Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions.
Isothermal titration calorimetry (ITC) is now routinely used to directly characterize the thermodynamics of biopolymer binding interactions and the kinetics of enzyme-catalyzed reactions. This is the result of improvements in ITC instrumentation and data analysis software. Modern ITC instruments make it possible to measure heat effects as small as 0.1 microcal (0.4 microJ), allowing the determination of binding constants, K's, as large as 10(8) - 10(9)M(-1). Modern ITC instruments make it possible to measure heat rates as small as 0.1 microcal/sec, allowing for the precise determination of reaction rates in the range of 10(-12) mol/sec. Values for K(m) and k(cat), in the ranges of 10(-2) - 10(3) microM and 0.05 - 500 sec(-1), respectively, can be determined by ITC. This chapter reviews the planning of an optimal ITC experiment for either a binding or kinetic study, guides the reader through simulated sample experiments, and reviews analysis of the data and the interpretation of the results.
Freyer
Matthew W
MW
Department of Chemistry and Biochemistry, Northern Arizona University, Flagstaff, Arizona 86011, USA.
Lewis
Edwin A
EA
D002151:Calorimetry / Q000379:methods*; D003198:Computer Simulation; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances / Q000378:metabolism*; D013816:Thermodynamics
D008024:Ligands; D046911:Macromolecular Substances