2.16. Tryptophan Fluorescence Spectroscopy and Acrylamide Quenching Assay

The Trp fluorescence emission spectra of the peptides were monitored in 10 mM sodium phosphate buffer in the presence of small unilamellar vesicles (SUVs) composed of either PE–PC–PI–ergosterol (5:4:1:2, w/w/w/w) or PC–CH–SM (1:1:1, w/w/w). In these studies, SUVs were used in order to avoid or minimize differential light scattering effects [46]. The Trp fluorescence measurements were made using a spectrofluorometer (Perkin-Elmer LS55, Mid Glamorgan, UK). Each peptide was added to 1 mL of PBS (pH 7.2) containing 200-μM liposomes, and the peptide–liposome mixture (a molar ratio of 1:100) was allowed to interact for 10 min at 25 °C. The Trp fluorescence was excited at 280 nm, and the emission was scanned from 300 to 400 nm.

The fluorescence quenching experiments were performed using acrylamide as the Trp fluorescence quencher, the concentration of which was between 0.04 to 0.20 M in the cuvette. The effect of acrylamide on the fluorescence of each peptide was recorded and then analyzed with the Stern–Volmer equation:

where F₀ is the fluorescence intensity in the absence of acrylamide, F represents the fluorescence intensity in the presence of acrylamide, K_SV is the Stern–Volmer quenching constant and (Q) is the concentration of the Trp fluorescence quencher.