Vidal-Madjar C, Jaulmes A, Sébille B, González M J, Jiménez B, Hernández L M
Laboratoire de Physico-Chimie des Biopolymères, CNRS UM 27, Université Paris Val de Marne, Thiais, France.
J Chromatogr. 1992 Dec 11;584(1):11-6. doi: 10.1016/0378-4347(92)80004-a.
The direct zonal on-column injection method was applied to a high-performance liquid chromatographic study of pollutant-protein binding interactions in solution. The protein and the protein-ligand complex are excluded on the basis of the size from the diol support, and the free ligand penetrates into the pores and is more retained. The pattern of the ligand elution profile depends on the protein-ligand dissociation constant. This effect was quantitatively analysed by developing a numerical simulation algorithm in which the column is divided into slices of given thickness. The column length, flow-rate and shape of the injection signal are given as input parameters. A global dispersion coefficient accounts for peak broadening. A rapid equilibrium is assumed with the hypothesis that a monovalent ligand interacts with a single binding site on the protein. The interaction of bovine serum albumin with pentachlorophenol was studied, and an apparent dissociation constant for the protein-ligand complex was determined by fitting the theoretical profile to the experimental one. The effect of the acetonitrile content in the solvent was studied. An important decrease of the dissociation constant is observed that affects the chromatographic elution pattern.
直接区域柱上注射法被应用于溶液中污染物 - 蛋白质结合相互作用的高效液相色谱研究。基于大小，蛋白质和蛋白质 - 配体复合物被二醇载体排斥，而游离配体渗透到孔中并被更多保留。配体洗脱曲线的模式取决于蛋白质 - 配体解离常数。通过开发一种数值模拟算法对这种效应进行了定量分析，在该算法中，将柱分成给定厚度的切片。柱长、流速和注射信号的形状作为输入参数给出。一个全局分散系数用于解释峰展宽。假设单价配体与蛋白质上的单个结合位点相互作用，存在快速平衡。研究了牛血清白蛋白与五氯苯酚的相互作用，并通过将理论曲线与实验曲线拟合来确定蛋白质 - 配体复合物的表观解离常数。研究了溶剂中乙腈含量的影响。观察到解离常数显著降低，这影响了色谱洗脱模式。
