Quantifying binding affinity between species with multiple binding sites can present a significant challenge to many biophysical characterization techniques. Composition-gradient multi-angle light scattering (CG-MALS) provides direct measurement of affinity and absolute stoichiometry for a wide variety of interactions, including multivalent interactions, without the need for surface immobilization or tagging.
A model fusion protein, Y, was engineered with two binding sites, one with high affinity and one with low affinity for its ligand, X. The interaction and two distinct binding affinities were readily quantified using CG-MALS, which also confirmed that no additional higher order species were formed. Results were essentially identical to other solution-based biophysical methods such as sedimentation equilibrium and isothermal titration calorimetry.