Requirements for reliable determination of binding affinity constants by kinetic approach.

The nonspecific binding (equilibrium coefficient kn) of ligand (L) and/or the incomplete recovery (alpha < 1) of the receptor-ligand (RL) complex in binding measurements, could hamper accurate determination of the association and dissociation rate constants of the R/L system. For the simplest model of R/L interaction, characterized by a bimolecular association process (rate constant k1) and a monomolecular dissociation process (rate constant k2), the consequences of kn and/or alpha neglect on k1 and k2 determination were investigated. Various situations that are especially relevant for k1 determination, were examined in which nonspecific binding was: (i) negligible relative to specific binding, or (ii) developed progressively or very rapidly in association kinetics. When only the initial kinetic phase was used, according to the situation (i.e. the nonspecific binding characteristics, and the fact that kn and/or alpha were or were not taken into account to correct the binding measurements), k1 could be accurately determined or generally slightly overestimated or slightly underestimated (in the two latter cases by factors involving mainly kn and/or alpha but not the R concentration or the R/L equilibrium association constant, K), whereas k2 should always be fairly well estimated. Consequently, for the simplest R/L systems, the k1/k2 ratio derived from such kinetic experiments should be much less susceptible to substantial underestimation than K derived from R saturation experiments [Borgna, J. Steroid Biochem. Mol. Biol. (2004)]. Kinetic experiments could also be more appropriate than R saturation experiments to detect cooperative--positive or negative--binding of L to R.