12 research outputs found
Comparison of Free-Solution and Surface-Immobilized Molecular Interactions Using a Single Platform
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