Isothermal Titration Calorimetry: Experimental Design, Data Analysis, and Probing Macromolecule/Ligand Binding and Kinetic Interactions

Abstract

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 0.1μcal (0.4 μJ), allowing the determination of binding constants, K's, as large as 10⁸–10⁹M⁻¹. Modern ITC instruments make it possible to measure heat rates as small as 0.1μcal/sec, allowing for the precise determination of reaction rates in the range of 10⁻¹²mol/sec. Values for K_m and k_cat, in the ranges of 10⁻²–10³μM and 0.05–500sec⁻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.

Introduction

In biology, particularly in studies relating the structure of biopolymers to their functions, two of the most important questions are (i) how tightly does a small molecule bind to a specific interaction site and (ii) if the small molecule is a substrate and is converted to a product, how fast does the reaction take place?

Perhaps the first question we need to ask here is why calorimetry? The calorimeter, in this case an isothermal titration calorimeter (ITC), can be considered a universal detector. Almost any chemical reaction or physical change is accompanied by a change in heat or enthalpy. A measure of the heat taken up from the surroundings (for an endothermic process) or heat given up to the surroundings (for an exothermic process) is simply equal to the amount of the reaction that has occurred, n (in moles, mmoles, μmoles, nmoles, etc.) and the enthalpy change for the reaction, ΔH (typically in kcal/mol or kJ/mol). A measure of the rate at which heat is exchanged with the surroundings is simply equal to the rate of the reaction, ∂n/∂t (in moles/sec, mmoles/sec, μmoles/sec, nmoles/sec) and again the enthalpy change, ΔH. A calorimeter is therefore an ideal instrument to measure either how much of a reaction has taken place or the rate at which a reaction is occurring. In contrast to optical methods, calorimetric measurements can be done with reactants that are spectroscopically silent (a chromophore or fluorophore tag is not required), can be done on opaque, turbid, or heterogeneous solutions (e.g., cell suspensions), and can be done over a range of biologically relevant conditions (temperature, salt pH, etc.). Although not a topic covered in this chapter, calorimetric measurements have been used to follow the metabolism of cells or tissues in culture over long periods of time and under varying conditions (e.g., anaerobic or aerobic) (Bandman 1975, Monti 1986).

Titration calorimetry was first described as a method for the simultaneous determination of K_eq and ΔH about 40 years ago by Christensen and Izatt (Christensen 1966, Hansen 1965). The method was originally applied to a variety of weak acid–base equilibria and to metal ion complexation reactions (Christensen 1965, Christensen 1968, Eatough 1970). These systems could be studied with the calorimetric instrumentation available at the time which was limited to the determination of equilibrium constant, K_eq, values less than about 10⁴–10⁵ M⁻¹ (Eatough et al., 1985). The determination of larger association constants requires more dilute solutions and the calorimeters of that day were simply not sensitive enough.

Beaudette and Langerman published one of the first calorimetric binding studies of a biological system using a small volume isoperibol titration calorimeter (Beaudette and Langerman, 1978). In 1979, Langerman and Biltonen published a description of microcalorimeters for biological chemistry, including a discussion of available instrumentation, applications, experimental design, and data analysis and interpretation (Biltonen 1979, Langerman 1979). This was really the beginning of the use of titration calorimetry to study biological equilibria. It took another 10 years before the first commercially available titration calorimeter specifically designed for the study of biological systems became available from MicroCal (Wiseman et al., 1989). This first commercial ITC was marketed as a device for “Determining K in Minutes” (Wiseman et al., 1989).

ITC is now routinely used to directly characterize the thermodynamics of biopolymer binding interactions (Freire et al., 1990). 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 μcal (0.4 μJ), allowing the determination of binding constants, Ks, as large as 10⁸–10⁹ M⁻¹.

Spink and Wadso (1976) published one of the first calorimetric studies of enzyme activity. Improvements in modern microcalorimeters including higher sensitivity, faster response, and the ability to make multiple additions of substrate (or inhibitors) has brought us to the point where ITC is now also routinely used to directly characterize the kinetic parameters (K_m and k_cat) for an enzyme (Todd 2001, Williams 1993). Kinetic studies take advantage of the fact that the calorimetric signal (heat rate, e.g., μcal/sec) is a direct measure of the reaction rate and the ΔH for the reaction. Modern ITC instruments make it possible to measure heat rates as small as 0.1 μcal/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 10⁻²–10³ μM and 0.05–500 sec⁻¹, respectively, can be determined by ITC.

Ladbury has published a series of annual reviews on ITC, describing the newest applications and a year‐to‐year survey of the literature on ITC applications (Ababou 2006, Cliff 2004). In order to take full advantage of the powerful ITC technique, the user must be able to design the optimum experiment, understand the data analysis process, and appreciate the uncertainties in the fitting parameters. ITC experiment design and data analysis have been the subject of numerous papers (Bundle 1994, Chaires 2006, Fisher 1995, Freiere 2004, Indyk 1998, Lewis 2005). 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.