Steady State and Multifrequency Phase Fluorometry Studies of Binding Site Flexibility in Related Antibodies

The fluorescence properties of related antibodies were characterized from proteins representing different time points in the murine antibody immune response (the period of time during which the selectivity and sensitivity to the small molecule hapten is enhanced several 100−1000-fold). Two of the antibodies studied are catalytic and bind covalently to a substrate analogue used to induce antibody production. The remaining three represent antibodies produced after primary, secondary, and tertiary immunizations with hapten. In the first series of experiments, steady-state fluorescence spectroscopy and multifrequency phase fluorometry were used to examine the fluorescence properties of the proteins' tryptophan residues. Though unique interpretations regarding the binding site differences are confounded by the intrinsic complexity of the system, analysis of the data reveals that while the lifetimes are similar for the different antibodies, the lifetime distributions seem to narrow as a function of antibody maturity. Hapten binding results in little change in the lifetimes but a marked narrowing of the lifetime distributions. In the second series of experiments, the steady state and multifrequency phase fluorometry data of Prodan [6-propionyl-2-(dimethylamino)naphthalene], a fluorescent hapten analogue, were analyzed for binding site differences. Excitation and emission spectra, lifetimes, and lifetime distributions were measured and demonstrated differences in the binding sites of antibodies collected at different stages of the immune response.