Cholestatrienol time resolved fluorescence in phosphatidylcholine bilayers

Multifrequency phase and modulation fluorometry and a fluorescent sterol analogue, p 5, 7, 9(11)cholestatrien-3ß-ol (CTE), were used to examine properties of sterols in 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV). The fluorescence decay of CTE in POPC SUV was examined both by sum of exponentials and by distributional analyses. The data best fit a continuous distribution of lifetimes with a two component Lorentzian function. The centers of lifetime distribution were near c1 =0.86 ns and c2 =3.24 ns, fractional intensities f1 =0.96 and f2 =0.04, and peak widths were very narrow. The centers of lifetime distribution, fractional intensities, and peak width at half-height were highly dependent on cholesterol content and vesicle curvature. In the range 0-6 mole %, CTE underwent a concentration dependent transition characterized by red shifted wavelengths of absorption maxima as well as altered ratios of absorbance maxima and fluorescence excitation maxima at 338nm/325nm. Fluorescence intensity of CTE increased up to 6 mole % CTE in POPC SUV while other parameters remained relatively constant. In contrast, between 6-33 mole % CTE, the CTE interacted to self-quench thereby decreasing fluorescence intensity, quantum yield, steady state anisotropy, limiting anisotropy, and rotational relaxation time without decreasing lifetime. The results were consistent with the interpretation that below 6 mole % sterol, the sterols behaved as monomers exposed to some degree to the aqueous solvent in POPC SUV. At higher concentrations the sterol partially segregated. At low mole %, CTE was an excellent probe molecule for determination of the motional properties of sterols in POPC membranes.