Hindered depolarizing rotations of perylene in lipid bilayers. Detection by lifetime-resolved fluorescence anisotropy measurements

Abstract

Oxygen quenching of perylene fluorescence was used to vary its fluorescence lifetime. Steady-state fluorescence anisotropy measurements under these quenching conditions were used to investigate the diffusive motions of perylene in the isotropic solvent propylene glycol and in lipid bilayers. These lifetime-resolved anisotropy measurements indicate that the anisotropy of perylene in propylene glycol decays to zero at times long compared to its fluorescence lifetime. In contrast, the asymptotic or "limiting" anistropy values at these long times (r infinity) are nonzero in vesicles of dimyristoyl-phosphatidylcholine (DMPC). r infinity values are largest at temperatures below the DMPC phase transition temperature of 23 degrees C. Representative values of r infinity for perylene in DMPC vesicles are 0.16 and 0.02 at 5 and 47 degrees C, respectively. Thus, in contrast to the free rotations observed for perylene in propylene glycol, perylene rotations are hindered in lipid bilayers. Less marked, yet significant, rotational hindrance was observed in dioleoylphosphatidylcholine (DOPC) vesicles. Representative values for r infinity in this unsaturated lipid are 0.05 and 0.01 at 2 and 45 degrees C, respectively. Steady-state anisotropy measurements with short-wavelength excitations were used to investigate whether the in-plane or out-of-plane rotations of perylene were responsible for the observed r infinity values. In DMPC vesicles we conclude that both rotations are partially hindered. In DOPC vesicles we can only conclude that one or both of these rotations are partially hindered, but both are not free. Most importantly, the existence of fundamentally different diffusive behavior for perylene in solvents and in lipids calls into question the meaning of membrane microviscosities which are derived via such comparisons.

FIGURE 1:

Effect of rotational anisotropy on the apparent limiting anisotropies. The solid lines represent the lifetimes and anisotropies which may be obtained by using oxygen quenching of perylene fluorescence, i.e., a range in τ from 8 to ~0.2 ns. The dashed lines illustrate the inaccessible lifetime range from 500 to 8 ns. The dotted lines illustrate how anisotropic rotations can yield apparent nonzero anisotropy values.

FIGURE 2:

Fluorescence emission spectra of perylene in DMPC vesicles.

FIGURE 3:

Stern–Volmer plots for oxygen quenching of perylene in DMPC vesicles.

FIGURE 4:

Separation of the static and dynamic oxygen quenching constants for perylene in DMPC vesicles.

FIGURE 5:

Effect of rotational anisotropy on the lifetime-resolved fluorescence anisotropy of perylene in propylene glycol. The anisotropic rotor model was used to fit this solvent data in a least-squares sense. The error bars indicate the effect of a ±0.2-ns error in τ and a ±0.005 error in anisotropy. Although the best fit was isotropic (R_ip/R_op = 1), ratios of R_ip/R_op up to about 10 are within error estimates.

FIGURE 6:

Lifetime-resolved anisotropies for perylene in DMPC and in DOPC vesicles. The dashed line represents similar data for perylene in propylene glycol. Note that the r-axis intercepts are clearly nonzero in the bilayers and especially at temperatures below the phase transition temperature of DMPC. These intercepts indicate hindered depolarizing rotations.

FIGURE 7:

Limiting (r_∞) and steady-state (r) anisotropies for perylene in lipid vesicles. The open squares are the observed r values and the solid squares are the r_∞ values for perylene in propylene glycol.

FIGURE 8:

Arrhenius plots for the perylene rotational rates derived from the free and hindered models. The open square is for perylene in propylene glycol at 26 °C. In this case, r_∞ = 0, and the same rotational rate is obtained with either model.

FIGURE 9:

Lifetime-resolved fluorescence anisotropies of perylene. These data, for perylene in propylene glycol and in lipid bilayers, were fit to that expected for a hindered rotator (eq 11). The symbols represent actual data, and the solid lines are the theoretical curves.

FIGURE 10:

Steady-state fluorescence anisotropies obtained at short-wavelength excitation. r₀ = 0.1 and r₀ = −0.165 were obtained by using 314- and 252-nm excitation, respectively.