title: Using affinity capillary electrophoresis to evaluate average binding constant of 18-mer diphosphotyrosine peptide to antiphosphotyrosine Fab.
abstract: We used affinity electrophoresis in capillaries to investigate the interaction between a monovalent antiphosphotyrosine antibody fragment, antigen-binding fragment (Fab), and a divalent antigen (dAg), an 18-mer diphosphopeptide phosphorylated on two-site tyrosine residues. The migration shift behavior of Fab in electrophoretic solution was observed and the quantitative expression was presented to estimate the arithmetical average value of the intrinsic affinities for two epitopes on the dAg with the Ag binding site on the Fab. In dAg excess, based on measurement of mobility changes of Fab analytes at different dAg concentrations, the experimental average dissociation constant (Kd = 27.7 microM) was calculated. It was also found that the structural variation of the two epitopes for binding specificity to the Ag-binding domain of Fab is not apparent. Moreover, the Kd values of Fab-dAg complexes were measured at higher electric fields and shown to be independent of changes in the electric field. Thus, under conditions where the total dAg concentration is in excess of the total Fab concentration, the method and quantitative expression which we developed is generally useful for the understanding of molecular interaction for an unlabeled monovalent receptor and its divalent ligand in free solution.
authors.affiliation: Laboratory of Molecular Recognition, Optoelectronic Biomedicine Center, National Taiwan University College of Medicine, Taipei.
authors.forename: S
authors.initials: S
authors.lastname: Lin
authors.forename: P
authors.initials: P
authors.lastname: Tang
authors.forename: S M
authors.initials: SM
authors.lastname: Hsu
mesh_terms: D000595:Amino Acid Sequence; D004560:Electricity; D019075:Electrophoresis, Capillary / Q000379:methods*; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D019000:Phosphotyrosine / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D007140:Immunoglobulin Fab Fragments; D019000:Phosphotyrosine