ABC of Kd Accuracy

The equilibrium dissociation constant (Kd) is a major characteristic of affinity complexes and one of the most frequently determined physicochemical parameters. Despite its significance, the values of Kd obtained for the same complex under similar conditions often exhibit considerable discrepancies and sometimes vary by orders of magnitude. These inconsistencies highlight the susceptibility of Kd determination to large systematic errors, ultimately leading to misconceptions and significant misallocation of research and development resources. It is imperative to both minimize and quantitatively assess the systematic errors inherent in Kd determination. Traditionally, Kd values are determined through nonlinear regression of binding isotherms. This analysis utilizes three variables: concentrations of two reactants and a fraction R of unbound limiting reactant. The systematic errors in Kd arise directly from systematic errors in these variables. In this study, we thoroughly analyze the sources of systematic errors within these variables, aiming to mitigate their impact on Kd accuracy. Through this analysis, we illustrate how each source contributes to inaccuracies in Kd determination. Additionally, we propose a method for quantitatively assessing the confidence interval of systematic errors in concentrations, a crucial step towards quantitatively evaluating Kd accuracy. While presenting original findings, this paper also reiterates the fundamentals of Kd determination, hence, guiding researchers across all proficiency levels. By shedding light on the sources of systematic errors and offering strategies for their mitigation, our work will help researchers to enhance the accuracy of Kd determination thereby making binding studies more reliable.