A New Phase Boundary in Phosphatidylcholine/Cholesterol Bilayers (In the Dimyristoylphosphatidylcholine/Cholesterol Bilayer): EPR and DSC Studies

Abstract

A new phase boundary, at cholesterol/dimyristoylphosphatidylcholine (Chol/DMPC) mixing ratio of ∼1, was observed by saturation-recovery electron paramagnetic resonance (EPR) spin-labeling method in a multilamellar suspension of DMPC and Chol prepared using a rapid solvent exchange method. With spin-labeled cholesterol analogues (androstane spin label [ASL] and cholestane spin label [CSL]) it was shown that the upper limit of the cholesterol concentration in the liquid-ordered phase of the DMPC membrane is ∼50 mol%, above which the excess of cholesterol forms the pure cholesterol bilayer domain (CBD). Thus, the phase boundary at a Chol/DMPC mixing ratio of ∼1 separates the region with a single liquid-ordered phase from the region with a coexisting liquid-ordered phase and the CBD. Because the pure cholesterol cannot form the free bilayer in the buffer, the CBD has to be supported by the surrounding phospholipid bilayer saturated with cholesterol. The next phase boundary is defined by the cholesterol solubility threshold (CST) that indicates the amount of cholesterol which can saturate the DMPC bilayer and form the CBD. The excess of cholesterol above the CST forms monohydrate cholesterol crystals which precipitate outside of the lipid bilayer. It was shown by differential scanning calorimetry (DSC) that the CST, which separates the region with a coexisting liquid-ordered phase saturated with cholesterol and the CBD from the region in which cholesterol crystals are formed, is located at the Chol/DMPC mixing ratio of ∼2 (∼66 mol% cholesterol). The extended phase diagram for the DMPC/cholesterol membrane, covering the region where the membrane is saturated and oversaturated with cholesterol, is proposed.