DETECTION OF COEXISTING FLUID PHOSPHOLIPID PHASES BY EQUILIBRIUM CA2+ BINDING - PEPTIDE-POOR L-ALPHA, AND PEPTIDE-RICH H-II PHASE COEXISTENCE IN GRAMICIDIN A'/PHOSPHOLIPID DISPERSIONS

The isothermal phase behavior of three gramicidin A'/phospholipid mixtures was investigated by an equilibrium Ca2+-binding technique. The phospholipid component was 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), or POPS/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at a constant mole ratio of 1/4. The bulk aqueous free Ca2+ concentration, [Ca2+]*(f), in equilibrium with one or two gramicidin A'/phospholipid fluid phases and a small amount of the Ca(phosphatidylserine)(2) gel phase, was measured as a function of composition at 20 degrees C by use of chromophoric high-affinity Ca2+ chelators. The coexistence of two gramicidin A'/phospholipid fluid phases was detected by an invariance in [Ca2+]*(f) over the range of compositions throughout which the two phases coexist. The compositions of the two coexisting phases are determined by the compositions at which the invariance in [Ca2+]*(f) begins and ends. With each of the gramicidin A'/phospholipid mixtures, we estimate that the composition of the gramicidin-poor phase is 0.03-0.04 mole fraction gramicidin A' and the composition of the gramicidin-rich phase is 0.13-0.14 mole fraction gramicidin A'. Characterization of these phases by low-angle X-ray diffraction revealed that, in each case, the gramicidin-poor phase is an L(alpha) phase and the gramicidin-rich phase is an HII phase. The isothermal phase behavior of gramicidin A'/POPC mixtures at similar to 23 degrees C, as determined by low-angle X-ray diffraction, was found to be similar to that of the other gramicidin A'/phospholipid mixtures. P-31 nuclear magnetic resonance spectroscopy failed to detect the gramicidin-rich H-II phase in all cases except when the phospholipid component was DOPS. The general applicability of the equilibrium Ca2+-binding technique to the detection of phospholipid phase separations, including L(alpha)(1)/L(alpha)(2) phase separations, is discussed.