Deterministic error propagation in ITC: Revealing multi-fold errors in Kd values under standard conditions

Abstract

Accurate determination of the equilibrium dissociation constant (K_d) is essential in fields such as drug discovery and molecular diagnostics, where a rigorous understanding of molecular interactions drives critical decisions. Among established techniques, isothermal titration calorimetry (ITC) is highly valued for its ability to directly capture binding thermodynamics without the need for labeling or immobilization. However, while ITC is often praised for its precision, potential inaccuracies due to the systematic errors in experimental variables (analyte concentrations and measured heat) are frequently overlooked. One key reason for this oversight is the lack of a deterministic framework that explicitly demonstrates how ITC-derived K_d values can be affected by these errors. To address this gap, we derived a closed-form mathematical model for error propagation in ITC-based K_d determination, quantifying the impact of inaccuracies in analyte concentrations and measured heat on the resulting K_d. This framework provides a robust foundation for understanding and predicting the influence of these systematic errors on K_d accuracy. Using this solution, we demonstrate that even within the conventionally recommended c-value range of 10–100, expected systematic errors in concentrations and heat can potentially lead to significant multi-fold deviations in K_d. These findings underscore the need for quantitative accuracy assessments in ITC experiments and highlight the importance of developing practical tools to support such evaluations.