Belew M, Yip T T, Andersson L, Porath J
Institute of Biochemistry, University of Uppsala, Sweden.
J Chromatogr. 1987 Aug 21;403:197-206. doi: 10.1016/s0021-9673(00)96353-2.
The interaction of lysozyme, ovalbumin, bovine and pig serum albumins with Cu2+ immobilized on Chelating Sepharose Fast Flow or TSK gel chelate-5PW was studied by frontal analysis at various initial concentrations of these solutes. The chromatographic data so obtained served as a basis for evaluating some relevant affinity chromatography parameters by adapting previously reported equations to this system. The TSK-based adsorbent had lower adsorption capacity for all the model proteins compared to the agarose-based adsorbent, due primarily to its lower porosity which has a marked influence on the accessibility of the immobilized ligand to the proteins. On the other hand, the TSK-based adsorbent offers almost ideal conditions for studying adsorption equilibria under column chromatographic conditions. The adsorption capacity of these adsorbents for the model proteins ranges from about 0.6 to 7 mumol/ml, equivalent to 40-100 mg/ml, of adsorbent. The following equilibrium constants for the interaction of the proteins with immobilized Cu2+ were obtained: lysozyme, 1.8.10(4); ovalbumin, 1.5.0(5); BSA, 1.7.10(5); PSA, 3.7.10(5) and imidazole, 8.10(3) M-1. Despite the comparatively low affinity of imidazole for the adsorbent, it is an effective competing ligand, at comparatively high concentrations, for adsorbed proteins primarily because all adsorption sites are available to it. The results obtained suggest that about 1/3 to 1/2 of the potential adsorption sites on the model proteins are involved in forming coordination complexes with Cu2+ immobilized to covalently bound iminodiacetate groups on insoluble gel matrices.
通过前沿分析法，研究了溶菌酶、卵清蛋白、牛血清白蛋白和猪血清白蛋白在不同初始浓度下与固定在螯合琼脂糖凝胶快速流动柱或TSK凝胶螯合物-5PW上的Cu2+之间的相互作用。通过将先前报道的方程应用于该系统，所获得的色谱数据成为评估一些相关亲和色谱参数的基础。与基于琼脂糖的吸附剂相比，基于TSK的吸附剂对所有模型蛋白的吸附容量较低，这主要是由于其较低的孔隙率，孔隙率对固定化配体与蛋白质的可及性有显著影响。另一方面，基于TSK的吸附剂为在柱色谱条件下研究吸附平衡提供了几乎理想的条件。这些吸附剂对模型蛋白的吸附容量范围约为0.6至7 μmol/ml，相当于每毫升吸附剂40 - 100 mg。获得了蛋白质与固定化Cu2+相互作用的以下平衡常数：溶菌酶为1.8×10⁴；卵清蛋白为1.5×10⁵；牛血清白蛋白为1.7×10⁵；猪血清白蛋白为3.7×10⁵；咪唑为8×10³ M⁻¹。尽管咪唑对吸附剂的亲和力相对较低，但在相对较高浓度下，它是吸附蛋白的有效竞争配体，主要是因为所有吸附位点对它都是可利用的。所得结果表明，模型蛋白上约1/3至1/2的潜在吸附位点参与了与固定在不溶性凝胶基质上共价结合的亚氨基二乙酸基团上的Cu2+形成配位络合物。
