Emission wavelength-dependent decay of the 9-anthroyloxy-fatty acid membrane probes

Using the phase-modulation technique, we have measured the fluorescence decay of 2- and 12-(9-anthroyloxy)-stearic acid (2- and 12-AS) and 16-(9-anthroyloxy)-palmitic acid (16-AP) bound to egg phosphatidylcholine vesicles or dissolved in nonpolar Solvents. Heterogeneity analysis demonstrates that the decay is generally not monoexponential and exhibits large component variations across it emission spectrum. The mean decay time increases (and in parallel, the steady-state polarization decreases) monotonically with increasing wavelength from values at the blue end. The decay at the red side of the emission spectrum contains an exponential term with a negative amplitude, indicating that emission occurs from intermediates created in the excited-state. This behavior is interpreted as arising from intramolecular fluorophore relaxation occurring on the time scale of the fluorescence lifetime. We believe this to be the first study of wavelength-dependent fluorescent emission which is dominated by an intramolecular relaxation process. Although the three probes exhibit qualitatively similar effects, the emission band variations are greatest for 2-AS and smallest for 16-AP. The differences among the probes are not entirely due to environmental factors as demonstrated, for example, by the emission polarization differences observed in the isotropic solvent paraffin oil. In summary, while these findings point out some of the complexities in the 9-anthroyloxy-fatty acids as membrane probes, they also indicate how these complexities might be used as a sensitive measure of lipid-probe interaction.