Skip to main content
SpringerLink
Log in

Isothermal Titration Calorimetry Characterization of Drug-Binding Energetics to Blood Proteins

  • Protocol
Optimization in Drug Discovery

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

Techniques such as calorimetry, spectroscopy, and hydrodynamic methods can be used to investigate the binding energetics of drugs bound to macromolecules. In this chapter, the authors describe the use of isothermal titration calorimetry (ITC) to measure the binding energetics of drugs bound to blood proteins (i.e., human serum albumin [HSA] and α-acid glycoprotein [AGP]). The stoichiometry (n), the association-binding constant (K a), and the enthalpy (ΔH 0) of binding can be rapidly, directly, and precisely measured using ITC. Because the free energy (ΔG 0) and the entropy (ΔS 0) are readily calculated from K a and ΔH 0, a complete thermodynamic characterization of binding can be acquired in a single experiment.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Shargel, L. and Yu, A. B. C. (1993) Applied Biopharmaceutical and Pharmacokinetics. 3rd ed. Appleton & Lange, Stamford, CT.

    Google Scholar 

  2. Benet, L. Z. and Hoener, B. (2002) Changes in plasma protein binding have little clinical relevance. Clin. Pharm. Ther. 71, 115–121.

    Article  CAS  Google Scholar 

  3. Uetrecht, J. P. (1997) Current trends in drug-induced autoimmunity. Toxicology 119, 37–43.

    Article  PubMed  CAS  Google Scholar 

  4. Harding, S. E. and Chowdhry, B. Z. (2001) Protein-Ligand Interactions: Hydrodynamics and Calorimetry. Oxford University Press, Oxford, UK.

    Google Scholar 

  5. Leavitt, S. and Freire, E. (2001) Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr. Opin. Struct. Bio. 11, 560–566.

    Article  CAS  Google Scholar 

  6. Cooper, A. (2000) Microcalorimetry of protein-DNA interactions, in DNA-Protein Interactions, (Travers, A. and Buckle, M., eds.), Oxford University Press, Oxford, UK, pp. 125–139.

    Google Scholar 

  7. Alexander, K. A. and Phelps, W. C. (1996) A fluorescence anisotropy study of DNA binding by HPV-11 E2C protein: a hierarchy of E2-binding sites. Biochemistry 35, 9864–9872.

    Article  PubMed  CAS  Google Scholar 

  8. Tanaka, M., Asahi, Y., Masuda, S., and Ota, T. (1991) Interaction between drugs and water-soluble polymers: IV. Binding position of azathioprine with bovine serum albumin determined by measuring nuclear magnetic resonance relaxation time. Chem. Pharm. Bull. 39, 2771–2774.

    PubMed  CAS  Google Scholar 

  9. Neuhofen, S., Theyssen, H., Reinstein, J., Trommer, W. E., and Vogel, P. D. (1996) Nucleotide binding to the heat-shock protein DnaK as studied by ESR spectroscopy. Eur. J. Biochem. 240, 78–82.

    Article  PubMed  CAS  Google Scholar 

  10. Ascoli, G., Bertucci, C., and Salvadori, P. (1995) Stereospecific and competitive binding of drugs to human serum albumin: a difference circular dichroism approach. J. Pharm. Sci. 84, 737–741.

    Article  PubMed  CAS  Google Scholar 

  11. Frostell-Karlsson, A., Remaeus, A., Roos, H., Andersson, K., Borg, P., Haemaelaeinen, M., et al. (2000) Biosensor analysis of the interaction between immobilized human serum albumin and drug compounds for prediction of human serum albumin binding levels. J. Med. Chem. 43, 1986–1992.

    Article  PubMed  CAS  Google Scholar 

  12. Banker, M. J., Clark, T. H., and Williams, J. A. (2003) Development and validation of a 96-well equilibrium dialysis apparatus for measuring plasma protein binding. J. Pharm. Sci. 92, 967–974.

