In biology, particularly in studies relating the structure of biopolymers to their functions, two of the most important questions are: (1) how tightly does a small molecule bind to a specific interaction site? and (2) if the small molecule is a substrate and is converted to a product, how fast does the reaction take place? Because almost any chemical reaction or physical change is accompanied by a change in heat or enthalpy, an isothermal titration calorimeter is 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).

Key Concepts:

  • Almost all chemical reactions are accompanied by a change in heat or enthalpy.

  • Calorimetry can determine the complete set of thermodynamic parameters that characterise binding reactions, for example K, ΔG°, ΔH°, ΔS° and ΔCp.

  • Binding reactions are typically pH, salt and temperature dependant.

Keywords: binding; thermodynamics; isothermal titration calorimetry (ITC)

Figure 1.

Proposed structures are shown for the two 1:1 TMPyP4/c‐MYC promoter sequence G‐quadruplex complexes. The ITC binding isotherm indicates that there are at least two binding processes or modes, that the saturation stoichiometry is 4:1, and that the heat change data are well fit with a binding model that includes two overlapping equilibria, the first being entropy driven and the second being enthalpy driven. The higher affinity TMPyP4 binding site (Mode 1) is attributed to end‐binding whereas the lower affinity‐binding site (Mode 2) has been attributed to intercalation. Two moles of TMPyP4 are bound via each mode.

Figure 2.

Simulated results from isothermal titration calorimetry experiments showing the effect of different binding constants on the heat evolved for an injection versus injection number. In the simulation, it was assumed that the concentration of M was 0.1 mmol L−1 and the concentration of L was 1.0 mmol L−1. The ΔH° of binding was set at −50 kJ, the cell volume was set to 1.5 mL and the injection volume was 10 μL. The different colours correspond to different binding constants as follows: grey 104; blue 105; red 106; orange 107 and green 108.

Figure 3.

ITC titrations for the addition of Netropsin to the target hairpin DNAs. The data for the titration of the GCG target hairpin are shown in panel (a). The symbols – filled circle, filled triangle, filled square and filled inverted triangle indicate data points taken in each of the four separate Netropsin titrations of the GCG hairpin. The line is calculated from the average of the best‐fit parameters (K1,K2, ΔH1, ΔH2,n1 and n2) shown for the GCG oligo in Table . All titrations were conducted at pH 6.5 in 10 mM cacodylic acid buffer, 100 mM NaCl and 1 mM EDTA with 25 μM DNA hairpin concentration. Typical ITC titrations are shown for each of the four different target hairpins in panel (b). The symbols, open circle, filled inverted triangle, filled triangle and filled circle are for the GCG, CGC, CG and GC target hairpins, respectively. The line through each set of data is calculated from the best‐fit parameters (K1,K2, ΔH1, ΔH2, n1 and n2) for these individual experiments. The average best‐fit parameters for each of the four target hairpins are listed in Table . The experimental conditions are the same as above. Reproduced from Lewis et al. with permission from Oxford University Press.

Figure 4.

Tetrahedral zinc coordination in the mononuclear metal binding site in human carbonic anhydrase II. Image is based on the high resolution (0.9 Å) structure 3KS3.pdb.

Figure 5.

ITC data for Zn2+ titration into 100 μM apo‐CA. (a) 20 mM PIPES at pH 7.0. (b) 20 mM MOPS at pH 7.0. (c) 20 mM ACES at pH 7.0. (d) 20 mM Tris at pH 7.0. The best‐fit lines associated with the data are generated from a one‐site model. (e) Plot of experimental enthalpies versus enthalpies of ionisation for common biological buffers (Goldberg et al., ).

Figure 6.

Representative ITC kinetic data for a direct injection experiment performed in Tris buffer (50 mM Tris–HCl pH 8.0, 1 mM DTT) in a MicroCal VP‐ITC (Microcal). Each experiment was comprised of two injections (10 μL), spaced 30 min apart at 37°C (with stirring at 200 rpm). The thermogram represents the instrument response to the heat produced by the phosphorylation reaction. The integrated area of the peaks in the thermogram represent the enthalpy change for 2‐deoxythymidine phosphorylation by VVTK. These data were then integrated and analysed to obtain the kinetic parameters for the enzymatic phosphorylation reaction. Reproduced from Smith et al. with permission from Elsevier.



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Further Reading

Lewis EA and Murphy KP (2005) Isothermal titration calorimetry. In: Methods in Molecular Biology, vol 305: Protein‐Ligand Interactions: Methods & Applications, pp. 1–16. Totowa, NJ: Humana Press, Inc.

Salim NN and Feij AL (2009) Isothermal titration calorimetry of RNA. Methods 47(3): 198–205.

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Emerson, Joseph P, Le, Vu H, and Lewis, Edwin A(Nov 2012) Calorimetry. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0003010.pub3]