INFLUENCE OF CHOLESTEROL AND BETA-SITOSTEROL ON THE DYNAMIC BEHAVIOR OF DPPC AS DETECTED BY TMA-DPH AND PYRPC FLUORESCENCE - A FLUORESCENCELIFETIME DISTRIBUTION AND TIME-RESOLVED ANISOTROPY STUDY

Fluorescence lifetime and time-resolved anisotropy measurements on rim ethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and oyl-2-[1 0-(1-pyrenyl)decanoyl]-phosphatidylcholine (PyrPC) in lipid vesicles o f 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) with and wit hout cholesterol and beta-sitosterol, using multifrequency cross-corre lation 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 h ave investigated the influence of sterol structure on the lipid dynami cs. The experiments were carried out at two temperatures, one below an d one above the main phase transition temperature of DPPC. Data were a nalyzed in terms of Lorentzian distribution functions for the fluoresc ence lifetime results and in terms of hindered rotation for the time-r esolved anisotropy results. We noticed, that addition of cholesterol a nd beta-sitosterol results in a distinct increase in average fluoresce nce 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 in crease in the contribution of fast rotational motions of TMA-DPH is fo und as the temperature is increased through the main transition. No si gnificant differences in rotational rates of TMA-DPH exist between sam ples with embedded cholesterol or beta-sitosterol at T=55 degrees C. T he 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, how ever. 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-% ster ol. The incorporation of either sterol into DPPC vesicles leads to an increase in fluorescence lifetime of PyrPC in both lipid phases. The r otational rates of PyrPC in the bilayer are an order of magnitude smal ler than those of TMA-DPH. Also the contributions of fast rotational m otions significantly differ for the two fluorophores. The motional fre edom in the deeper acyl chain region of DPPC in its fluid-like state a t T=55 degrees C is scarcely affected by addition of either sterol. Ho wever, differences in the rotational rates of the two sterols are obse rved for high sterol concentrations.