Pimplikar S W, Reithmeier R A
J Biol Chem. 1986 Jul 25;261(21):9770-8.
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.
使用一系列与各种抑制剂配体和间隔臂合成的亲和基质对带3进行亲和层析。长度大于四个原子的亲水性间隔臂对于带3的结合至关重要。发现通过使4-乙酰氨基-4'-异硫氰基芪-2,2'-二磺酸盐（Ki = 10 microM）与Affi-Gel 102反应制备的亲和树脂是所测试系列中最有效的树脂。将来自人红细胞膜的溶解蛋白与亲和树脂孵育，并通过用4-苯甲酰胺基-4'-氨基芪-2,2'-二磺酸盐（BADS；Ki = 2 microM）洗脱来回收纯带3。带3在其芪二磺酸盐结合位点特异性结合到树脂上，并且在pH 8和高离子强度下实现最佳结合。在4℃下，高达80%的结合带3可以用1 mM BADS洗脱，而其余部分可以在变性条件下使用1%十二烷基硫酸锂洗脱。在22或37℃下，BADS可洗脱的带3量减少，同时十二烷基硫酸锂洗脱物中的带3量增加。因此，为了成功进行亲和层析，实验必须在4℃下快速进行。该方法也用于从小鼠、马、猪和鸡的红细胞中纯化带3蛋白。
