European Biophysics Journal

, Volume 37, Issue 1, pp 11–18 | Cite as

Protonation linked equilibria and apparent affinity constants: the thermodynamic profile of the α-chymotrypsin–proflavin interaction

  • Gilles Bruylants
  • René Wintjens
  • Yvan Looze
  • Christina Redfield
  • Kristin BartikEmail author
Original Paper


Protonation/deprotonation equilibria are frequently linked to binding processes involving proteins. The presence of these thermodynamically linked equilibria affects the observable thermodynamic parameters of the interaction (K obs, ΔH obs 0 ). In order to try and elucidate the energetic factors that govern these binding processes, a complete thermodynamic characterisation of each intrinsic equilibrium linked to the complexation event is needed and should furthermore be correlated to structural information. We present here a detailed study, using NMR and ITC, of the interaction between α-chymotrypsin and one of its competitive inhibitors, proflavin. By performing proflavin titrations of the enzyme, at different pH values, we were able to highlight by NMR the effect of the complexation of the inhibitor on the ionisable residues of the catalytic triad of the enzyme. Using ITC we determined the intrinsic thermodynamic parameters of the different equilibria linked to the binding process. The possible driving forces of the interaction between α-chymotrypsin and proflavin are discussed in the light of the experimental data and on the basis of a model of the complex. This study emphasises the complementarities between ITC and NMR for the study of binding processes involving protonation/deprotonation equilibria.


Nuclear Magnetic Resonance Isothermal Titration Calorimetry Protonation State Affinity Constant Titratable Group 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



G.B. thanks the Belgian FRIA for a PhD fellowship and the Foundation Wiener-Anspach for a post-doctoral fellowship. C.R. thanks the Wellcome Trust for a “Value in People” award. The authors acknowledge Professor Jacques Reisse for very helpful discussions. This work was supported by the Belgian FNRS (LEA CNRS-FNRS), the “Communauté Française de Belgique” (ARC 2002–2007).

