Outer membrane permeability assay.

The outer membrane permeability activity of the peptides was determined using the fluorescent dye N-phenyl-1-napthylamine (NPN) assay, as previously described (20). Briefly, E. coli UB1005 in mid-log phase was diluted to 1 × 10⁵ CFU/ml in HEPES buffer (pH 7.2, containing 5 mM glucose). A final concentration of 10 mM NPN was added, and the background fluorescence was recorded (excitation k = 350 nm, emission k = 420 nm). Changes in the fluorescence were recorded with an F-4500 fluorescence spectrophotometer (Hitachi, Japan). The peptides were added, and the fluorescence was recorded as a function of time until no further increase in fluorescence was observed. Values were converted to percent NPN uptake using the following equation:

where F_obs is the observed fluorescence at a given peptide concentration, F₀ is the initial fluorescence of NPN with E. coli cells in the absence of peptide, and F₁₀₀ is the fluorescence of NPN with E. coli cells upon addition of 10 mg/ml polymyxin B, which was used as a positive control in this assay.