Requirements for reliable determination of binding affinity constants by kinetic approach

Abstract The nonspecific binding (equilibrium coefficient k n ) of ligand (L) and/or the incomplete recovery ( α k 1 ) and a monomolecular dissociation process (rate constant k 2 ), the consequences of k n and/or α neglect on k 1 and k 2 determination were investigated. Various situations that are especially relevant for k 1 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 k n and/or α were or were not taken into account to correct the binding measurements), k 1 could be accurately determined or generally slightly overestimated or slightly underestimated (in the two latter cases by factors involving mainly k n and/or α but not the R concentration or the R/L equilibrium association constant, K ), whereas k 2 should always be fairly well estimated. Consequently, for the simplest R/L systems, the k 1 / k 2 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.