Thermodynamic Characterization of the Association of Cholesterol with Phospholipids with Varying Degrees of Unsaturation

The preferential association of cholesterol with saturated phospholipids, especially sphingomyelins, is responsible for the formation of coexisting fluid phases in lipid bilayers, and is believed to play a critical role in the existence of more ordered membrane domains in biological systems, sometimes termed lipid rafts. The coexistence of separate fluid phases in model bilayer constructs must be an equilibrium phenomenon, and titration of bilayer cholesterol content allows for thermodynamic characterization of cholesterol partitioning. To achieve a more complete understanding of the parameters governing phase separation, high sensitivity isothermal titration calorimetry was employed to investigate the effects of degree of unsaturation at the sn-2 position of a series of biologically relevant phospholipids. Cholesterol was either extracted from or added to bilayers of a single phospholipid component, allowing determination of a partition constant and enthalpy of transfer. By varying the relative cholesterol content of the bilayer, the non-ideal partitioning behavior was also investigated. It was found that the relative affinity of cholesterol for the bilayer decreased in a generally monotonic manner in response to increasing unsaturation, with the exception that both 20:4n6 and 22:6n3 acyl chains at the sn-2 position showed similar partition constants and enthalpies of transfer. The partitioning of cholesterol was also measured in membranes composed of mixtures of 18:0,22:6 PC and sphingomyelin. Values of both the partition constant and enthalpy of transfer extrapolated to pure sphingomyelin matched those previously reported for mixtures of 16:0,18:1 PC and sphingomyelin. This result suggests that the association of cholesterol and sphingomyelin is largely unaffected by other phospholipids in the bilayer.