Evaluation and Minimization of Uncertainty in ITC Binding Measurements: Heat Error, Concentration Error, Saturation, and Stoichiometry.
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.
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0736, USA.
Samuel A
SA
Kantonen
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0736, USA.
Niel M
NM
Henriksen
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0736, USA. Electronic address: mgilson@ucsd.edu.
Michael K
MK
Gilson
D055592:Biophysical Phenomena / Q000502:physiology; D002151:Calorimetry / Q000379:methods*; D006358:Hot Temperature; D016018:Least-Squares Analysis; D009010:Monte Carlo Method; D011485:Protein Binding / Q000502:physiology; D015203:Reproducibility of Results; D012107:Research Design; D012984:Software; D013816:Thermodynamics; D035501:Uncertainty
Data analysis; Error evaluation; Error minimization; ITC