Peptide interactions with di-8-ANEPPS labeled LUV and cell membranes.
(A–C) Differential spectra of di-8-ANEPPS bound to LUV (A) erythrocytes (B) and PBMC (C) membranes in the presence of C34-cholesterol and in its absence. Spectra were obtained by subtracting the excitation spectrum (normalized to the integrated areas) of labeled cells in the presence of peptide from the spectrum in the absence of the respective peptide. The shift to the red (decrease in dipole potential) is peptide concentration-dependent. For LUV, spectra traces represent C34-cholesterol 4 µM, in the presence of different lipid compositions: POPC, POPC:Chol 2∶1 and POPC:Chol 1∶1. For cells, spectra traces represent different C34-cholesterol concentrations: 0 µM, 0.25 µM, 1 µM and 5 µM. (D) Binding profiles of C34-cholesterol to LUV of POPC, POPC:Chol 2∶1 and POPC:Chol 1∶1, by plotting the di-8-ANEPPS excitation ratio, R (I455/I525, normalized to the initial value), as a function of the peptide concentration. DMSO, cholesterol and C34 (unconjugated) were also tested, as controls, and no significant changes on the dipole potential were observed (data not shown). (E–F) Binding profiles of C34-cholesterol, C34 and cholesterol to erythrocytes (E) and PBMC (F) cell membranes. Controls for DMSO (empty circles) and DMSO:ethanol (empty square) were also performed. Dashed curves represent the fitting to the single binding site equation (eq. 5). Affinity parameters for the cells are indicated in Table 3. Error bars represent SEM, with n = 5 for C34-cholesterol curves in cells and n = 3 for the control curves and LUV.