Antibody-hapten interactions: Circular and linear polarization of the fluorescence of dansyl bound to anti-dansyl antibodies

Abstract

The fluorescence spectra of several dansyl derivatives (dansylamide, ϵ- N-dansyl- l-lysine, dansyl- l-alanine, and α- N-dansyl- l-alanine amide) bound to anti-dansyl antibodics (induced by an α- N-dansyl-poly d,l-alanine-poly l-lysine conjugate) are shifted by about 60 nm to the blue, and the quantum yields are markedly enhanced, compared to their respective fluorescence properties in water. The light emitted by the bound haptens is partly circularly polarized, reflecting the asymmetry induced in the bound chromophores by the antibody combining site. In contradistinction, the fluorescence spectrum of 1-dansyl-2-alanine diaminoethane bound to anti-alanine antibodies is similar to that of the free fluorophore in water and lacks circular polarization. These results imply that in this case the fluorophore of the hapten protrudes out of the site into the aqueous solvent. No circular dichroism is observed in the 300 to 400 nm region for the dansyl-anti-dansyl complex. Thus a change in the mode of interaction between the chromophore and its binding site takes place upon electronic excitation. The heterogeneity of the antibody binding sites is expressed by the dependence of the circular polarization of fluorescence on excitation wavelength. Differences in the circular polarization of luminescence were also observed when the residues attached to the dansyl group have been varied. This may reflect differences in the alignment of the fluorophore within the binding sites for the different dansyl derivatives. The linear polarization of dansylamide dissolved in glycerol is not constant across the emission band, indicating that the transition dipole moments related to the various vibronic states do not have the same spatial directions. Vibronic mixing of the emitting excited state with higher electronic states is thus indicated. Dansyl- l-alanine bound to anti-dansyl antibodies exhibitsan even more pronounced variation of the linear polarization across the emission band. In this case, the dependence of the linear polarization of the emitted light on excitation wavelength is anomalous, which is again a reflection of the heterogeneity of the population of the antibody molecules. The implications of these results to the studies of the fluorescence polarization of dansyl-protein complexes are discussed.