Vidal-Madjara Claire, Cañada-Cañada Florentina, Jaulmes Alain, Pantazaki Anastasia, Taverna Myriam
Laboratoire de Recherche sur les Polymères, CNRS, 2 rue Henry Dunant, 94320 Thiais, France.
J Chromatogr A. 2005 Sep 16;1087(1-2):95-103. doi: 10.1016/j.chroma.2005.01.008.
A numerical simulation of the direct zonal liquid chromatographic method is described for studying the binding of a ligand to a macromolecule by quantification of the interacting species present in a sample at equilibrium. The algorithm accounts for both the kinetic exchanges in solution and the dispersion effects depicted by the Fick law. Dimensionless variables are used for the concentrations which are expressed as a function of the equilibrium constant, KD. The free ligand concentration was varied in the injected samples from 0.1 to 20 KD, while that of the macromolecule was kept constant. An apparent binding isotherm was obtained from the total ligand chromatogram generated by the simulation run, when the amount emerging at almost column dead volume is plotted against that eluting at the free ligand retention time. As a continuous dissociation of the complex may occur during its migration, the apparent binding curve and the theoretical binding isotherm coincide at extremely low dissociating rates. At larger dissociation rates (0.001 s(-1) < kd <0.1 s(-1), for a first peak eluting in 1 min) the simulations were used to test various chromatographic conditions. The flow rate (or column volume) is the major effect which influences the on-column dissociation process as an exponential decay was found when the apparently bound fraction is plotted against the analysis time. The apparent equilibrium coefficient is close to the theoretical one for a binding curve generated with an initial solution containing a relatively low total concentration of binding sites (< or = KD). The apparent stoichiometric term is largely underestimated as its value decreases exponentially at increasing dissociation rates. An extrapolation at extremely short analysis times could be used to determine the stoichiometric coefficient characterizing the binding interaction.
本文描述了一种直接区域液相色谱法的数值模拟，用于通过对平衡时样品中存在的相互作用物种进行定量研究配体与大分子的结合。该算法考虑了溶液中的动力学交换以及菲克定律所描述的扩散效应。浓度使用无量纲变量表示，其作为平衡常数KD的函数。注入样品中的游离配体浓度在0.1至20 KD之间变化，而大分子的浓度保持恒定。当将几乎在柱死体积处出现的量与在游离配体保留时间洗脱的量作图时，从模拟运行生成的总配体色谱图中获得表观结合等温线。由于复合物在迁移过程中可能会持续解离，因此在极低的解离速率下，表观结合曲线与理论结合等温线重合。在较大的解离速率下（对于在1分钟内洗脱的第一个峰，0.001 s(-1) < kd <0.1 s(-1)），使用模拟来测试各种色谱条件。流速（或柱体积）是影响柱上解离过程的主要因素，因为当将表观结合分数与分析时间作图时发现呈指数衰减。对于由初始溶液中结合位点总浓度相对较低（<或 = KD）生成的结合曲线，表观平衡系数接近理论值。表观化学计量项在很大程度上被低估，因为其值在解离速率增加时呈指数下降。在极短的分析时间进行外推可用于确定表征结合相互作用的化学计量系数。
