Evaluation and Minimization of Uncertainty in ITC Binding Measurements: Heat Error, Concentration Error, Saturation, and Stoichiometry

• Published in: Biochimica et biophysica acta Vol. 1861; no. 2; pp. 485 - 498
• Main Authors: Kantonen, Samuel A., Henriksen, Niel M., Gilson, Michael K.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2017
• Subjects: Biophysical Phenomena - physiology; calorimetry; Calorimetry - methods; computer simulation; computer software; Data analysis; enthalpy; Error evaluation; Error minimization; experimental design; Gibbs free energy; guidelines; heat; Hot Temperature; ITC; least squares; Least-Squares Analysis; Monte Carlo Method; Protein Binding - physiology; Reproducibility of Results; Research Design; Software; stoichiometry; Thermodynamics; titration; Uncertainty; ITC; Error minimization; Data analysis; Error evaluation; MC; LS
• ISSN: 0304-4165; 0006-3002; 1872-8006; 1878-2434
• DOI: 10.1016/j.bbagen.2016.09.002

Isothermal titration calorimetry (ITC) is uniquely useful for characterizing binding thermodynamics, because it straightforwardly provides both the binding enthalpy and free energy. However, the precision of the results depends on the experimental setup and how thermodynamic results are obtained from the raw data. Experiments and Monte Carlo analysis are used to study how uncertainties in injection heat and concentration propagate to binding enthalpies in various scenarios. We identify regimes in which it is preferable to fix the stoichiometry parameter, N, and evaluate the reliability of uncertainties provided by the least squares method. The noise in the injection heat is mainly proportional in character, with ~1% and ~3% uncertainty at 27C and 65C, respectively; concentration errors are ~1%. Simulations of experiments based on these uncertainties delineate how experimental design and curve fitting methods influence the uncertainty in the final results. In most cases, experimental uncertainty is minimized by using more injections and by fixing N at its known value. With appropriate technique, the uncertainty in measured binding enthalpies can be kept below ~2% under many conditions, including low C values. We quantify uncertainties in ITC data due to heat and concentration error, and identify practices to minimize these uncertainties. The resulting guidelines are important when ITC data are used quantitatively, such as to test computer simulations of binding. Reproducibility and further study are supported by free distribution of the new software developed here. •The uncertainty in injection heat for a VP-ITC microcalorimeter is roughly proportional to heat release•Over a wide range of experimental conditions, fixing the stoichiometry, N, reduces uncertainty in calorimetry results, relative to letting N float•Reducing the number of titration steps tends to degrade the precision of ITC results•Binding thermodynamics can often be precisely fit from low C value plots, given that N is held fixed