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-α-phosphatidylcholine (DMPC), dipalmitoyl-L-α-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 Δ_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°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 Δ measurements of DPH in DMPC and DPPC bilayers is characterized by a rapid increase of tan Δ at the transition temperature (T_c), followed by a gradual decline in tan Δ at temperatures above T_c. The observed tanΔ_max values are only 62 and 43% of the theoretical maximum. This defect in tanΔ_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 Δ values under conditions where the probe's rotational motions are restricted to a limiting anisotropy value, r_∞. 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_∞ at this same temperature. The sensitivity of the tan Δ values to both R and r_∞ indicates that differential phase fluorometry will provide a method to describe more completely the depolarizing motion of probes in lipid bilayers.