What Does the Accuracy of the Equilibrium Dissociation Constant of Affinity Complexes Depend on Fundamentally?

The equilibrium dissociation constant (Kd) characterizes stability of binding complexes. The classic way of Kd determination involves finding the dependence of a fraction of unbound ligand on the total concentration of target (T0) when the total concentration of ligand (L0) remains constant. It is known that Kd determination for highly stable complexes is notoriously inaccurate; however, what the accuracy of Kd depends on fundamentally, i.e., method-independently, is largely unknown. Here we present an error-propagation analysis that answers this question in detail. This analysis explains the critical importance of the L0/Kd value for the accuracy of Kd and allows one to define the range of L0/Kd values required for accurate Kd determination. Our analysis creates a theoretical foundation for improving the accuracy of Kd determination.