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

The isothermal phase behavior of three gramicidin A'/phospholipid mixt ures was investigated by an equilibrium Ca2+-binding technique. The ph ospholipid component was 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS ), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), or S/1-palm itoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at a constant mole r atio of 1/4. The bulk aqueous free Ca2+ concentration, [Ca2+](f), in equilibrium with one or two gramicidin A'/phospholipid fluid phases an d a small amount of the Ca(phosphatidylserine)(2) gel phase, was measu red as a function of composition at 20 degrees C by use of chromophori c high-affinity Ca2+ chelators. The coexistence of two gramicidin A'/p hospholipid fluid phases was detected by an invariance in [Ca2+](f) o ver the range of compositions throughout which the two phases coexist. The compositions of the two coexisting phases are determined by the c ompositions at which the invariance in [Ca2+](f) begins and ends. Wit h each of the gramicidin A'/phospholipid mixtures, we estimate that th e 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 gramicid in-poor phase is an L(alpha) phase and the gramicidin-rich phase is an HII phase. The isothermal phase behavior of gramicidin A'/POPC mixtur es at similar to 23 degrees C, as determined by low-angle X-ray diffra ction, was found to be similar to that of the other gramicidin A'/phos pholipid 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 equ ilibrium Ca2+-binding technique to the detection of phospholipid phase separations, including L(alpha)(1)/L(alpha)(2) phase separations, is discussed.