Phase fluorometric studies of spectral relaxation at the lipid-water interface of phospholipid vesicles

We examined the dynamic properties of the lipid-water interface region of model membranes using the probe 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS). For comparison we also examined the temperature-dependent spectral properties of TNS in the viscous solvent glycerol. Fluorescence phase shift and demodulation measurements were used to prove that the membranes relax around the excited state of TNS on the ns time scale. The rate of spectral relaxation is thought to reflect the mobility of the polar interface region of the membranes on this same time scale. The spectral relaxation times were estimated by the use of phase-sensitive detection of fluorescence. Using this method one may directly record, in an approximate fashion, the emission spectra of the relaxed and the initially excited states of TNS. The relative intensities of these phase-sensitive spectra, in combination with the measured phase and modulation values on the short and long wavelength sides of the emission, yield the spectral relaxation times. For saturated and unsaturated phosphatidylcholines, at temperatures ranging from 5 to 50°C, the relaxation times ranged from 5 to 1 ns. The activation energies for spectral relaxation were near 4 kcal/mol. Surprisingly, the relaxation times decreased smoothly with increasing temperature, and did not change abruptly at the phase transition temperatures. These results indicate that the small molecular motions of the interface region of membranes, which are responsible for spectral relaxation, are not dramatically influenced by the phase state of the acyl side chain region of the membranes.