Hindered depolarizing rotations of perylene in lipid bilayers. Detection by lifetime-resolved fluorescence anisotropy measurements.
Oxygen quenching of perylene fluorescence was used to vary its fluorescence lifetime. Steady-state fluorescence anisotropy measurements under these quenching conditions were used to investigate the diffusive motions of perylene in the isotropic solvent propylene glycol and in lipid bilayers. These lifetime-resolved anisotropy measurements indicate that the anisotropy of perylene in propylene glycol decays to zero at times long compared to its fluorescence lifetime. In contrast, the asymptotic or "limiting" anistropy values at these long times (r infinity) are nonzero in vesicles of dimyristoyl-phosphatidylcholine (DMPC). r infinity values are largest at temperatures below the DMPC phase transition temperature of 23 degrees C. Representative values of r infinity for perylene in DMPC vesicles are 0.16 and 0.02 at 5 and 47 degrees C, respectively. Thus, in contrast to the free rotations observed for perylene in propylene glycol, perylene rotations are hindered in lipid bilayers. Less marked, yet significant, rotational hindrance was observed in dioleoylphosphatidylcholine (DOPC) vesicles. Representative values for r infinity in this unsaturated lipid are 0.05 and 0.01 at 2 and 45 degrees C, respectively. Steady-state anisotropy measurements with short-wavelength excitations were used to investigate whether the in-plane or out-of-plane rotations of perylene were responsible for the observed r infinity values. In DMPC vesicles we conclude that both rotations are partially hindered. In DOPC vesicles we can only conclude that one or both of these rotations are partially hindered, but both are not free. Most importantly, the existence of fundamentally different diffusive behavior for perylene in solvents and in lipids calls into question the meaning of membrane microviscosities which are derived via such comparisons.