Supplementary material


  1. Bachovchin WW (1985) Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme. Proc Natl Acad Sci USA 82:7948–7951CrossRefADSGoogle Scholar
  2. Bachovchin WW (1986) Nitrogen-15 NMR spectroscopy of hydrogen-bonding interactions in the active site of serine proteases: evidence for a moving histidine mechanism. Biochemistry 25:7751–7759CrossRefGoogle Scholar
  3. Bachovchin WW (2001) Contributions of NMR spectroscopy to the study of hydrogen bonds in serine protease active sites. Magn Reson Chem 39:S199–S213CrossRefGoogle Scholar
  4. Baker BM, Murphy KP (1996) Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry. Biophys J 71:2049–2055Google Scholar
  5. Baker BM, Murphy KP (1997) Dissecting the energetics of a protein–protein interaction: the binding of ovomucoid third domain to elastase. J Mol Biol 268:557–569CrossRefGoogle Scholar
  6. Barratt E, Bingham RJ, Warner DJ, Laughton CA, Phillips SEV, Homans SW (2005) Van der Waals interactions dominate ligand–protein association in a protein binding site occluded from solvent water. J Am Chem Soc 127:11827–11834CrossRefGoogle Scholar
  7. Bender ML, Kezdy FJ (1964) The mechanism of action of proteolytic enzymes. XXXII. The current status of the α-chymotrypsin mechanism. J Am Chem Soc 86:3704–3714CrossRefGoogle Scholar
  8. Bernhard SA, Lee BF, Tashjian ZH (1966) On the interaction of the active side of alpha-chymotrypsin with chromophores: proflavin binding and enzyme conformation during catalysis. J Mol Biol 18:405–420Google Scholar
  9. Blow DM, Birktoft JJ, Hartley BS (1969) Role of a buried acid group in the mechanism of action of chymotrypsin. Nature 221:337–340CrossRefADSGoogle Scholar
  10. Boutonnet NS, Rooman MJ, Ochagavia M-E, Richelle J, Wodak SJ (1995) Optimal protein structure alignments by multiple linkage clustering: application to distantly related proteins. Protein Eng 8:647–662Google Scholar
  11. Bruylants G, Redfield C, Bartik K (2007) Developments in the characterisation of the catalytic triad of α-chymotrypsin: effect of the protonation state of Asp102 on the 1H NMR signals of His57. ChemBioChem 8:51–54CrossRefGoogle Scholar
  12. Caplow M (1969) Chymotrypsin catalysis. Evidence for a new intermediate. J Am Chem Soc 91:3639–3645CrossRefGoogle Scholar
  13. Conti E, Rivetti C, Wonacott A, Brick P (1998) X-ray and spectrophotometric studies of the binding of proflavin to the S1 specificity pocket of human alpha-thrombin. FEBS Lett 425:229–233CrossRefGoogle Scholar
  14. Dauber-Osguthorpe P, Roberts VA, Osguthorpe DJ, Wolff J, Genest M, Hagler AT (1988) Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system. Proteins 4:31–47CrossRefGoogle Scholar
  15. Doyle ML, Louie G, Dal Monte PR, Sokoloski TD (1995) Tight binding affinities determined from thermodynamic linkage to protons by titration calorimetry. Methods Enzymol 259:183–194CrossRefGoogle Scholar
  16. Dullweber F, Stubbs MT, Musil D, Stuerzebecher J, Klebe G (2001) Factorising ligand affinity: a combined thermodynamic and crystallographic study of trypsin and thrombin inhibition. J Mol Biol 313:593–614CrossRefGoogle Scholar
  17. Eftink MR, Anusiem AC, Biltonen RL (1983) Enthalpy–entropy compensation and heat capacity changes for protein–ligand interactions: general thermodynamic models and data for the binding of nucleotides to ribonuclease A. Biochemistry 22:3884–3896CrossRefGoogle Scholar
  18. Feinstein G, Feeney RE (1967) Binding of proflavine to α-chymotrypsin and trypsin and its displacement by avian ovomucoids. Biochemistry 6:749–753CrossRefGoogle Scholar
  19. Fersht AR (1972) Mechanism of the α-chymotrypsin-catalyzed hydrolysis of specific amide substrates. J Am Chem Soc 94:293–295CrossRefGoogle Scholar
  20. Fersht AR, Renard M (1974) pH Dependence of chymotrypsin catalysis. Appendix. Substrate binding to dimeric α-chymotrypsin studied by X-ray diffraction and the equilibrium method. Biochemistry 13:1416–1426CrossRefGoogle Scholar
  21. Fersht AR, Requena Y (1971) Mechanism of the α-chymotrypsin-catalyzed hydrolysis of amides. pH dependence of k c and k m kinetic detection of an intermediate. J Am Chem Soc 93:7079–7087CrossRefGoogle Scholar
  22. Fersht AR, Sperling J (1973) Charge relay system in chymotrypsin and chymotrypsinogen. J Mol Biol 74:137–149CrossRefGoogle Scholar
