Examining the Effects of Cholesterol: Laurdan and Patman see it Differently

Abstract

The liquid-ordered phase induced by cholesterol in phosphatidylcholine bilayers can be detected with Laurdan fluorescence by an increase in the associated Generalized Polarization (GP) value. This increase in GP is usually interpreted as a reduction in the access of water molecules to the bilayer at depths approaching the phospholipid glycerol backbones. Comparisons of Laurdan fluorescence over a broad range of temperatures with various saturated and unsaturated phosphatidylcholines demonstrated that cholesterol has little effect on GP at temperatures below the melting point of the pure lipid (tm). However, above tm, increasing cholesterol concentrations monotonically raised the value of GP. The resulting GP increments generally did not vary with temperature above tm. In contrast, the observations were more complex with Patman, a charged derivative of Laurdan. First, cholesterol raised the value of Patman GP at temperatures below tm, and its effect as a function of concentration was bimodal. Second, although cholesterol increased Patman GP above tm (similar to Laurdan), the effect size was smaller and was bimodal as a function of cholesterol concentration. Finally, the GP values at high versus low cholesterol concentration converged as temperature was raised well beyond tm. In some cases, such as with unsaturated lipids ≥54°C above tm, this convergence of Patman GP values reached a cross-over point; Patman GP was reduced by the presence of cholesterol beyond this point. These results suggest that the charge associated with Patman, or possibly the slightly deeper location of the probe in the bilayer, provides a means for identifying additional effects of cholesterol on the membrane that are not visible with Laurdan.