Obtaining precise and accurate results by ITC.

Obtaining precise and accurate results by ITC.
Eur Biophys J. 2019 Sep 25;:
Authors: Hansen LD, Quinn C
Abstract
Acquisition of precise and accurate results by isothermal titration calorimetry (ITC) can be achieved through thoughtful experimental design and modeling and careful experimental operations. Large reported errors in ITC results in determinations of stoichiometries, equilibrium constants and enthalpy changes for ligand binding to proteins are the consequence of poor experiment design, failure to properly calibrate and test instruments and protocols, lack of controls, errors in solution preparation, and incorrect data analyses. Analysis of a recent report that claimed to have determined the "repeatability, precision, and accuracy of the enthalpies and Gibbs energies of a protein-ligand binding reaction" by ITC is used to illustrate how to improve ITC operations and results. The analysis shows that the reported results are misleading because calorimeters were not calibrated, operating parameters were not optimized, errors were made in solution preparations, and data analysis was not optimized. As a consequence, the results do not provide a valid comparison of the capabilities of the calorimeters included in the study. A proposal that reaction of acetazolamide with carbonic anhydrase II be used as a comparison standard for testing ITCs and procedures is problematic because the binding constant is too large and for several othe...

Source: European Biophysics Journal : EBJ - September 24, 2019 Category: Physics Authors: Hansen LD, Quinn C Tags: Eur Biophys J Source Type: research