4 years ago

Measuring Rapid Time-Scale Reaction Kinetics Using Isothermal Titration Calorimetry

Measuring Rapid Time-Scale Reaction Kinetics Using Isothermal Titration Calorimetry
Justin M. Di Trani, Anthony K. Mittermaier, Nicolas Moitessier
Isothermal titration calorimetry (ITC) is a powerful tool for acquiring both thermodynamic and kinetic data for biological interactions including molecular recognition and enzymatic catalysis. ITC-based kinetics measurements typically focus on reactions taking place over long time scales (tens of minutes or hours) in order to avoid complications due to the finite length of time needed detect heat flow in the calorimeter cell. While progress has been made toward analyzing more rapid reaction kinetics by ITC, the capabilities and limitations of this approach have not been thoroughly tested to date. Here, we report that the time resolution of commercial instruments is on the order of 0.2 s or less. We successfully performed rapid ITC kinetics assays with durations of just tens of seconds using the enzyme trypsin. This is substantially shorter than previous ITC enzyme measurements. However, we noticed that for short reaction durations, standard assumptions regarding the ITC instrument response led to significant deviations between calculated and measured ITC peak shapes. To address this issue, we developed an ITC empirical response model (ITC-ERM) that quantitatively reproduces ITC peak shapes for all reaction durations. Applying the ITC-ERM approach to another enzyme (prolyl oligopeptidase), we unexpectedly discovered non-Michaelis–Menten kinetics in short time-scale measurements that are absent in more typical long time-scale experiments and are obscured in short time-scale experiments when standard assumptions regarding the instrument response are made. This highlights the potential of ITC measurements of rapid time scale kinetics in conjunction with the ITC-ERM approach to shed new light on biological dynamics.

Publisher URL: http://dx.doi.org/10.1021/acs.analchem.7b00693

DOI: 10.1021/acs.analchem.7b00693

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