Large Domain Formation in POPC/POPE Lipid Bilayers at the Maximum Solubility Limit of Cholesterol

Abstract

Lipid bilayers composed of POPC and POPE usually stay as 1-phase mixtures at room temperature, with or without incorporation of modest amount of cholesterol. However, at the maximum solubility limit of cholesterol, possibility of domain formation has been suggested based on the detail molecular packing patterns of phospholipids with cholesterol. The maximum solubility of cholesterol in a lipid bilayer is the highest mole fraction of cholesterol that can be incorporated into a lipid bilayer before cholesterol crystals precipitate. Previously, the maximum solubility of cholesterol in mixtures of POPC/POPE lipid bilayers has been measured using a cholesterol oxidase (COD) reaction rate assay. The maximum solubility of cholesterol was determined to be 67 mol % in POPC bilayers and 50 mol % in POPE bilayers. In mixtures of POPE/POPC, the maximum solubility of cholesterol increases linearly as a function of the ratio POPC/(POPE+POPC). In this study, planar lipid bilayers of POPC and POPE at their cholesterol solubility limits were incubated on mica surfaces inside hydrated chambers. Although the bilayers appeared having uniform fluorescence intensity initially, we found that large lipid domains became visible by fluorescence microscopy after several days of incubation. Previously, it has been suggested that cholesterol may form a “hexagonal” regular distribution pattern at the maximum solubility limit in POPE bilayers and a “maze” pattern at the maximum solubility in POPC bilayers. Thus, it is possible that the lipid mixtures separated into POPC-rich and POPE-rich lipid domains, each having their signature lipid packing pattern and mole fraction of cholesterol.