Time-Resolved Fluorescence Anisotropics of Diphenylhexatriene and Perylene in Solvents and Lipid Bilayers Obtained from Multifrequency Phase-Modulation Fluorometry

Time-resolved decays of fluorescence anisotropy were obtained from frequency-domain measurements of the phase angle difference between the parallel and perpendicular components of the polarized emission and the ratio of the modulated amplitudes. These data were measured at modulation frequencies ranging from 1 to 200 MHz. To demonstrate the general applicability of this method, we describe the resolution of both simple and complex decays of anisotropy. In particular, we resolved single, double, and triple exponential decays of anisotropy and the hindered rotational motions of fluorophores within lipid bilayers. The ease and rapidity with which these results were obtained indicate that frequency-domain measurements are both practical and reliable for the determination of complex decays of anisotropy. Tme-resolved decays of fluorescence anisotropy reveal the time-dependent rotational motions of fluorophores on the nanosecond time scale. These motions are generally dependent Supported by National Science Foundation Grants PCM 80-41 320, 81-06910, and 82-10878 to J.R.L. and PCM 79-18646 to E.G. *On leave from Nicholas Copernicus University, Torun, Poland. 0006-2960/85/0424-0376$0 1 .50/0 upon the fluorophore and its surrounding environment. For instance, time-resolved anisotropies can reveal the torsional motions of tryptophan and tyrosine residues in proteins (Ichiye & Karplus, 1983; Levy & Szabo, 1982; Lipari & Szabo 1980; Munro et al., 1979; Ross et al., 1981a,b), which in turn may reflect the extent of structural fluctuations within the protein matrix. Additionally, time-resolved anisotropies have provided