FIG. 7.

Permeabilization of calcein-loaded liposomes by purified S protein. (A) Kinetics of dye release by holin and alpha-hemolysin. Total release is effected by adding 0.05% Triton X-100 (○). Relative fluorescence was monitored continuously for the first 5 min. Single point measurements were taken between 10 and 20 min postaddition of protein. Amounts of protein added to initiate the assay were as follows: 8.75 μg of alpha-hemolysin (□), 15.2 μg of S105H94 (⧫), 3.8 μg of S105H94 (◊), and 17.6 μg of S105H94a52v (▿). (B) Sequential amounts (1.9 μg) of S105H94 were added to calcein-loaded liposomes, as indicated by the vertical arrows. Fluorescence was monitored continuously. (C) Pretreatment of calcein-loaded liposomes with a S105H94a52v. Maximum release of dye was established by addition of 0.05% Triton X-100 (⧫). A baseline signal was established by adding an equal volume of 6 M GdnHCl in TBS (◊). At point a, 7.6 μg of S105H94 protein (+) or 8.8 μg of S105H94a52v protein (○) was mixed separately with calcein-loaded liposomes, and fluorescence was monitored for 1 h. At point b, 7.6 μg of S105H94 was then added to both S105 (+) and S105H94 (⊕) samples, and fluorescence was monitored.