (Ki = 10 microM) with Affi-Gel 102 was found to be the most effective resin of the series tested. Solubilized proteins from human erythrocyte membranes were incubated with the affinity resin, and pure Band 3 was recovered by eluting with 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS; Ki = 2 microM). Band 3 bound to the resin specifically in its stilbene disulfonate binding site, and optimal binding was achieved at pH 8 and at high ionic strength. At 4 degrees C, up to 80% of the bound Band 3 could be eluted by 1 mM BADS, whereas the remainder could be eluted under denaturing conditions using 1% lithium dodecyl sulfate. At 22 or 37 degrees C, the amount of BADS-elutable Band 3 was reduced with a concomitant increase of Band 3 in the lithium dodecyl sulfate elute. Thus, for successful affinity chromatography, the experiment must be carried out rapidly at 4 degrees C. This procedure was also used to purify the Band 3 protein from mouse, horse, pig, and chicken erythrocytes.