一种研究生物大分子与其小分子配基相互作用的毛细管电泳新方法

A Novel Method of Capillary Electrophoresis for Studying the Interaction of Macromolecule with Its Small Molecule Ligand

建立了一种定量研究大分子与小分子间相互作用的毛细管电泳新方法，即在线微透析三电要和毛细管电泳法。本法可测定小分子的自由浓度而不受蛋白质大分子的干扰。详细介绍了实验装置的制作，探讨了进样机制。作为实例，了生理条件下ｐＨ７．４，５０ｍｍｏｌ／Ｌ磷酸介质中，牛血清中蛋白与磺胺甲基异恶唑、Ｌ－色氨酸的亲和常数的测定，其值分别为５．８×１０＾－４Ｌ／ｍｏｌ，２．３×１０＾４Ｌ／ｍｏｌ，１．７７×１０＾３Ｌ／

A novel capillary electrophoresis method for studying the interaction of biomacromolecule with its small molecule ligand has been presented. In the method a home-made capillary electrophoresis set-up coupled with on-line microdialysis by an attached electrode was introduced. The method can be used to determine the free concentration of small molecule without the interference of protein macromolecule. The procedure making of the set-up was described in detail and the sampling mechanism was discussed. As examples the binding constants of sulfamethoxazolum (SMZ) with bovine serum albumin (\), BSA with L-tryptophane(Trp) and BSA with D-trytophane were studied under the physiological conditions (pH 7.4, 50 mmol/L phosphate buffer). The values were 5. 8 x 104 L/mol, 2. 3 x 104 L/mol, 1 .77 x 103 L/mol, respectively. The proposed method is simple and has small sample consuming. It can be used in the desired physiological conditions.