Laurdan

Laurdan (Sigma, Taufkirchen, Germany) is a fluorescent dye that incorporates into the lipid bilayer and allows quantifying membrane lipid order (Parasassi et al., 1991 (link); Parasassi and Gratton, 1995 (link)). The dye was added to 0.5 mM lipid solutions prior to liposome formation at a molar ratio of 1:500. After addition of the protein and incubation for 30 min at room temperature, Laurdan fluorescence emission was determined at 25°C upon excitation of the dye at 350 nm using a FluoroMax-4 fluorescence spectrometer from Horiba Scientific, Kyoto, Japan. The fluorescence emission spectrum of Laurdan depends on the physical state of the surrounding lipid bilayer. The Laurdan generalized Polarization (GP) value reflects the lipid order (Parasassi et al., 1991 (link)) and is calculated according to equation 1, where I₄₄₀ and I₄₉₀ are the fluorescence emission intensities at 440 and 490 nm, respectively.

The stock suspension of 100 μM LUV₁₀₀ was labeled with 0.2 μM concentration of 2-dimethylamino-6-lauroyl naphthalene (Laurdan, Molecular Probes, dissolved in dimethylsulfoxide, DMSO) at 37 °C for 60 min, the final ratio of Laurdan per lipid was 1 : 500. Final concentration of DMSO was 0.2% in all samples. The LUV suspension was split to individual aliquots of 0.1 mL. To study the possible shift in packing of the lipids in membranes with two different compositions (DOPE : DOPG and DOPG : DOPE, 2 : 1, w/w), we measured the generalized polarization (GP) of Laurdan-labeled LUVs under stable temperature 27 °C using FluoroMax-3 spectrofluorometer (Jobin Yvon, Horiba). Fluorescence spectra were measured from 400 to 600 nm (after excitation at 365 nm), with 4 nm bandpass. GP values were calculated according to Parasassi et al. (1990).^{28 (link)}