title: Proximity of antibody binding sites studied by fluorescence energy transfer.
abstract: Fluorescence energy transfer experiments by steady-state and nanosecond monophoton techniques were carried out with a covalently linked hybrid rabbit IgG antibody containing one antilactose site and one anti-Dns [5-(dimethylamino)-1-naphthalenesulfonyl] site. The hybrid antibody was prepared from antilactose and anti-Dns antibody by mild reduction, dissociation into half-molecules in acid, and random reassociation with re-formation, to the extent of 80%, of the single disulfide bond between the heavy chains. Fractionation with an antilactose-specific immunoadsorbent yielded a population in which each IgG molecule contained no more than one anti-Dns site per antibody. The acceptor molecules used for intramolecular energy transfer were derivatives of p-aminophenyl beta-lactoside (PAPL): (dimethyl-amino)benzeneazo-PAPL and N-fluorescyl-PAPL. The fluorescence lifetime (24 ns) and quantum yield (0.57) of the bound Dns group were unaffected by the presence of the acceptor in the adjacent site. Three models were used to calculate the minimum distance between the adjacent sites of the IgG antibody based on the overlap in the emission and absorption spectra of the donor-acceptor pairs and the segmental flexibility of the immunoglobulin molecule. The calculations yielded values in the range of 5.5-7.0 nm for the minimum distance of separation between the antibody sites in solution and demonstrated a substantial energy barrier to the closer approach of the sites.
mesh_terms: D000818:Animals; D001666:Binding Sites, Antibody; D003619:Dansyl Compounds; D004735:Energy Transfer; D005453:Fluorescence; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007700:Kinetics; D007785:Lactose; D008433:Mathematics; D011817:Rabbits / Q000276:immunology; D013050:Spectrometry, Fluorescence
chemical_list: D003619:Dansyl Compounds; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007785:Lactose