Calorimetry

Joseph P Emerson, Mississippi State University, Starkville, Mississippi, USA
Vu H Le, Mississippi State University, Starkville, Mississippi, USA
Edwin A Lewis, Mississippi State University, Starkville, Mississippi, USA

Published online: November 2012

DOI: 10.1002/9780470015902.a0003010.pub3

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, G°, H°, S° and Cp.
  • 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 H° of binding was set at –50 kJ, the cell volume was set to 1.5 mL and the injection volume was 10 L. 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, H1, H2, n1 and n2) shown for the GCG oligo in Table 1. All titrations were conducted at pH 6.5 in 10 mM cacodylic acid buffer, 100 mM NaCl and 1 mM EDTA with 25 M 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, H1, H2, n1 and n2) for these individual experiments. The average best-fit parameters for each of the four target hairpins are listed in Table 1. The experimental conditions are the same as above. Reproduced from Lewis et al. (2011) 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 M 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., 2002).
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 L), 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. (2007) with permission from Elsevier.
close
 References
    Awaru BS, Kim CU, Sippel KH et al. (2010) A short, strong hydrogen bond in the active site of human carbonic anhydrase II. Biochemistry 49: 249–251.
    Birringer MS, Perozzo R, Kut E et al. (2006) High-level expression and purification of human thymidine kinase 1: quaternary structure, stability, and kinetics. Protein Expression and Purification 47: 506–515.
    Black ME and Hruby D (1992) Site directed mutagenesis of a conserved domain in vaccinia virus thymidine kinase: evidence for a potential role in magnesium binding. Journal of Biological Chemistry 267: 6801–6806.
    Cashman D, Buscaglia R, Freyer MW et al. (2008) Molecular Modeling and Biophysical Analysis of the c-MYC NHE-III1, Silencer Element. Journal of Molecular Modeling 14(2): 93–101.
    Chaires JB (2006) A thermodynamic signature for drug–DNA binding mode. Archives of Biochemistry and Biophysics 453(1): 26–31.
    Christensen JJ, Izatt RM, Hansen LD and Partridge JA (1966) Entropy titration. A calorimetric method for the determination of G, H and S from a single thermometric titration. Journal of Physical Chemistry 70: 2003–2010.
    Cox JD, Hunt JA, Compher KM, Fierke CA and Christianson DW (2000) Structural influence of hydrophobic core residues on metal binding and specificity in carbonic anhydrase II. Biochemistry 39: 13687–13694.
    Degtyareva NN, Fresia MJ and Petty JT (2007) DNA conformational effects on the interaction of netropsin with A-tract sequences. Biochemistry 46: 15136–15143.
    book Dettler JM and Lewis EA (2011) "Biophysical studies of the structure, stability, and ligand binding properties of g-quadruplex DNA: thoughts and comparisons of the k-RAS, c-MYC, and BCL-2 oncogene promoter sequence quadruplexes, Chapter 3". In: Sheardy RD and Winkle SA (eds) Frontiers in Nucleic Acids, pp. 33–50, Washington, DC: American Chemical Society. ACS Symposium Series, DOI: 10.1021/bk-2011-1082.
    DiTusa CA, Christensen T, McCall KA, Fierke CA and Toone EJ (2001) Thermodynamics of metal ion binding. 1. Metal ion binding by wild-type carbonic anhydrase. Biochemistry 40: 5338–5344.
    Doyle ML (1997) Characterization of binding interactions by isothermal titration calorimetry. Current Opinion in Biotechnology 8: 31–35.
    book Eatough DJ, Lewis EA et al. (1985) "Determination of H and Keq values". In: Grime K (ed.) Analytical Solution Calorimetry, pp. 137–161. New York: Wiley.
    Fan X, Zhang Z, Zhou L et al. (2006) 5-dimethoxymethyl 2¢ deoxyuridine: a novel gem diether nucleoside with anti-orthopox virus activity. Journal of Medicinal Chemistry 49: 3377–3382.
    Freire E, Mayorga OL and Straume M (1990) Isothermal titration calorimetry. Analytical Chemistry 62: 950A–959A.
    Freyer MW, Buscaglia R, Cashman D et al. (2007b) Binding of netropsin to several DNA constructs: evidence for at least two different 1:1 complexes formed from an -A2T2-containing ds-DNA construct and a single minor groove binding ligand. Biophysical Chemistry 126: 186–196.
    Freyer MW, Buscaglia R, Hollingsworth A et al. (2007a) Break in the heat capacity change at 303 K for complex binding of netropsin to AATT containing hairpin DNA constructs. Biophysical Journal 92: 2516–2522.
    Freyer MW, Buscaglia R, Kaplan K et al. (2007c) Biophysical studies of the c-MYC NHE III1 promoter: model quadruplex interactions with a cationic porphyrin. Biophysical Journal 92(6): 1–9.
    Freyer MW, Buscaglia R, Nguyen B, Wilson WD and Lewis EA (2006) Binding of netropsin and 4, 6-diamidino-2-phenylindole to an A2T2 DNA hairpin: a comparison of biophysical techniques. Analytical Biochemistry 355: 259–266.
