Determination of the binding of ligands containing the N-2,4-dinitrophenyl group to bivalent monoclonal rat anti-DNP antibody using affinity capillary electrophoresis.
Determination of the binding of ligands containing the N-2,4-dinitrophenyl group to bivalent monoclonal rat anti-DNP antibody using affinity capillary electrophoresis.
复制标题
使用亲和毛细管电泳测定含有 N-2,4-二硝基苯基的配体与二价单克隆大鼠抗 DNP 抗体的结合。
DOI：
10.1021/ac00115a023
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发表时间：
1995
影响因子：
7.4
通讯作者：
Whitesides,GM
中科院分区：
文献类型：
--
作者：
Mammen,M;Gomez,FA;Whitesides,GM
本文用亲和毛细管电泳法测定了抗DNP鼠单克隆抗体IgG_（2b）与含N-二硝基苯基的配体形成复合物的两个解离常数。用单电荷和多电荷配体研究了电荷对IG与配体形成的复合物迁移率的影响。两性离子缓冲添加剂减少蛋白质吸附到毛细管壁。结合数据的一种分析形式是衍生的，这是更有用的比Scatchard分析某些多价系统中的结合的协同性是有问题的，但它也是可能的，使似是而非的approprip-tions有关蛋白质和蛋白质-配体复合物的电泳迁移率。将该分析的不确定性和假设与Scatchard分析的不确定性和假设进行了对比。对于该抗体和这些单价配体，配体从抗体上的解离非协同地发生。pH 8.3时IgG^ B上的电荷估计为-8.0 0. 2;该值通过分析复合物IgGzbLz的电泳迁移率获得。其中配体L在结构上相似但具有不同的电荷（配体上的电荷也通过CE确定）.自然界使用多价-具有多个识别位点的受体或聚集体与含有多个互补配体的分子的合作缔合-来介导许多种类的生物相互作用：在两个表面之间（细胞-细胞，* 1 234细胞-病原体2 -4）;表面和可溶性物质之间（细胞-蛋白，5病原体-蛋白，6· 7病原体-多糖，8细胞-多糖9）;和两种可溶性物质之间（蛋白质-蛋白，10药物-配体11 *）。我们希望定量的贡献，同时，多价结合这些相互作用。作为制定分析方法的第一个初步步骤，
Affinity capillary electrophoresis has been used to determine the two dissociation constants of the complex between anti-DNP rat monoclonal IgG2b antibody and charged ligands that contained a iV-dinitrophenyl group. Singly and multiply charged ligands were used to establish the influence of the charge on the mobilityof the complex between Ig and its ligand (s). Zwitterionic buffer additives lessened adsorption of protein to the walls of the capillary. A form of analysis of the binding data is derived that is more useful than Scatchard analysis for certain multivalent systems where cooperativity of binding is in question, but where it is also possible to make plausible assump-tions about electrophoretic mobilities of protein and protein-ligand complexes. The uncertainties and as-sumptions of this analysis are contrasted with those of Scatchard analysis. For this antibody and these monova-lent ligands, the dissociation of the ligands from the antibody occurred noncooperatively. The charge on IgG^ b at pH 8.3 is estimatedto be-8.0 ą 0.2; this value is obtained by analysis of the electrophoretic mobilities of complexes IgGzbLz. where the ligands L are structurally similar but have different charges (the charges on the ligands were also determined by CE).Nature uses polyvalency—the cooperative association of a receptor, or aggregate, having multiple recognition sites with a molecule containing multiple complementary ligands—to mediate many classes of biological interactions: between two surfaces (cell-cell,* 1 234cell-pathogen2-4); between a surface and a soluble species (cell-protein, 5 pathogen-protein, 6· 7 pathogen-polysac-charide, 8 cell-polysaccharide9); and between two soluble species (protein-protein, 10 drug-ligand11*). We wish to quantitate the contribution of simultaneous, multivalent binding to these interac-tions. As a first, preliminary step in developing analytical methods