Quantitation of hindered rotations of diphenylhexatriene in lipid bilayers by differential polarized phase fluorometry

Diffusional motions of 1,6-diphenyl-1,3,5-hexatriene (DPH) were observed by differential polarized phase fluorometry. The measurements indicated that the depolarizing rotations of DPH in propylene glycol are isotropic. The results in vesicles of dimyristoyl-L-α-phosphatidylcholine indicated that diffusional rotations of DPH are dominated by hindered torsional motions. Combined use of both differential phase and steady-state anisotropy measurements showed that the average rotational angle of DPH, at times long compared to the fluorescence lifetime, is limited to about 23° at temperatures below the transition temperature of the lipid and that these rotations become less hindered above the transition temperature. The evidence that the depolarizing rotations of DPH in a lipid bilayer are different from those in an isotropic solvent calls into question the meaning of membrane microviscosity as determined by fluorescence anisotropy.