Illustra nap 25 g25

Proton transport induced by lugdunin and its analogs was analyzed using the pH-sensitive dye pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid). LUVs were filled with 100 mm KCl, 10 mm HEPES, and 0.5 mm pyranine (pH = 7.4), and extravesicular dye was removed after extrusion via size exclusion chromatography (Illustra NAP-25 G25, GE Healthcare, Chalfont St Giles, UK). The vesicles were diluted in the same buffer without pyranine and pH = 6.4 to a final lipid concentration of 50 µm. Pyranine fluorescence was monitored in a time-dependent manner with λ_ex = 458 nm, λ_em = 512 nm, and band widths of 3 nm using an FP 6500 spectrofluorometer (Jasco Germany, Groß-Umstadt, Germany; Spectra Manager V. 1.54.03) under constant stirring. After acquiring a baseline for 100 s, peptide stock solution in isopropanol was added to a nominal peptide-to-lipid ratio (n/n) of 1:250. Acidification of the lumen resulted in fluorescent quenching and was monitored over the course of 500 s. Afterward, the vesicles were lysed by the addition of N,N-dimethyl-n-dodecylamine N-oxide (LDAO) leading to a disruption of the pH gradient. All data points were normalized to the fluorescence intensity directly before the addition of the peptide and after vesicle lysis.

The potential transport of chloride ions by lugdunin was assessed using a lucigenin-based vesicle assay. LUVs were prepared as described above and filled with 225 mm KNO₃, 10 mm HEPES, and 0.8 mm lucigenin (pH 7.4). Free dye molecules were removed via size exclusion chromatography (Illustra NAP-25 G25, GE Healthcare, Chalfont St Giles, UK). Before the experiment, vesicles were diluted to a volume of 800 µL and a lipid concentration of 50 µm and incubated for 3 min in the presence of 0.2 µm lugdunin under constant stirring (peptide-to-lipid ratio of 1:250). Lucigenin fluorescence was excited at λ_ex = 430 nm and monitored at λ_em = 505 nm with 3 nm slit widths. A baseline was recorded for 100 s, before the chloride gradient was established by the addition of 10 mm KCl to the cuvette from a 1 m stock solution. Changes in fluorescence intensity were monitored over the course of 10 min before the addition of LDAO resulted in vesicle lysis and the disruption of the chloride gradient. All data were normalized to the fluorescence intensity directly before the establishment of the chloride gradient and after vesicle lysis. For control experiments without lugdunin, pure isopropanol was used instead.