Cardiolipin cl

Mixtures of 1–3 mg of egg PC (Avanti Polar Lipids Inc.), cardiolipin (CL; Avanti Polar Lipids Inc.) to a final molar PC:CL molar ratio of 4:1 (when present), and the fluorescent lipid probes diphenyl-1,3,5-hexatriene (DPH; Sigma-Aldrich) or 1-(4(trimethylammonium)-phenyl)-6-DPH (TMA-DPH; Sigma-Aldrich), at a lipid:probe molar ratio of 1:100, were dissolved in 200 μL of chloroform:methanol (2:1) and dried under flow of nitrogen for at least 60 min. The resulting dry lipid film was then resuspended in 1.5 mL of Milli-Q-degree water and vortexed vigorously for 1 min. This suspension of multilamellar vesicles was then incubated for 1 h at 37 °C. Large unilamellar vesicles were then prepared by extrusion of this lipid dispersion through polycarbonate filters of controlled 100-nm diameter pore size. These vesicles were maintained at 37 °C for no more than 3 h before being used^{52 (link)}. The steady-state fluorescence emission anisotropy experiments were carried out on a SLM Aminco 8000C spectrofluorimeter (SLM Aminco), as previously described^{53 (link)}. Fluorescence anisotropy (r) of DPH or TMA-DPH incorporated into the liposomes described above was recorded at 37 °C using excitation and emission wavelengths of 350 or 356 and 452 or 451 nm, respectively.