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

Abstract

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.

Fig. 1.

Fluorescence lifetimes and differential polarized phase lifetimes of DPH in propylene glycol.

Fig. 2.

Rotational rate of DPH in propylene glycol. Rotational rates were calculated by both steady-state polarization mea surements (A) and differential polarized phase fluorometry (B). The duplicate points between the bars in (B) result from the two possible solutions to Eq. 1. Near Δτ = Δτ_max the choice of the proper solution is not clear. However, the value chosen is often unimportant since the rotational rates are similar.

Fig. 3.

Fluorescence lifetimes (○) and differential polarized phase lifetimes (□) of DPH in DMPC vesicles. he Δτ_max indicates the maximum differential lifetime predicted for a free isotropic rotator with r₀ = 0.392 and τ = 8.7 nsec.

Fig. 4.

Limiting anisotropy values for DPH in DMPC vesicles. Limiting anisotropy values (□) were calculated from Eqs. 9 to 11, using data from Fig. 3 at regular temperature intervals, and from the steady-state anisotropy values (○). By using data read off Fig. 3, we effectively smoothed the data and minimized errors resulting from single data points. There are two solutions for r_∞. at each point because Eq. 11 is quadratic. However, in each in stance one of the rotational rates was negative and thus inadmissible.