Proton induced vesicle fusion and the isothermal L α→H II phase transition of lipid bilayers: a 31P-NMR and titration calorimetry study

Abstract

The proton-induced isothermal fusion of unilamellar lipid vesicles (Duzgunes et al., Biochemistry 24 (1985) 3091–3098) is compared with the lamellar (L α)→hexagonal (H II) phase transition of multilamellar lipid dispersions. Both lipid systems are composed of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) and oleic acid (OA) at a 7:3 molar ratio. Using solid-state phosphorus-31 nuclear magnetic resonance ( 31P-NMR) it is demonstrated that the multilamellar lipid dispersions are in the bilayer state at physiological pH and undergo a L α→H II phase transition between pH 6.3 and 5.7. This phase transition can also be induced at constant pH by increasing the temperature. The midpoint of the temperature-induced L α→H II transition is T h=56°C (at pH 7.4) and the corresponding transition enthalpy is Δ H=0.7±0.1 kcal/mol as determined with differential scanning calorimetry. Both the proton-induced and the temperature-induced phase transition can be completely inhibited by addition of 30 mol% of 1-palmitoyl-2-hydroxy- sn-glycero-3-phosphocholine (LPC). In a second set of experiments unilamellar vesicles are prepared either by sonication or by extrusion through polycarbonate filters at pH 7.4 and are titrated into buffer at pH 5.7. The proton-induced fusion of the lipid vesicles is monitored with isothermal titration calorimetry, light scattering and fluorescence spectroscopy. The fusion reaction is characterized by an endothermic enthalpy of Δ H=0.5±0.2 kcal/mol (at 28°C). The fusion enthalpy is independent of the vesicle diameter and is only slightly reduced by an increase in temperature to 50°C. Vesicle fusion is accompanied by an increase in light scattering, indicating the formation of larger lipid structures. The transition from unilamellar vesicles to fused lipid structures occurs in the same narrow pH range of 6.3–5.7 as observed for the L α→H II transition of multilamellar dispersions. Vesicle fusion can be inhibited with 30% LPC. The virtually identical set of parameters found for the L α→H II phase transition and the vesicle fusion reaction suggests that vesicle fusion also entails a L α→H II phase transition.