A fluorescence study of dehydroergosterol in phosphatidylcholine bilayer vesicles

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Abstract
The fluorescent sterol .DELTA.5,7,9(11),22-ergostatetraen-3.beta.-ol (dehydroergosterol) was incorporated into 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV) with and without cholesterol in order to monitor sterol-sterol interactions in model membranes. In the range 0-5 mol% fluorescent sterol, dehydroergosterol underwent a concentration-dependent relaxation characterized by red-shifted wavelengths of maximum absorption as well as altered ratios of absorbance maxima and fluorescence excitation maxima at 338 nm/324 nm. Fluorescence intensity per mole of dehydrogergosterol increased up to 5 mol % in POPC vesicles. In contrast, quantum yield, steady-state anisotropy, limiting anisotropy, lifetime, and rotational rate remained relatively constant in this concentration range. Similarly, addition of increasing cholesterol in the range 0-5 mol % in the presence of 3 mol% dehydroergosterol also increased the fluorescence intensity per mole of dehydroergosterol, red-shifted wavelengths of maximum absorption, and altered ratios of absorbance maxima. In POPC vesicles containing between 5 and 33 mol % dehydroergosterol, the fluorescent dehydroergosterol interacted to self-quench, thereby decreasing the fluorescence intensity, quantum yield, steady-state anisotropy, and limiting anisotropy and increasing the rotational rate (decreased rotational relaxation time) of the fluorescent sterol. The fluorescence lifetime of dehydroergosterol remained unchanged. The results were in accord with the interpretation that below 5 mol % sterol, the sterols behaved as monomers exposed to some degree to the aqueous solvent in POPC bilayers. At higher concentrations, the sterol laterally segregated. Above 33 mol % sterol, only a single type of sterol phase existed within the POPC bilayer. At low mole percent fluorescent sterol, dehydroergosterol was an excellent probe molecule for determination of the motional properties (dynamic and static) of sterols in POPC membranes. Differential polarized phase fluorescence spectroscopy indicated that the dehydroergosterol had a rapid rotational rate and a high degree of order at 24.degree. C in POPC vesicles. Between 7 and 50 mol % cholesterol, the limiting anisotropy, quantum yield, and lifetime increased while the rotational rate decreased. In summary, in POPC vesicles containing mixtures of dehydroergosterol and cholesterol, the two sterols appear similar in motional as well as dielectric properties.