Quantification of tight binding to surface-immobilized phospholipid vesicles using surface plasmon resonance: Binding constant of phospholipase A(2)

A new and sensitive method for quantifying high affinity binding of protein s land potentially other ligands) to the surfaces of phospholipid bilayered vesicles is demonstrated A planar, high-density streptavidin monolayer is first immobilized onto a surface plasmon resonance (SPR) sensor slide conta ining a mixed monolayer of biotin-terminated and hydroxyl-terminated poly(e thylene oxide) alkylthiolates tethered to the gold surface. Phospholipid ve sicles containing 0.3% biotin-functionalized headgroups are then bound to t his streptavidin monolayer to make a high-density, planar layer of intact v esicles. The absolute amount of protein binding to the vesicle layer can be monitored by SPR in real time to extract equilibrium and kinetic informati on under flowing solutions. Coupled with a catalytic assay to monitor the s olution-phase concentration of enzyme, the value of the dissociation equili brium constant for the complex of cobra venom phospholipase A(2) (PLA(2)) b ound to phosphatidylcholine vesicles was determined to be 6 +/- 2 x 10(-7) M. In principle, the method could be extended to determine dissociation con stants as low as 10(-10) M. The vesicles bind a maximum of 1 PLA(2) per 74 +/- 16 outer leaflet phospholipids. The method provides a practical solutio n to a number of problems encountered with previous methods to quantify tig ht interfacial binding of proteins to vesicles. Spectral probes attached to enzymes or vesicles, which may perturb interfacial binding, are not requir ed. Furthermore, surface-supported vesicles offer improved sensitivity over planar bilayers and are physiologically more relevant.