title: Comparison of free-solution and surface-immobilized molecular interactions using a single platform.
abstract: While it is generally accepted that surface immobilization affects the binding properties of proteins, it has been difficult to quantify these effects due to the lack of technology capable of making affinity measurements with species tethered and in free solution on a single platform. Further, quantifying the interaction of binding pairs with widely differing masses has also been challenging, particularly when it is desirable to tether the high molecular weight protein. Here we describe the use of backscattering interferometry (BSI) to quantify the binding affinity of mannose and glucose to concanavalin A (ConA), a 106 KDa homotetramer protein, in free solution using picomoles of the protein. Using the same platform, BSI, we then studied the effect on the binding constants of the ConA-carbohydrate interactions upon chemically immobilizing ConA on the sensor surface. By varying the distances (0, 7.17, and 20.35 nm) of the ConA tether and comparing these results to the free-solution measurements, it has been possible to quantify the effect that protein immobilization has on binding. Our results indicate that the apparent binding affinity of the sugar-lectin pair increases as the distance between ConA and the surface decreases. These observations could lend insight as to why the affinity values reported in the literature sometimes vary significantly from one measurement technique to another.
authors.affiliation: Department of Chemistry and the Vanderbilt Institute for Chemical Biology, Vanderbilt University, 4226 Stevenson Center, Nashville, Tennessee 37235, USA.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Olmsted
authors.forename: Amanda
authors.initials: A
authors.lastname: Kussrow
authors.forename: Darryl J
authors.initials: DJ
authors.lastname: Bornhop
mesh_terms: D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D011485:Protein Binding / Q000502:physiology; D012996:Solutions / Q000737:chemistry / Q000378:metabolism*; D013499:Surface Properties
chemical_list: D055767:Immobilized Proteins; D012996:Solutions