Isothermal Titration Calorimetry

Adrián Velázquez‐Campoy1, Hiroyasu Ohtaka1, Azin Nezami1, Salman Muzammil1, Ernesto Freire1

1 Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.8
DOI:  10.1002/0471143030.cb1708s23
Online Posting Date:  September, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


In the last two decades, isothermal titration calorimetry (ITC) has become the preferred technique to determine the binding energetics of biological processes, including protein‐ligand binding, protein‐protein binding, DNA‐protein binding, protein‐carbohydrate binding, protein‐lipid binding, and antigen‐antibody binding. In this unit several protocols are presented, ranging from the basic ones that are aimed at characterizing binding of moderate affinity to advanced protocols that are aimed at determining very high or very low affinity binding processes. Also, alternate protocols for special cases (homodimeric proteins and unstable proteins) and additional information accessible by ITC (heat capacity and protonation/deprotonation processes coupled to binding) are presented.

Keywords: isothermal titration calorimetry; thermodynamics; binding; macromolecule‐macromolecule interactions

PDF or HTML at Wiley Online Library

Table of Contents

  • Principles of the Technique
  • Instrumentation
  • Basic Protocol 1: Isothermal Titration Calorimetry
  • Support Protocol 1: Data Analysis for ITC Experiments
  • Basic Protocol 2: Macromolecule/Ligand Interaction with Moderate Affinity
  • Basic Protocol 3: Macromolecule/Macromolecule Interaction with Moderate Affinity
  • Alternate Protocol 1: Macromolecule/Ligand Binding with High Affinity
  • Alternate Protocol 2: Macromolecule/Ligand Binding with Low Affinity
  • Alternate Protocol 3: Binding of Homodimeric Proteins
  • Alternate Protocol 4: Binding of Unstable Proteins
  • Alternate Protocol 5: Measuring the Heat Capacity Change Associated with Binding
  • Alternate Protocol 6: Measuring Protonation/Deprotonation Processes Coupled to Binding
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Isothermal Titration Calorimetry

  • Reactant solutions: macromolecule and ligand
  • Methanol
  • VP‐ITC calorimeter (e.g., Microcal LLC or equivalent)
  • Vacuum pump
  • 2.5‐ml long‐needle syringe (e.g., Hamilton)
  • 12 × 75–mm and 6 × 50–mm glass tubes

Support Protocol 1: Data Analysis for ITC Experiments

  • RNase A, lyophilized powder (Sigma)
  • 15 mM potassium acetate buffer, pH 5.5
  • 2′CMP, lyophilized powder (Sigma)
  • 12 × 75–mm and 6 × 50–mm glass tubes
  • 6‐kDa MWCO dialysis membrane
  • 0.22‐µm filter

Basic Protocol 2: Macromolecule/Ligand Interaction with Moderate Affinity

  • Porcine pancreatic trypsin (PPT), lyophilized powder (Sigma)
  • 25 mM potassium acetate/10 mM calcium chloride, pH 4.5
  • Soybean trypsin inhibitor (STI), lyophilized powder (Sigma)
  • 10‐kDa MWCO dialysis tubing
  • 0.22‐µm filter

