Influence of cholesterol and beta-sitosterol on the dynamic behaviour of DPPC as detected by TMA-DPH and PyrPC fluorescence - A fluorescence lifetime distribution and time-resolved anisotropy study

Fluorescence lifetime and time-resolved anisotropy measurements on 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1-palnitoyl-2-[10-(1-pyrenyl)decanoyl]-phosphatidylcholine (PyrPC) in lipid vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) with and without cholesterol and beta-sitosterol, using multifrequency cross-correlation phase fluorometry, are presented. We used TMA-DPH and PyrPC as fluorescence probe for detecting rotational dynamics. Contrary to TMA-DPH, which is tethered to the bilayer interface and therefore detects wobbling rotational dynamics in the interfacial and headgroup region, PyrPC senses the deeper acyl chain dynamics of the lipid bilayer. We have investigated the influence of sterol structure on the lipid dynamics. The experiments were carried out at two temperatures, one below and one above the main phase transition temperature of DPPC. Data were analyzed in terms of Lorentzian distribution functions for the fluorescence lifetime results and in terms of hindered rotation for the time-resolved anisotropy results. We noticed, that addition of cholesterol and beta-sitosterol results in a distinct increase in average fluorescence lifetime of TMA-DPH in both lipid phases. The rotational rates of TMA-DPH depolarizing motions are only modestly increased in going from the gel to the liquid-crystalline phase of DPPC. However, a marked increase in the contribution of fast rotational motions of TMA-DPH is found as the temperature is increased through the main transition. No significant differences in rotational rates of TMA-DPH exist between samples with embedded cholesterol or beta-sitosterol at T=55 degrees C. The rotational rate of TMA-DPH in the gel state of the lipid bilayer at T=35 degrees C is slightly larger in the case of beta-sitosterol, however. Cholesterol and beta-sitosterol, then, have a similar effect on the contribution of fast rotational dynamics in the interfacial region of the bilayer. It decreases about 40% upon addition of 50 mol-% sterol. The incorporation of either sterol into DPPC vesicles leads to an increase in fluorescence lifetime of PyrPC in both lipid phases. The rotational rates of PyrPC in the bilayer are an order of magnitude smaller than those of TMA-DPH. Also the contributions of fast rotational motions significantly differ for the two fluorophores. The motional freedom in the deeper acyl chain region of DPPC in its fluid-like state at T=55 degrees C is scarcely affected by addition of either sterol. However, differences in the rotational rates of the two sterols are observed for high sterol concentrations.