Functional immobilization of biomembrane fragments on planar waveguides for the investigation of side-directed ligand binding by surface-confined fluorescence

A method for the functional immobilization of Na,K-ATPase-rich membrane fra gments on planar metal oxide waveguides has been developed. A novel optical technique based on the highly sensitive detection of surface-confined fluo rescence in the evanescent held of the waveguide allowed us to investigate the interactions of the immobilized protein with cations and ligands. For s pecific binding studies, a FITC-Na,K-ATPase was used, which had been labell ed covalently within the ATP-binding domain of the protein. Fluorophore lab els of the surface-bound enzyme can be selectively excited in the evanescen t field. A preserved functional activity of the immobilized enzyme was only found when a phospholipid monolayer was preassembled onto the hydrophobic chip surface to form a gentle, biocompatible interface. In situ atomic forc e microscopy (AFM) was used to examine and optimize the conditions for the lipid and membrane fragment assembly and the quality of the formed layers. The enzyme's functional activity was tested by selective K+ cation binding, interaction with anti-fluorescein antibody 4-4-20, phosphorylation of the protein and binding of inhibitory ligand ouabain. The comparison with corre sponding fluorescence intensity changes found in bulk solution provides inf ormation about the side-directed surface binding of the Na,K-ATPase membran e fragments. The affinity constants of K+ ions to the Na,K-ATPase was the s ame for the immobilized and the non-immobilized enzyme, providing evidence for the highly native environment on the surface. The method for the functi onal immobilization of membrane fragments on waveguide surfaces will be the basis for future applications in pharmaceutical research where advanced me thods for exploring the molecular mechanisms of membrane receptor targets a nd drug screening are required.