    Article  PubMed  CAS  Google Scholar 

  13. Lebowitz, J., Lewis, M. S., and Schuck, P. (2002) Modern analytical ultracentrifugation in protein science: a tutorial review. Protein Sci. 11, 2067–2079.

    Article  PubMed  CAS  Google Scholar 

  14. Lee, K.-J., Mower, R., Hollenbeck, T., Castelo, J., Johnson, N., Gordon, P., et al. (2003). Modulation of nonspecific binding in ultrafiltration protein binding studies. Pharm. Res. 20, 1015–1021.

    Article  PubMed  CAS  Google Scholar 

  15. Gell, D., Kong, Y., Eaton, S. A., Weiss, M. J., and Mackay, J. P. (2002) Biophysical characterization of the α-globin binding protein α-hemoglobin stabilizing protein. J. Bio. Chem. 277, 40,602–40,609.

    Article  CAS  Google Scholar 

  16. Blom, K. F., Larsen, B. S., and McEwen, C. N. (1999) Determining affinityselected ligands and estimating binding affinities by online size exclusion chromatography/liquid chromatography-mass spectrometry. J. Combin. Chem. 1, 82–90.

    Article  CAS  Google Scholar 

  17. McDonnell, P. A., Caldwell, G. W., and Masucci, J. A. (1998) Using capillary electrophoresis/frontal analysis to screen drugs interacting with human serum proteins. Electrophoresis 19, 448–454.

    Article  PubMed  CAS  Google Scholar 

  18. O’Brien, R., Ladbury, J. E., and Chowdhry, B. Z. (2001) Isothermal titration calorimetry of biomolecules, in Protein-Ligand Interactions: Hydrodynamics and Calorimetry (Harding, S. E. and Chowdhry, B. Z., eds.), Oxford University Press, Oxford, UK, pp. 263–286.

    Google Scholar 

  19. Todd, M. J. and Gomez, J. (2001) Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Anal. Biochem. 296, 179–187.

    Article  PubMed  CAS  Google Scholar 

  20. Voet, D. and Voet, J. G. (1995) Biochemistry. 2nd ed. John Wiley, New York.

    Google Scholar 

  21. Schulze, A. and Heremans, C. (1966) Molecular Biology of Human Proteins with Special Reference to Plasma Proteins. Elsevier, Amsterdam, 1966.

    Google Scholar 

  22. Kragh-Hansen, U. (1981) Molecular aspects of ligand binding to serum albumin. Pharmacol. Rev. 33, 17–53.

    PubMed  CAS  Google Scholar 

  23. Fournier, T., Medjoubi-N, N., and Porquet, D. (2000) Alpha-1-acid glycoprotein. Biochimica et Biophysica Acta 1482, 157–171.

    Article  PubMed  CAS  Google Scholar 

  24. Horn, J. R., Brandts, J. F., and Murphy, K. P. (2002) van’t Hoff and calorimetric enthalpies: II. Effects of linked equilibria. Biochem. 41, 7501–7507.

    Article  CAS  Google Scholar 

  25. Wadso, I. (2000) Needs for standards in isothermal microcalorimetry. Thermochimica Acta 347, 73–77.

    Article  CAS  Google Scholar 

  26. Olofsson, G., Berling, D., Markova, N., and Molund, M. (2000) The dissolution of propan-1-ol and dilution of 10wt. % propan-1-ol solution in water as calibration and test reactions in solution chemistry. Thermochimica Acta 347, 31–36.

    Article  CAS  Google Scholar 

  27. Izatt, R. M., Terry, R. E., Nelson, D. P., Chan, Y., Eatough, D. J., Bradshaw, J. S., et al. (1976) Calorimetric titration study of the interaction of some uni-and bivalent cations with benzo-15-crown-5, 18-crown-6, dibenzo-24-crown-8, and dibenzo-27-crown-9 in methanol-water solvents, at 25°C and µ = 0.1. J. Am. Chem. Soc. 98, 7626–7630.

