COOPERATIVITY IN THE BINDING OF AVIDIN TO BIOTIN-LIPID-DOPED LANGMUIR-BLODGETT-FILMS

Monolayers of arachidic acid (AA) doped with either biotinylated DPPE (B-DPPE) or a chain extended biotinylated DPPE (B-x-DPPE) were deposited onto alkylsilane treated surfaces of quartz evanescent fiber optic sensors (EFO) by the Langmuir-Blodgett (LB) technique. The surface-modified EFOs were used to obtain binding isotherms of fluorescein-labeled avidin to the biotin-lipid-doped LB films. Hyperbolic binding isotherms were observed for all B-DPPE doped LB films and for B-x-DPPE doped films with < 0.63 mol % biotin lipid. Sigmoid or positively cooperative binding isotherms were observed for all LB films with greater-than-or-equal-to 0.63 mol % B-x-DPPE. A mathematical expression for protein binding to a two-dimensional array of receptors that takes protein-protein interaction into account was used to quantitatively assess the cooperativity observed in the isotherms. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to address speculation that cooperativity resulted from a conformational change in avidin. ATR-FTIR results show that avidin experienced significant conformational changes when bound to biotin lipids in the LB films, wheras no conformational change was observed for avidin nonspecifically bound to biotin-free LB films.