Kinetics of Amphiphile Association with Two-Phase Lipid Bilayer Vesicles

We examined the consequences of membrane heterogeneity for the association of a simple amphiphilic molecule with phospholipid vesicles with solid-liquid and liquid-liquid phase coexistence. To address this problem we studied the association of a single-chain, fluorescent amphiphile with dimyristoylphosphatidylcholine (DMPC) vesicles containing varying amounts of cholesterol. DMPC bilayers containing 15 mol% cholesterol show a region of solid–liquid-ordered ( s- ℓ o) coexistence below the T m of pure DMPC (23.9°C) and a region of liquid-disordered–liquid-ordered coexistence ( ℓ d- ℓ o) above the T m. We first examined equilibrium binding and kinetics of amphiphile insertion into single-phase vesicles ( s, ℓ d, and ℓ o phase). The data obtained were then used to predict the behavior of the equivalent process in a two-phase system, taking into account the fractions of phases present. Next, the predicted kinetics were compared to experimental kinetics obtained from a two-phase system. We found that association of the amphiphile with lipid vesicles is not influenced by the existence of ℓ d- ℓ o phase boundaries but occurs much more slowly in the s- ℓ o phase coexistence region than expected on the basis of phase composition.