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

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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.