Detection of hindered rotations of 1,6-diphenyl-1,3,5-hexatriene in lipid bilayers by differential polarized phase fluorometry.

Differential polarized phase fluorometry has been used to investigate the depolarizing motions of 1,6-diphenyl-1,3,5-hexatriene (DPH) in the isotropic solvent propylene glycol and in lipid bilayers of dimyristoyl-L-alpha-phosphatidylcholine (DMPC), dipalmitoyl-L-alpha-phosphatidylcholine (DPPC), and other phosphatidylcholines. Differential phase fluorometry is the measurement of differences in the phase angles between the parallel and perpendicular components of the fluorescence emission of a sample excited with sinusoidally modulated light. The maximum value of the tangent of the phase angle (tan Delta(max)) is known to be a function of the isotropy of the depolarizing motions. For DPH in propylene glycol the maximum tangent is observed at 18 degrees C, and this tangent value corresponds precisely with the value expected for an isotropic rotator. Additionally, the rotational rates determined by steady-state polarization measurements are in precise agreement with the differential phase measurements. These results indicate that differential phase fluorometry provides a reliable measure of the probe's rotational rate under conditions where these rotations are isotropic and unhindered.Rotational rates of DPH obtained from steady-state polarization and differential phase measurements do not agree when this probe is placed in lipid bilayers. The temperature profile of the tan Delta measurements of DPH in DMPC and DPPC bilayers is characterized by a rapid increase of tan Delta at the transition temperature (T(c)), followed by a gradual decline in tan Delta at temperatures above T(c). The observed tanDelta(max) values are only 62 and 43% of the theoretical maximum. This defect in tanDelta(max) is too large to be explained by any degree of rotational anisotropy. However, these defects are explicable by a new theory that describes the tan Delta values under conditions where the probe's rotational motions are restricted to a limiting anisotropy value, r(infinity). Theoretical calculations using this new theory indicate that the temperature dependence of the depolarizing motions of DPH in these saturated bilayers could be explained by a rapid increase in its rotational rate (R) at the transition temperature, coupled with a simultaneous decrease in r(infinity) at this same temperature. The sensitivity of the tan Delta values to both R and r(infinity) indicates that differential phase fluorometry will provide a method to describe more completely the depolarizing motion of probes in lipid bilayers.