Phase transitions and phase separations in phospholipid membranes induced by changes in temperature, pH, and concentration of bivalent cations.

Differential scanning calorimetry (DSC) and fluorescence polarization of embedded probe molecules were used to detect phase behavior of various phospholipids. The techniques were directly compared for detecting the transition of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidic acid (DPPA) dispersed in aqueous salt solutions. Excellent agreement occurred in the case of phosphatidylcholine; however, in the case of phosphatidic acid, at pH 6.5, transitions detected by fluorescence polarization using the disc-like perylene molecule occurred about 10 degrees lower than those detected by DSC. Discrepancy between fluorescence and DSC methods is eliminated by using a rod-like molecule, diphenylhexatriene (DPH). Both techniques show that doubly ionizing the phosphate group reduces the Tc by about 9 degrees. Direct pH titration of fluidity can be accomplished and this effect is most dramatic when membranes are in their transition temperature range (ca. 50 degrees). Phosphatidic acid transitions occur at higher temperatures, and have appreciably lower transition enthalpies and entropies than phosphatidylcholine. These effect could not be explained simply on the basis of double layer electrostatics and several other factors were discussed in an attempt to rationalize the results. Addition of monovalent cations (0.01-0.5 M) is shown to increase the Tc of dipalmitoylphosphatidylglycerol by less than 3 degrees. However, addition of (1 x 10-3 M) Ca2+ abolishes the phase transition of both phosphatidyglycerol and phosphatidylserine in the range 0-70 degrees. Preliminary X-ray evidence indicates the phosphatidylserine-Ca2+ bilayers are in a crystalline state at 24 degrees. In contrast, 5 x 10-3 M Mg2+ only broadens the transition and increases the Tc indicating a considerable difference between the effects of Ca2+ and Mg2+. Neutralization of PS increases the Tc from 6 degrees (at pH 7.4) to 20-26 degrees (at pH 2.5-3.0) but does not abolish the transition, suggesting the Ca2+ effect involves more than charge neutralization. Addition of Ca2+ to mixed phosphatidylserine-phosphatidylcholine dispersions, induces a phase separation of the dipalmitoyl- (and also distearoyl-) phosphatidylcholine as seen by the appearance of a new endothermic peak at 41 degrees (58 degrees). Similarly, in mixed (dipalmitoyl) phosphatidic acid-phosphatidylcholine (2:1) dispersions, Ca2+ again can separate the phosphatidylcholine component.