Miscalculating Equilibrium Constants for Multivalent Protein Complexes: Site-Binding Concentration or Protein Molecular Concentration?

Because the authors continue to note instances in the scientific literature of failure to use the correct receptor binding site concentration for determining binding constants, herein we discuss the fundamental concepts that need to be considered to determine correct binding constants or conversely calculate accurate reactant concentrations with known equilibrium constants. We also show the derivation and analytical solutions of the cubic and quartic equations that give the exact free ligand concentration in bivalent and trivalent receptor systems at equilibrium as a function of the macroscopic equilibrium dissociation constants and the total concentrations of ligand and multivalent protein. These equations and solutions strongly reemphasize the critical dependency of deriving the correct concentrations of bound or free ligand and multivalent protein on the choice of the correct concentration basis for the multivalent protein, which is in turn dependent upon the type of equilibrium constant used.

Biacore surface matrix effects on the binding kinetics and affinity of an antigen/antibody complex.

To characterize a proprietary therapeutic monoclonal antibody (mAb) candidate, a rigorous biophysical study consisting of 53 Biacore and kinetic exclusion assay (KinExA) experiments was undertaken on the therapeutic mAb complexing with its target antigen. Unexpectedly, the observed binding kinetics depended on the chip used, suggesting that the negatively charged carboxyl groups on CM5, CM4, and C1 chips were adversely affecting the Biacore kinetic results. To study this hypothesis, Biacore solution-phase and KinExA equilibrium titrations, as well as KinExA kinetic measurements, were performed to establish accurate values for the affinity and kinetic rate constants of the binding reaction between antigen and mAb.

A strategic and systematic approach for the determination of biosensor regeneration conditions.

Determining the optimal conditions for surface regeneration is fundamental for performance of efficient and robust protein-protein interaction kinetic studies. We devised a systematic methodology comprised of an automated seven-cycle analyte and buffer injection Biacore scheme and data interpretation algorithm. The efficiency and utility is illustrated using an antigen/monoclonal antibody interaction that required ultimately six pulses of acid for regeneration.