Fig. 2. (A and B) UV-vis absorption and fluorescence spectra of RHG in the presence of DOPC/DOPE/TOCL LUVs at binding saturation concentrations (DOPC : DOPE = 2 : 1; TOCL, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, and 25% of total lipids) at pH 6.5: (A) UV-vis absorption and (B) fluorescence spectra. ε represents the molar extinction coefficient of RHG, and ε0539 represents ε at 539 nm at pH 3.0. F represents the fluorescence intensity, and F0560 represents F at pH 3.0. The intensity changes upon increasing the TOCL ratio in LUVs are shown by arrows. (C and D) Plots of Xo-RHG ([o-RHG]/[RHG]) against bulk pH under LUV-binding saturation conditions (red, DOPC/DOPE/TOCL = 2 : 1 : 1; purple, DOPG): analysed with (C) absorption and (D) fluorescence spectra. The Xo-RHG plots for RHG are also shown (gray, with 58% (w/w) ethanol; black, without ethanol). The solid lines represent the least-squares fitted curves of the plots with sigmoidal-Boltzmann equations. Measurement conditions: RHG, (A and C) 2.0 μM and (B and D) 1.0 μM; total lipid, (A and C) 2 mM and (B and D) 1 mM; buffer: citrate–phosphate buffer, pH 3.5–5.0, 10 mM cacodylate buffer, pH 5.0–6.0, or 10 mM HEPES buffer, pH 6.0–8.5; temperature, 25 °C.