  23. Glazer AN (1965) Spectral studies of the interaction of alpha-chymotrypsin and trypsin with proflavine. Proc Natl Acad Sci USA 54:171–176CrossRefADSGoogle Scholar
  24. Goldberg RN, Kishore N, Lennen RM (2002) Thermodynamic quantities for the ionization reactions of buffers. J Phys Chem Ref Data 31:231–370CrossRefADSGoogle Scholar
  25. Gomez J, Freire E (1995) Thermodynamic mapping of the inhibitor site of the aspartic protease endothiapepsin. J Mol Biol 252:337–350CrossRefGoogle Scholar
  26. Hagler AT, Huler E, Lifson S (1974) Energy functions for peptides and proteins. I. Derivation of a consistent force field including the hydrogen bond from amide crystals. J Am Chem Soc 96:5319–5327CrossRefGoogle Scholar
  27. Havsteen BH (1967) Kinetics of the two-step interaction of chymotrypsin with proflavine. J Biol Chem 242:769–771Google Scholar
  28. Hendrickson HS (1999) Binding of proflavin to chymotrypsin: an experiment to determine protein–ligand interactions by direct nonlinear regression analysis of spectroscopic titration data. Biochem Educ 27:118–121CrossRefGoogle Scholar
  29. 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. Chem Biol 3:973–980CrossRefGoogle Scholar
  30. Lucas EC, Caplow M, Bush KJ (1973) Chymotrypsin catalysis. Evidence for a new intermediate. III. J Am Chem Soc 95:2670–2673CrossRefGoogle Scholar
  31. Plateau P, Gueron M (1982) Exchangeable proton NMR without base-line distorsion, using new strong-pulse sequences. J Am Chem Soc 104:7310–7311CrossRefGoogle Scholar
  32. Robillard G, Shulman RG (1972) High resolution nuclear magnetic resonance study of the histidine–aspartate hydrogen bond in chymotrypsin and chymotrypsinogen. J Mol Biol 71:507–511CrossRefGoogle Scholar
  33. Robillard G, Shulman RG (1974) High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the “charge relay” system. J Mol Biol 86:519–540CrossRefGoogle Scholar
  34. Shiao DDF, Sturtevant JM (1969) Calorimetric investigations of the binding of inhibitors to α-chymotrypsin. I. Enthalpy of dilution of α-chymotrypsin and of proflavine, and the enthalpy of binding of indole, N-acetyl-D-tryptophan, and proflavine to α-chymotrypsin. Biochemistry 8:4910–4917CrossRefGoogle Scholar
  35. Sturgill TW, Johnson RE, Biltonen RL (1978) Thermal perturbation techniques in characterizing ligand–macromolecule interactions: theory and application to the proflavin-α-chymotrypsin system. Biopolymers 17:1773–1792CrossRefGoogle Scholar
  36. Sturtevant JM, Beres L (1971) Calorimetric studies of the activation of chymotrypsinogen A. Biochemistry 10:2120–2126CrossRefGoogle Scholar
  37. Tame JRH, Sleigh SH, Wilkinson AJ, Ladbury JE (1996) The role of water in sequence-independent ligand binding by an oligopeptide transporter protein. Nat Struct Biol 3:998–1001CrossRefGoogle Scholar
  38. Tulinsky A, Blevins RA (1987) Structure of a tetrahedral transition state complex of alpha-chymotrypsin dimer at 1.8-Å resolution. J Biol Chem 262:7737–7743Google Scholar
  39. Waelbroeck M (1982) The pH dependence of insulin binding. A quantitative study. J Biol Chem 257:8284–8291Google Scholar
  40. Wallace RA, Kurtz AN, Niemann C (1963) Interaction of aromatic compounds with alpha-chymotrypsin. Biochemistry 128:824–836CrossRefGoogle Scholar
  41. Weber G (1972) Ligand binding and internal equilibiums in proteins. Biochemistry 11:864–878CrossRefGoogle Scholar
  42. Wyman J (1965) The binding potential, a neglected linkage concept. J Mol Biol 11:631–644CrossRefGoogle Scholar
  43. Zhong S, Haghjoo K, Kettner C, Jordan F (1995) Proton magnetic resonance studies of the active center histidine of chymotrypsin complexed to peptideboronic acids: solvent accessibility to the Nδ and Nε sites can differentiate slow-binding and rapidly reversible inhibitors. J Am Chem Soc 117:7048–7055CrossRefGoogle Scholar

Copyright information

© EBSA 2007

Authors and Affiliations

  • Gilles Bruylants
    • 1
  • René Wintjens
    • 2
  • Yvan Looze
    • 2
  • Christina Redfield
    • 3
  • Kristin Bartik
    • 1
    Email author
  1. 1.Ingénierie Moléculaire et Biomoléculaire, CP 165/64Université Libre de BruxellesBruxellesBelgium
  2. 2.Service de Chimie Générale, CP 206/4, CP 165/64Université Libre de BruxellesBruxellesBelgium
  3. 3.Department of BiochemistryUniversity of OxfordOxfordUK

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