    book Freyer MW and Lewis EA (2008) "Isothermal titration calorimetry: experimental design, data analysis and probing macromolecule/ligand binding interactions. Chapter 4". In: Correia JJ and Detrich HW (eds) Biophysical Tools for Biologists, 82, pp. 79–113. San Diego, CA: Academic Press.
    Goldberg RN, Kishore N and Lennen RM (2002) Thermodynamic quantities for the ionization reactions of buffers. Journal of Physical and Chemical Reference Data 31: 231–370.
    Grossoehme NE, Spuches AM and Wilcox DE (2010) Application of isothermal titration calorimetry in bioinorganic chemistry. Journal of Biological Inorganic Chemistry 15: 1183–1191.
    Henry CM (2001) Structure-based drug design. Chemical and Engineering News 79: 69–74.
    Hruby DE (1985) Inhibition of vaccinia virus thymidine kinase by the distal products of its own metabolic pathway. Virus Research 151–156.
    Krishnamurthy VM, Kaufman GK, Urbach AR et al. (2008) Carbonic anhydrase as a model for biophysical and physical-organic studies of proteins and protein-ligand binding. Chemical Reviews 108: 946–1051.
    Ladbury J (2004) Application of isothermal titration calorimetry in the biological sciences: things are heating up! Biotechniques 37: 885–887.
    Ladbury JE (1996) Just add water! The effect of water on the specificity of protein–ligand binding sites and its potential application to drug design. Chemistry and Biology 3: 973–980.
    Ladbury JE (2001) Isothermal titration calorimetry: application to structure-based drug design. Thermochimica Acta 380: 209–215.
    Lah J, Drobnak I, Dolinar M and Vesnaver G (2008) What drives the binding of minor groove-directed ligands to DNA hairpins. Nucleic Acids Research 36: 897–904.
    Lewis EA, Munde M, Wang S et al. (2011) Complexity in the Binding of Minor Groove Agents, Netropsin has Two Thermodynamically Different DNA Binding Modes at a Single Site. Nucleic Acids Research 39(22): 9649–9658.
    Lindskog S (1963) Effects of pH and inhibitors on some properties related to metal binding in bovine carbonic anhydrase. Journal of Biological Chemistry 238: 945–951.
    McCall KA and Fierke CA (2004) Probing determinants of the metal ion selectivity in carbonic anhydrase using mutagenesis. Biochemistry 43: 3979–3986.
    Morin PE and Freire E (1991) Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase. Biochemistry 30: 8494–8500.
    other NIST Standard Reference Data Base 46, version 7.0; National Institute of Standards and Technology, Gaithersburg, MD.
    Perozzo R, Jelesarov I, Bosshard HR, Folkers G and Scapozza L (2000) Compulsory order of substrate binding to herpes simplex virus type I thymidine kinase: a calorimetric study. Journal of Biological Chemistry 275: 16139–16145.
    Pilger B, Perozzo R, Alber F et al. (1999) Substrate diversity of herpes simplex viral thymidine kinase. Journal of Biological Chemistry 274: 31967–31973.
    Smith RF, Freyer MW and Lewis EA (2007) Biophysical characterization of vaccinia virus thymidine kinase substrate utilization. Journal of Virological Methods 142(1-2): 151–158.
    other Song H, Wilson DL, Farquhar ER, Lewis EA and Emerson JP (2012) Revisiting zinc coordination in human carbonic anhydrase II. Inorganic Chemistry. doi:10.1021/ic301645j.
    Spink C and Wadso I (1976) Calorimetry as an analytical tool in biochemistry and biology. Methods of Biochemical Analysis 23: 1–159.
    Todd MJ and Gomez J (2001) Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Analytical Biochemistry 296: 179–187.
    Velazquez-Campoy A and Freire E (2006) Isothermal titration calorimetry to determine association constants for high-affinity ligands. Nature Protocols 1: 186–191.
    Watt GD (1990) A microcalorimetric procedure for evaluating the kinetic parameters of enzyme-catalyzed reactions: kinetic measurements of the nitrogenase system. Analytical Biochemistry 187: 141–146.
    Williams BA and Toone EJ (1993) Calorimetric evaluation enzyme kinetics parameters. Journal of Organic Chemistry 58: 3507–3510.
    Wiseman T, Williston S, Brandts JF and Lin L-N (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Analytical Biochemistry 179: 131–137.
    book Wyman J and Gill SJ (1990) Binding and Linkage: The Functional Chemistry of Biological Macromolecules. Mill Valley, CA: University Science Books.
 Further Reading
    book 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.

Related Sub-Topics:

General Structural Biology | Biochemical and Biophysical Techniques | Biomolecular Interactions | Bioenergetics
Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

Article Title: Calorimetry
 
How to Cite close
Emerson, Joseph P, Le, Vu H, and Lewis, Edwin A(Nov 2012) Calorimetry. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003010.pub3]