Solvent perturbation of the fluorescence of fluorescein bound to specific antibody. Fluorescence quenching of the bound fluorophore by iodide.

Differential accessibility of liganded, high affinity rabbit anti-fluorescyl IgG antibody combining sites to the aqueous milieu has been investigated by solvent perturbation of the extrinsic fluorescence of bound fluorophore. Iodide, a dynamic quencher of fluorescein, was selected for use in these studies after examination of a number of water-soluble fluorescence quenchers. Quenching of antibody-bound fluorophore by iodide was measured with a number of liganded anti-fluorescyl IgG preparations, demonstrating partial solvent exposure of the fluorophore as well as heterogeneity of the high affinity antibody populations. Fluorescence quenching, lifetime, and absorption spectroscopy provided evidence that the antibody-bound fluorophore quenched by iodide interacted with it directly and that anomalous binding of the anion to the surface of the protein, resulting in ground state perturbations of the immunoglobulin, could not explain the observed results.