Basic Protocol 3: Macromolecule/Macromolecule Interaction with Moderate Affinity

  • Acetyl pepstatin (Bachem)
  • 9 mM NaOH
  • Nelfinavir (Viracept; or any other clinical or experimental inhibitor)
  • 100% DMSO
  • HIV‐1 protease (Todd et al., and Velazquez‐Campoy et al., )
  • 10 mM sodium acetate, pH 5.0/2% (v/v) DMSO
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Amzel, L.M. 1997. Loss of translational entropy in binding, folding, and catalysis. Proteins 28:144‐149.
   Amzel, L.M. 2000. Calculation of entropy changes in biological processes: Folding, binding, and oligomerization. Methods Enzymol. 323:167‐177.
   Baker, B.M. and Murphy, K.P. 1996. Evaluation of linked protonation effects in protein binding using isothermal titration calorimetry. Biophys. J. 71:2049‐2055.
   Baker, B.M. and Murphy, K.P. 1997. Dissecting the energetics of a protein‐protein interaction: The binding of ovomucoid third domain to elastase. J. Mol. Bio. 268:557‐569.
   Burrows, S.D., Doyle, M.L., Murphy, K.P., Franklin, S.G., White, J.R., Brooks, I., McNulty, D.E., Scott, M.O., Knutson, J.R., Porter, D., Young, P.R., and Hensley, P. 1994. Determination of the monomer‐dimer equilibrium of interleukin‐8 reveals it is a monomer at physiological concentrations. Biochemistry 33:12741‐12745.
   Cantor, C.R. and Schimmel, P.R. 1980. Biophysical Chemistry: The Behavior of Biological Macromolecules. W.H. Freeman & Co. New York.
   D'Aquino, J.A., Gómez, J., Hilser, V.J., Lee, K.H., Amzel, L.M., and Freire, E. 1996. The magnitude of the backbone conformational entropy change in protein folding. Proteins 25:143‐156.
   D'Aquino, J.A., Freire, E., and Amzel, L.M. 2000. Binding of small organic molecules to macromolecular targets: Evaluation of conformational entropy changes. Proteins Suppl 4:93‐107.
   Doyle, M.L. 1997 Characterization of binding interactions by isothermal titration. Curr. Opin. Biotech. 8:31‐35.
   Doyle, M.L. and Hensley, P. 1998. Tight ligand binding affinities determined from thermodynamic linkage to temperature by titration calorimetry. Methods Enzymol. 295:88‐99.
   Doyle, M.L., Louie, G.L., Dal Monte, P.R., and Sokoloski, T.D. 1995. Tight binding affinities determined from linkage to protons by titration calorimetry. Methods Enzymol. 259:183‐194.
   El Harrous, M. and Parody‐Morreale, A. 1997. Measurement of biochemical affinities with a Gill titration calorimeter. Anal. Biochem. 254:96‐108.
   Freire, E. 2002. Designing drugs against heterogeneous targets. Nature Biotech. 20:15‐16.
   Freire, E., Mayorga, O.L., and Straume, M. 1990. Isothermal titration calorimetry. Anal. Chem. 62:950A‐959A.
   Gómez, J. and Freire, E. 1995. Thermodynamic mapping of the inhibitor site of the aspartic protease endothiapepsin. J. Mol. Biol. 252:337‐350.
   Gómez, J., Hilser, V.J., and Freire, E. 1995. The heat capacity of proteins. Proteins 22:404‐412.
   Indyk, L. and Fisher, H.F. 1998. Theoretical aspects of isothermal titration calorimetry. Methods Enzymol. 295:350‐364.
   Jaenicke, L. 1974. A rapid micromethod for the determination of nitrogen and phosphate in biological material. Anal. Biochem. 61:623‐627.
   Jelessarov, I. and Bosshard, H.R. 1999. Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition. J. Mol. Recog. 12:3‐18.
   Ladbury, J.E. 2001. Isothermal titration calorimetry: Application to structure‐based drug design. Thermochimica Acta 380:209‐215.
   Leavitt, S. and Freire, E. 2001. Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr. Opin. Struct. Biol. 11:560‐566.
   Lee, K.H., Xie, D., Freire, E., and Amzel, L.M. 1994. Estimation of changes in side chain configurational entropy in binding and folding: General methods and application to helix formation. Proteins 20:68‐84.
   Lovatt, M., Cooper, A., and Camilleri, P. 1996. Energetics of cyclodextrin‐induced dissociation of insulin oligomers. Euro. Biophys. J. 24:354‐357.
   Luque, I. and Freire, E. 1998. A system for the structure‐based prediction of binding affinities and molecular design of peptide ligands. Methods Enzymol. 295:100‐127.
   Murphy, K.P. and Freire, E. 1992. Thermodynamics of structural stability and cooperative folding behavior in proteins. Adv. Protein Chem. 43:313‐361.
