Solvent perturbation of the fluorescence of fluorescyl ligand bound to specific antibody fluorescence enhancement of antibody bound fluorescein (hapten) in deuterium oxide

Abstract

The relative fluorescence quantam yield (s) and fluorescence lifetime (τ) of fluorescein bound to specifically purified high affinity rabbit and chicken IgG antibody molecules was significantly increased in deuterium oxide (D 2O) with respect to the complex in aqueous solution. The degree of enhancement appeared to be dependent on the affinity of the antibody for the ligand, the highest affinity sites showing the smallest increases in quantum yield. Fluorescence enhancement in D 2O was identical for ligand bound to IgG and the F(ab)' 2 fragments derived from the same molecule. The isotope effect was apparently not due to ligand dissociation, as shown by a comparison of difference spectroscopy and equilibrium dialysis results in D 2O and H 2O buffers, and by the observation of similar enhancement with affinity labeled antibody. Fluorescence enhancement of antibody bound ligand in D 2O was temperature dependent. The relative s D 2O /s H 2O of free fluorescein increased, while that of bound ligand remained relatively constant with a lowering of the pH. Deuterium oxide did not affect the ground state of fluorophore-antibody complex. Identical extrinsic circular dichroism of the antibody bound fluorophore in H 2O and D 2O, coupled with the absence of a méasurable isotope effect on the quenching of fluorescein by l-tryptophan, supports the contention that the isotope effect is an excited state phenomenon.