Development of the Methodology for Dissociation Kinetic Analysis of Biomolecular Complexes with Capillary Electrophotetic Reactor

微芯片毛细管电泳反应器（MCER）是金属配合物解离动力学分析的一种实用而有力的工具。在金属配合物的离解动力学分析中，由于反应物（金属配合物）和产物（金属离子和自由配体）难以区分，不能直接测定金属配合物的离解动力学。相反，MCER自完成了对金属配合物离解速率常数的直接测量以来，彻底改变了金属配合物离解速率常数的测量。MCER的基本思想是由微芯片毛细管电泳（MCE）中尖锐条带轮廓的浓度跳跃特征引起的;即在MCE分离过程中，在电泳缓冲液中没有配体的金属络合物暴露于配体的急剧浓度跳跃引起的压倒性解离力。用内标和外标法研究了Ce^（3+）与多氨基羧酸配体8-氨基-2-[（2-氨基-5-甲基苯氧基）甲基]-6-甲氧基喹啉-N，N，N '，N'，-四乙酸（[Ce L]^-）配合物的时间依赖性解离反应。用毛细管电泳法直接测定了303 K时[CeL]^-的溶剂分解反应速率常数为6.2 × 10 ~<-3><-1>（-3）s ~（-1）。选择抗地高辛IgG与地高辛的免疫复合物和复杂的双链DNA与溴化乙锭作为分析模型化合物。不幸的是，解离反应速率常数的测定不能实现CER在这两个系统中，因为它们的小电泳迁移率和较低的动力学惰性的属性。

A microchip capillary electrophoretic reactor (MCER), a practical and powerful tool for dissociation kinetic analysis of metal complexes, has been proposed. In the dissociation kinetic analysis of metal complexes using conventional batch reaction techniques, owing to the difficulty in dividing between reactant (metal complex) and products (metal ion and free ligand), the direct measurement of the dissociation kinetics had not been achieved. On the contrary, MCER revolutionized the measurement of the dissociation rate constant of metal complexes since its accomplishment of the direct measurement. The basic idea of a MCER was provoked by the concentration jump character of the sharp band profiles in microchip capillary electrophoresis (MCE) ; i.e. metal complexes during the MCE separation processes, in which there had been no ligand in the electrophoretic buffer solution, were exposed to overwhelming force to dissociation caused by a steep concentration jump of the ligand. The time depending dissociation reaction profile of metal complex of Ce^<3+> with a polyaminocarboxylic ligand, 8-amino-2-[(2-amino-5-methylphenoxyl)methyl]-6-methoxyquinoline-N,N,N',N',-tetraacetic acid ([CeL]^-) was described using both internal and external standard. The solvolytic dissociation rate constant of [CeL]^-have been determined directly as 6.2 X 10^<-3> s^<-1> at 303 K with MCER.Capillary electrophoretic reactor (CER) was applied to the determination of dissociation reaction rate constant of bio-molecular complexes. Both an immunocomplex of anti digoxin IgG with digoxin and a complex double stranded DNA with ethidium bromide were chosen as analytical model compounds. Unfortunately, determination of dissociation reaction rate constants could not be achieved with CER in both systems, since their properties of small electrophoretic mobility and lower kinetically inertness.