Chemically Induced Phase Separation in Mixed Vesicles Containing phosphatidic Acid.An Optical Study

The possibility of chemically induced phase separation in lipid lamellae containing charged lipids is demonstrated. In mixed membranes of synthetic dipalmitoyllecithin (DPL) and dipalmitoylphosphatidic acid (DPA), a lipid domain structure may be triggered both by the addition of Ca²⁺ and of polylysine in random coil configuration. A new optical (excimer forming) probe, pyrenedecanoic acid, is introduced. The rate of excited complex formation is the crucial physical parameter yielding quantitative information on both the coefficient of lateral diffusion and on the lipid segregation. At all pH values bivalent ions bind strongly to DPA or mixed DPA-DPL lamellae. The number of ions bound per lipid molecule is one at pH 9 and about one-half at pH 5-7. The Ca²⁺ bound phosphatidic acid segregates into regions characterized by a rigid (or crystalline) structure. A quantitative analysis of the experiments shows that at pH 9 the number of DPA molecules segregated is about equal to the number of Ca²⁺ ions. At pH 9 polylysine attaches strongly to DPA membranes and triggers the formation of lipid domains that differ in their microviscosity from the rest of the membrane. At this pH the transition temperature T_t of the lysine-bound DPA is shifted from T_t = 47° to T_t = 61°, corresponding to a shift from pH 9 to 2. Strong evidence is provided that the polylysine assumes such a (random coil) configuration that the charged lysine groups point toward one direction and that one lysine group may bind one DPA molecule. The coefficient of lateral diffusion of pyrenedecanoic acid in DPA at pH 9 and at 60° has been determined as D_L = 1.7 × 10^-7 cm²/sec. The corresponding value for DPL at 60° is D_L = 0.8 × 10^-7 cm²/sec.