AVS 46th International Symposium
    Biomaterial Interfaces Group Tuesday Sessions
       Session BI-TuP

Paper BI-TuP5
A Circulating Flow Surface Plasmon Resonance System for Measuring Protein-Vesicle Binding Affinities

Tuesday, October 26, 1999, 5:30 pm, Room 4C

Session: Poster Session
Presenter: J. Shumaker-Parry, University of Washington
Authors: J. Shumaker-Parry, University of Washington
L.S. Jung, University of Washington
M.H. Gelb, University of Washington
C.T. Campbell, University of Washington
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We have developed a circulating flow surface plasmon resonance (SPR) system for measuring equilibrium binding of proteins to surface-bound vesicles. Traditional methods for measuring interfacial binding of proteins to membranes often require the use of a non-physiological reporter group such as a tryptophan residue. The addition of a tryptophan has been shown to perturb binding in some cases. The fluorescence-based methods also lack the sensitivity to measure high binding affinities (K@sub d@ < 10@super-7@M). Other methods that rely on centrifugation are plagued by protein loss through non-specific adsorption on tube surfaces. Our circulating flow SPR system measures equilibrium binding constants rapidly without a spectral probe and can be used to quantify values of K@sub d@ down to 10@super -10@M. The sensing surface contains a high density of vesicles immobilized via biotinylated lipids that bind to biotin sites in a streptavidin monolayer. Fluorescence studies using dye-filled vesicles show that the vesicles remain intact when attached to the surface. We have used the surface-bound vesicles to measure K@sub d@ values for phospholipase A@sub 2@ with phospholipid vesicles. A circulating flow system is used to introduce the protein to the vesicle surface and the protein concentration can be adjusted by adding protein or buffer to a reservoir. A binding isotherm is generated by performing equilibrium titrations and is used to obtain the K@sub d@ value directly without the need to fit association and dissociation rate curves. We have shown that vesicles provide a more biologically relevant surface for measuring binding affinities than planar hybrid bilayer membranes.