Optimizing isothermal titration calorimetry protocols for the study of 1:1 binding: Keeping it simple

Area of Science:

• Biophysical Chemistry
• Thermodynamics
• Biochemistry

Background:

• Isothermal Titration Calorimetry (ITC) relies on heat changes during molecular binding.
• Accurate quantification of binding constant (K), reaction enthalpy (ΔH°), and stoichiometry (n) is crucial for successful ITC experiments.

Purpose of the Study:

• To develop quantitative guidelines for optimizing ITC experiment design.
• To predict the precision of estimated thermodynamic parameters (K, ΔH°, n) based on experimental conditions.

Main Methods:

• Nonlinear least squares analysis in error-propagation mode was employed.
• Parameter precisions were estimated across a range of the dimensionless quantity c = K[M]0.
• Measurement precision (σq) was determined using water-water blanks.

Main Results:

• Relative standard errors for K, ΔH°, and n are proportional to σq/qtot when product conversion exceeds 90%.
• Specific error dependencies on 'c' were derived for K, ΔH°, and n.
• Fewer titrant injections (e.g., 10) can yield better results and higher productivity than traditional methods (20-40 injections).

Conclusions:

• New quantitative guidelines offer a data-driven approach to ITC experiment design, superseding empirical "c rules".
• These principles are demonstrated using the ITC-PLANNER program for experimental design.