Binding stoichiometry of DNA adducts with antibody studied by capillary electrophoresis and laser-induced fluorescence

摘要

Four oligonucleotides (fluorescently labeled and unlabeled 16- and 90-mer), each containing a single adduct of benzo[a]pyrene diol epoxide (BPDE), were synthesized and used to study the binding stoichiometry between the DNA adduct and its antibody. The free oligonucleotide and its complexes with mouse monoclonal antibody were separated using capillary electrophoresis and detected with laser-induced fluorescence (LIF). Two complexes, representing the 1:1 and 1:2 stoichiometry between the antibody and the DNA adduct, were clearly demonstrated. The stoichiometry depended upon the relative concentrations of the antibody and the DNA adducts. A new approach examining the binding of the antibody with a mixture of a tetramethylrhodamine (TMR)-labeled and unlabeled BPDE-16-mer revealed insights on ligand redistribution and exchange between the labeled and unlabeled BPDE-16-mer oligonucleotides in the complexes. The observation of this unique behavior has not been possible previously with other binding studies. A mixture of the antibody with the MR-labeled BPDE-16-mer and an unlabeled BPDE-90-mer further revealed the formation of three fluorescent complexes: antibody with one TMR-BPDE-16-mer molecule, antibody with two TMR-BPDE-16-mer molecules, and antibody with one TMR-BPDE-16-mer and one BPDE-90-mer. The three complexes clearly demonstrated binding stoichiometry and ligand redistribution/exchange. [References: 31]