Calorimetric and EPR studies of the thermotropic phase behavior of phospholipid membranes

Abstract

Transmission electron micrographs (TEM) showed that liposome vesicles prepared from DL-α-phosphatidylcholine dimyristoyl (1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine) (DMPC) by the modified reverse-phase evaporation method (mREV) were spherical in shape and in majority of them were less than 100 nm in diameter. Differential scanning calorimetry (DSC) method was used to determine the influence of cholesterol content and pH of Tris-HCl buffer used for the preparation of liposomes on the temperature of phase transition T C of phospholipids which form the investigated liposome vesicles. The use of DSC method made it possible to determine not only the temperature of the main phase transition of phospholipids but also the temperature of the phospholipid phase transition from the tilted gel phase(L β′) to the ripple gel phase(P β′). The results were compared with those obtained with EPR study. EPR study was carried out in the temperature range from 284 to 310 K i.e. below and above the phase transition temperature T C of DMPC. On the basis of EPR spectra of spin marker 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) incorporated into the liposome, the values of parameters f were determined. Hence TEMPO can be used to observe the change in partition between aqueous and fluid lipid regions. The change in the relative values of f determined for DMPC as a function of temperature shows that this phospholipid undergoes a transition from a ‘gel phase’ to a lamellar smectic liquid crystalline phase in the presence of excess water. The EPR study of TEMPO allowed us to determine the transition temperature T C. The results were compared with those obtained with DSC method.