Requirements for reliable determination of binding affinity constants by saturation analysis approach

Abstract Accurate calculation of the equilibrium association constant ( K ) and binding site concentration ( N ) related to a receptor (R)/ligand (L) interaction, via R saturation analysis, requires exact determination of the specifically bound L concentration ( B S ) and the unbound L concentration ( U ) at equilibrium. However, most binding determinations involve a procedure for separation of bound and unbound L. In such situations, it was previously shown that correct calculation of B S and U from binding data requires prior determination of α , i.e. the procedure parameter representing the proportion of equilibrium B S recovered after running the separation process, and of kn , i.e. the equilibrium nonspecific binding coefficient. For the simplest model of R/L interaction, the consequences of α neglect and/or kn neglect on determination of K and N , via R saturation analysis, are investigated. When α but not kn has been determined, B S can be accurately calculated, whereas U is overestimated by factor ( kn  + 1). Consequently the type (linear or hyperbolic) of theoretic curves obtained by usual representations (such as the Scatchard, the Lineweaver–Burk or the Michaelis–Menten plot) of the R/L binding is unchanged; these curves afford correct N and underestimation of K by factor ( kn  + 1). When α ( α B S and U are underestimated and overestimated, respectively. Then erroneous representations of the R/L binding result (e.g. instead of regular straight line segments, Scatchard plot and Lineweaver–Burk plot involve convex-upward and convex-downward hyperbola portions, respectively, suggestive of positive cooperativity of L binding), which leads to incorrect N and K . Errors in N and K would depend on (i) the binding ( K , N and kn ) and method ( α ) parameters and (ii) the expressions used to calculate approximate B S and U values. Simulations involving variable α , KN and kn values indicate that: (1) the magnitude of error in N determination (mainly involving moderate underestimation) directly depends on the α value; (2) the magnitude of K underestimation mainly depends on the KN value; it is moderate (usually  KN values  100-fold), when KN  > 10 2 . In this case, the K underestimation is modulated by the α and kn values. Practical situations which afford high KN and thus might result in very marked underestimation of K are discussed. A single R dilution method is proposed to assess the validity of K determinations using the R saturation analysis approach.