   Murphy, K.P., Xie, D., Garcia, K.C., Amzel, L.M., and Freire, E. 1993. Structural energetics of peptide recognition: Angiotensin II/antibody binding. Proteins 15:113‐120.
   Murphy, K.P., Xie, D., Thompson, K.S., Amzel, L.M., and Freire, E. 1994. Entropy in biological binding processes: Estimation of translational entropy loss. Proteins 18:63‐67.
   Myszka, D.G, Sweet, R.W., Hensley, P., Brigham‐Burke, M., Kwong, P.D., Hendrickson, W.A., Wyatt, R., Sodroski, J., and Doyle, M.L. 1997. Energetics of the HIV gp120‐CD4 binding reaction. Proc. Natl. Acad. Sci. U.S.A. 97:9026‐9031.
   Nezami, A., Luque, I., Kimura, T., Kiso, Y., and Freire, E. 2002. Identification and characterization of allophenylnorstatine‐based inhibitors of plasmepsin II, an antimalarial target. Biochemistry 41:2273‐2280.
   Nezami, A., Kimura, T., Hidaka, K., Kiso, A., Liu, J., Kiso, Y., Goldberg, D.E., and Freire, E. 2003. High‐affinity inhibition of a family of plasmodium falciparum proteases by a designed adaptive inhibitor. Biochemistry 42:8459‐8464.
   Ohtaka, H., Velázquez‐Campoy, A., Xie, D., and Freire, E. 2002. Overcoming drug resistance in HIV‐1 chemotherapy: The binding thermodynamics of amprenavir and TMC‐126 to wild‐type and drug‐resistant mutants of the HIV‐1 protease. Prot. Sci. 11:1908‐1916.
   Pace, C.N., Vaidos, F., Fee, L., Grimsley, G., and Gray, T. 1995. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4:2411‐2423.
   Parker, M.H., Lunney, E.A., Ortwine, D.F., Pavlovsky, A.G., Humblet, C., and Brouillette, C.G.. 1999. Analysis of the binding of hydroxamic acid and carboxylic acid inhibitors to the stromelysin‐1 (matrix metalloproteinase‐3) catalytic domain by isothermal titration calorimetry. Biochemistry 38:13592‐13601.
   Privalov, P.L. and Makhatadze, G.I. 1992. Contribution of hydration and non‐covalent interactions to the heat capacity effect on protein unfolding. J. Mol. Bio. 224:715‐723.
   Sigurskjold, B.W. 2000. Exact analysis of competition ligand binding by displacement isothermal titration calorimetry. Anal. Biochem. 277:260‐266.
   Straume, M. and Freire, E. 1992. Two‐dimensional differential scanning calorimetry: Simultaneous resolution of intrinsic protein structural energetics and ligand binding interactions by global linkage analysis. Anal. Biochem. 203:259‐268.
   Todd, M.J., Semo, N., and Freire, E. 1998. The structural stability of the HIV‐1 protease. J. Mol. Bio. 283:475‐488.
   Todd, M.J., Luque, I., Velázquez‐Campoy, A., and Freire, E. 2000. Thermodynamic basis of resistance to HIV‐1 protease inhibition: Calorimetric analysis of the V82F/I84V active site resistant mutant. Biochemistry 39:11876‐11883.
   Van Holde, K.E., Johnson, W.C., and Ho, P.S. 1998. Principles of Physical Biochemistry. Prentice Hall. Upper Saddle River, New Jersey.
   Velázquez‐Campoy, A. and Freire, E. 2001. Incorporating target heterogeneity in drug design. J. Cell. Biochem. S37:82‐88.
   Velázquez‐Campoy, A., Todd, M.J., and Freire, E. 2000a. HIV‐1 protease inhibitors: Enthalpic versus entropic optimization of the binding affinity. Biochemistry 39:2201‐2207.
   Velázquez‐Campoy, A., Luque, I., Todd, M.J., and Freire, E. 2000b. Thermodynamic dissection of the binding energetics of KNI‐272, a potent HIV‐1 protease inhibitor. Prot. Sci. 9:1801‐1809.
   Velázquez‐Campoy, A. Kiso, Y., and Freire, E. 2001. The binding energetics of first‐ and second‐generation HIV‐1 protease inhibitors: Implications for drug design. Arch. Biochem. Biophys. 390:169‐175.
   Velázquez‐Campoy, A., Vega, S., and Freire, E. 2002. Amplification of the effects of drug‐resistance mutations by background polymorphisms in HIV‐1 protease from African subtypes. Biochemistry 41:8613‐8619.
   Ward, W.H. and Holdgate, G.A. 2001. Isothermal titration calorimetry in drug discovery. Prog. Med. Chem. 38:309‐376.
   Wiseman, T., Williston, S., Brandts, J.F., and Nin, L.N. 1989. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179:131‐137.
   Wyman, J. and Gill, S.J. 1990. Binding and Linkage: Functional Chemistry of Biological Macromolecules. University Science Books Mill Valley California.
   Zhang, Y.‐L. and Zhang, Z.‐Y. 1998. Low‐affinity binding determined by titration calorimetry using a high‐affinity coupling ligand: A thermodynamic study of ligand binding to protein tyrosine phosphatase 1B. Anal. Biochem. 261:139‐148.
PDF or HTML at Wiley Online Library