Fluorescence properties of cholestatrienol in phosphatidylcholine bilayer vesicles

Abstract

The fluorescent sterol A5~7~g*~")-cholestatrien-3&ol (cholestatrienol) was incorporated into l-pahnitoyl-2-oleoyl-phosphatidylcholine (POPC) small unilamellar vesicles (WV) with and without cholesterol in order to monitor sterol-sterol interactions in model membranes. Previously another fluorescent sterol, dehydroergosterol (F. Schroeder, Y. Barenholz, E. Gratton and T.E. Thompson, Biochemistry 26 (1987) 2441), was used for this purpose. However, there is some concern that dehydroergosterol may not be the best analogue for cholesterol. Fluorescence properties of cholestatrienol in POPC SW were highly sensitive to cholestatrienol purity. The fluorescence decay of cholestatienol in the POPC SUV was analyzed by assuming either that the decay is comprised of a discrete sum of exponential components or that the decay is made up of one or more component's distribution of lifetimes. The decay for cholestatrienol in POPC SUV analyzed using distributions had a lower x2 value and was described by a two-component Lorentzian function with centers near 0.86 and 3.24 ns, and fractional intensities of 0.96 and 0.04, respectively. Both distributions were quite narrow, i.e., 0.05 ns full-width at half-maximum peak height. It is proposed that the two lifetime distributions are generated by separate continua of environments for the cholestatrienol molecule described by different dielectric constants. In the range O-6 mol% cholestatrienol, the cholestatrienol underwent a concentration-dependent relaxation. This process was characterized by red-shifted absorption and maxima and altered ratios of absorption and fluorescence excitation maxima. Fluorescence quantum yield, lifetime, steady-state anisotropy, limiting anisotropy and rotational rate remained constant. In contrast, in POPC vesicles containing between 6 and 33 mol% cholestatrienol, the fluorescent cholestatrienol partially segregated, resulting in quenching. Thus, below 6 mot% cholestatienol, the cholestatrienol appeared to behave in part as monomers exposed to some degree to the aqueous solvent in a sterol-poor domain within POPC bilayers. Sin= the lifetime did not decrease above 6 molX cholestatrienol, the fluorescence at high mol% values of cholestatrienol was due to cholestatrienol in the sterol-poor domain. The fluorescence intensity, quantum yield, steady-state anisotropy, and limiting anisotropy of cholestatrienol in the sterol-poor domain decreased to limiting, nonzero values while the rotational rate increased to a limiting value. Thus, the sterol-poor domain became more disordered when it coexisted with the sterol-rich domain. In POPC vesicles containing 3 mol% cholestatrienol plus increasing mol% cholesterol the two sterols codistributed, since fluorescence quenching was not observed. Above 6 mol% total sterol the cholestatrienol was sensitive to sterol motional properties in the laterally segregated sterol-rich domain. The sterol-rich domain became more rigid with increasing cholesterol content of the POPC SUV. The data are consistent with the presence of a r...

Comparative Cellular Toxicity of Hydrophilic and Hydrophobic Microcystins on Caco-2 Cells

“…This transfer of energy may occur depending, for example, on the orientation of the molecules and their distance from each other [37]. Cholestatrienol (CTL), a fluorescent analog of cholesterol, was used as an acceptor due to its suitable fluorescence properties [38]. Resonance energy transfer can occur if there is a spectral overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, provided that certain other criteria are met, such as the distance between the two molecules [35].…”

Cholesterol Reporter Molecules

“…Both fluorescent sterol analogues have been introduced at the same time as membrane and lipoprotein probes and can be used to measure the sterol exchange between membranes (Bergeron and Scott 1982;Nemecz et al 1988). In each case, cholestatrienol is regarded as a cholesterol analogue that mimics the membrane behavior of cholesterol quite well (Fischer et al 1984;Schroeder et al 1988;Hyslop et al 1990;Yeagle et al 1990;Scheidt et al 2003;Bjorkqvist et al 2005;Smutzer et al 1986). The reason why cholestatrienol has not been used as much as dehydroergosterol is probably ascribed to the fact that it is not commercially available up to now.…”

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

“…The usefulness of DHE and cholestatrienol (CTE) as fluorescent cholesterol analogs in model membranes in a series of investigations spanning several decades demonstrated the usefulness of these probes to monitor cholesterol structural (polarization, limiting anisotropy, order) and dynamic (lifetime, rotational rate) properties in membranes (12,65,70,95,96,108,(110)(111)(112)(113)(114)(115)(116)(117)(118)(119)(120). The lifetime studies resolved for the first time at least two DHE domains in model membranes, one less sensitive to the aqueous than the other, and that these domains were dependent on the lipid composition, temperature, and other properties of the membrane (54,119).…”