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-alpha-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 degrees 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.