The feasibility of determining kinetic constants from isothermal titration calorimetry data

Shih Chia Tso, Thomas A. Jowitt, Chad A. Brautigam

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Isothermal titration calorimetry (ITC) has long been established as an excellent means to determine the thermodynamic parameters of biomolecular interactions. More recently, efforts have focused on exploiting the power/time trace (the “thermogram”) resulting from ITC experiments to glean kinetic association and dissociation rates for these interactions. The success of such analyses rests on the ability of algorithms to simulate with high accuracy the output of the calorimeter. Thus, several critical factors must be taken into account: the injection protocol, the kinetics of the interaction, accurate discovery of the instrumental response to heat signals, and the addition of unrelated signals. All of these aspects of extracting kinetic constants from thermograms have been considered and addressed in the current work. To validate the resultant methods, we performed several ITC experiments, titrating small-molecule inhibitors into solutions of bovine carbonic anhydrase II or titrating lysozyme into solutions of anti-lysozyme nanobodies. We found that our methods could arrive at kinetic constants that were close to the known values for these interactions taken from other methods. Finally, the effort to improve ITC kinetic characterizations uncovered a set of best practices for both the calorimetric experiment and the subsequent analyses (termed “kinetically optimized ITC” or “KO-ITC”) that is detailed in this work.

Original languageEnglish (US)
Pages (from-to)2474-2484
Number of pages11
JournalBiophysical journal
Volume121
Issue number12
DOIs
StatePublished - Jun 21 2022

ASJC Scopus subject areas

  • Biophysics

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