Combined Use of Several Fluorescent Membrane Probes to Study the Subgel Phase and the Effects of Cholesterol Thereon

Abstract

Several lines of evidence have indicated that a highly ordered and condensed phase occurs for phosphatidylcholines at low temperatures. Generation of this lamellar subgel phase (Lc) with dipalmitoylphosphatidylcholine (DPPC) depends on the thermal history of the sample; vesicles must be cooled at ∼4°C for at least 48 h. Similarly, samples exit the Lc phase slowly at the sub-transition temperature (18°C). Consequently, vestiges of Lc behavior can still be detected at 25°C within 15 min of passing through the transition temperature. We used this hysteresis as an assay for detecting the Lc phase with fluorescent membrane probes by recording the emission spectrum or anisotropy on refrigerated vesicles at 15, 25, 35, and 45°C followed by a return to 35, 25, and finally 15°C. In each case, samples were incubated for 10 min at the new temperature before data acquisition. The presence of a Lc or Lc-like phase was recognized by lack of reversal of fluorescent properties at 25 or 15°C. The Lc phase was observed for pure DPPC vesicles by merocyanine 540, prodan, and patman but not laurdan or diphenylhexatriene; fluorescence properties of the latter two probes were completely reversible at all temperatures. Inclusion of cholesterol in the vesicles up to 20 mol% caused a linear reduction in the magnitude of the difference between membrane properties detected by these probes before and after vesicle heating. In contrast to previous reports using other techniques, the lack of reversal was not completely eradicated by cholesterol in the range of 20 to 45 mol% suggesting that an Lc-like phase exists for DPPC/cholesterol mixtures. The distinction between probes that can and cannot detect these phases has implications for interpreting the nature of them.