    Article  CAS  Google Scholar 

  28. Hasegawa, Y. and Date, H. (1988) Stability of strontium(II) and barium(II) complexes with 18-crown-6 in aqueous solution and their extractability as picrates. Sol. Extra Ion Exchange 6, 431–437.

    Article  CAS  Google Scholar 

  29. Briggner, L. E. and Wadsoe, I. (1991) Test and calibration processes for microcalorimeters, with special reference to heat conduction instruments used with aqueous systems. J. Biochem. Biophys. Methods 22, 101–118.

    Article  PubMed  CAS  Google Scholar 

  30. Liu, Y. and Sturtevant, J. M. (1995) Significant discrepancies between van’t Hoff and calorimetric enthalpies: II. Protein Sci. 4, 2559–61. See also Horn, J. R., Russell, D., Lewis, E. A., and Murphy, K. P. (2001) van’t Hoff and calorimetric enthalpies from isothermal titration calorimetry: are there significant discrepancies? Biochemistry 40, 1774-1778.

    Article  PubMed  CAS  Google Scholar 

  31. Gutte, B. and Merrifeild, R. B. (1969) The total synthesis of an enzyme with ribonuclease A activity. J. Am. Chem. Soc. 91, 501–502.

    Article  PubMed  CAS  Google Scholar 

  32. Zegers, I., Maes, D., Dao-Thi, M. H., Poortmans, F., Palmer, R., and Wyns, L. (1994) The structures of RNase A complexed with 3′-CMP and d(CpA): active site conformation and conserved water molecules. Protein Sci. 3, 2322–2339.

    Article  PubMed  CAS  Google Scholar 

  33. Garcia-Fuentes, L., Baron, C., and Mayorga, O. L. (1998) Influence of dynamic power compensation in an isothermal titration microcalorimeter. Anal. Chem. 70, 4615–4623.

    Article  PubMed  CAS  Google Scholar 

  34. Wiseman, T., Williston, S., Brandts, J. F., and Lin, L.-N. (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131–137.

    Article  PubMed  CAS  Google Scholar 

  35. Kotelnikov, G. V., Moiseyeva, S. P., Mezhburd, E. V., and Krayev, V. P. (2002) New isothermal titration calorimeter for investigation on very small samples: theoretical and experimental results. J. Therm. Anal. Calor. 68, 8003–8018.

    Article  Google Scholar 

  36. Aki, H. and Yamamoto, M. (1994) Thermodynamic characterization of drug binding to human serum albumin by isothermal titration microcalorimetry. J. Pharm. Sci. 83, 1712–1716.

    Article  PubMed  CAS  Google Scholar 

  37. Anders, J. C., Parten, B. F., Petrie, G. E., Marlowe, R. L., and McEntire, J. E. (2003) Using amino acid analysis to determine absorptivity constants: a validation case study using bovine serum albumin. Biopharm. Intern. 16, 30–32, 34–37.

    CAS  Google Scholar 

  38. Pace, C. N., Vajdos, 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.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Johnson & Johnson Pharmaceutical Research & Development, LLC, Drug Discovery, Spring House, PA

    Gary W. Caldwell & Zhengyin Yan

Authors
  1. Gary W. Caldwell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhengyin Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Johnson & Johnson Pharmaceutical Research & Development, Spring House, PA

    Zhengyin Yan PhD  & Gary W. Caldwell PhD  & 

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Caldwell, G.W., Yan, Z. (2004). Isothermal Titration Calorimetry Characterization of Drug-Binding Energetics to Blood Proteins. In: Yan, Z., Caldwell, G.W. (eds) Optimization in Drug Discovery. Methods in Pharmacology and Toxicology. Humana Press. https://doi.org/10.1385/1-59259-800-5:123

Download citation

  • DOI: https://doi.org/10.1385/1-59259-800-5:123

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-332-9

  • Online ISBN: 978-1-59259-800-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation