Skip to main content
SpringerLink
Log in

Thermal denaturation of α-chymotrypsinogen A in presence of polyols at pH 2.0 and pH 3.0

  • Published:
Journal of Thermal Analysis and Calorimetry Aims and scope Submit manuscript

Abstract

Osmolytes are cosolutes that induce stabilization of the structure of proteins. Polyols are osmolytes that have importance in a wide variety of biotechnological and industrial processes. In this work, a systematic study concerning the effect of polyols of different number of methylene and hydroxyl groups on the stability of α-chymotrypsinogen A is presented. Protein thermal stability measurements in buffer, ethylene glycol, glycerol, meso-erythritol, sorbitol and inositol was followed by fluorescence measurements. Under the selected conditions, the thermal denaturation of α-chymotrypsinogen A is a reversible transition between native and unfolded state that can be well described by a two-state model. Reversibility of the transition was confirmed by DSC, circular dichroism, UV–Vis and fluorescence measurements. The change in thermal stability of the protein in the presence of ethylene glycol, glycerol, erythritol, sorbitol and inositol shows that ethylene glycol is the only polyol that presents a destabilizing effect. The other cosolutes exert stabilizing effects that increase with the number of hydroxyl groups and depend on concentration.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Romero CM, Lozano JM, Sancho J, Giraldo GI. Thermal stability of beta-lactoglobulin in the presence of aqueous solution of alcohols and polyols. Int J Biol Macromol. 2007;40:423–8.

    Article  CAS  Google Scholar 

  2. Xie G, Timasheff S. Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured than for the native protein. Protein Sci. 1997;6:211–21.

    Article  CAS  Google Scholar 

  3. Davis-Searles PR, Saunders AJ, Erie DA, Winzor DJ, Pielak GJ. Interpreting the effects of small uncharged solutes on protein-folding equilibria. Annu Rev Biophys Biomol Struct. 2001;30:271–306.

    Article  CAS  Google Scholar 

  4. Kaushik JK, Bhat R. Thermal stability of proteins in aqueous polyol solutions: role of the surface tension of water in the stabilizing effect of polyols. J Phys Chem. 1998;102:7058–66.

    Article  CAS  Google Scholar 

  5. Gerlsma S. The effects of polyhydric and monohydric alcohols on the heat induced reversible denaturation of chymotrypsinogen A. Eur J Biochem. 1970;14:150–3.

    Article  CAS  Google Scholar 

  6. Xie G, Timasheff SN. The thermodynamic mechanism of protein stabilization by trehalose. Biophys Chem. 1997;64:25–43.

    Article  CAS  Google Scholar 

  7. Theodore BG, Herskovits T. On the structural stability and solvent denaturation of proteins. J Biol Chem. 1970;245:2588–98.

    Google Scholar 

  8. Hartley BS. Amino-acid sequence of bovine chymotrypsinogen-A. Nature. 1964;201:1284–7.

    Article  CAS  Google Scholar 

  9. Brandts JF. The thermodynamics of protein denaturation i. the denaturation of chymotrypsinogen. J Am Chem Soc. 1964;86:4291–301.

    Article  CAS  Google Scholar 

  10. Brandts JF. The thermodynamics of protein denaturation II. A model of reversible denaturation and interpretations regarding the stability of chymotrypsinogen. J Am Chem Soc. 1964;86:4302–14.

    Article  CAS  Google Scholar 

  11. Jackson WM, Brandts JF. Thermodynamics of protein denaturation. Calorimetric study of the reversible denaturation of chymotrypsinogen and conclusions regarding the accuracy of the two-state approximation. Biochemistry. 1970;9:2294–301.

    Article  CAS  Google Scholar 

  12. Poklar N, Vesnaver G, Lapanje S. Interactions of alpha-chymotrypsinogen A with alkylureas. Biophys Chem. 1996;57:279–89.

    Article  CAS  Google Scholar 

  13. Chalikian TV, Volker J, Anafi D, Breslauer KJ. The native and the heat-induced denatured states of α-chymotrypsinogen A: thermodynamic and spectroscopic studies. J Mol Biol. 1997;274:237–52.

    Article  CAS  Google Scholar 

  14. Romero CM, Albis A, Lozano JM, Sancho J. Thermodynamic study of the influence of polyols and glucose on the thermal stability of holo-bovine α-lactalbumin. J Therm Anal Calorim. 2009;98:165–71.

    Article  CAS  Google Scholar 

  15. Cooper A. Thermodynamics of protein folding and stability. Protein A Compr Treatise. 1999;2:217–70.

    CAS  Google Scholar 

  16. Brandts JF, Hunt L. Thermodynamics of protein denaturation III. Denaturation of ribonuclease in water and in aqueous urea and aqueous ethanol mixtures. J Am Chem Soc. 1967;89:4826–38.

    Article  CAS  Google Scholar 

  17. Haque I, Singh R, Moosavi-Movahedi AA, Ahmad F. Effect of polyol osmolytes on ∆GD, the Gibbs energy of stabilisation of proteins at different pH values. Biophys Chem. 2005;117:1–12.

    Article  CAS  Google Scholar 

  18. Sancho J. The stability of 2-state, 3-state and more-state proteins from simple spectroscopic techniques… plus the structure of the equilibrium intermediates at the same time. Arch Biochem Biophys. 2013;531:4–13.

    Article  CAS  Google Scholar 

  19. Miyawaki O, Tatsuno M. Thermodynamic analysis of alcohol effect on thermal stability of proteins. J Biosci Bioeng. 2011;111:198–203.

    Article  CAS  Google Scholar 

  20. Gekko K, Morikawa T. Thermodynamics of polyol-induced thermal stabilization of chymotrypsinogen. J Biochem. 1981;90:51–60.

    CAS  Google Scholar 

  21. Privalov PL. Stability of proteins: small globular proteins. Adv Protein Chem. 1979;33:167–241.

    Article  CAS  Google Scholar 

  22. Sturtevant JM. Biochemical applications of differential scanning calorimetry. Annu Rev Phys Chem. 1987;38:463–88.

    Article  CAS  Google Scholar 

  23. Privalov PL, Khechinashvili NN. A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. J Mol Biol. 1974;86:665–84.

    Article  CAS  Google Scholar 

  24. Greenfield NJ. Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc. 2006;1:2876–90.

    Article  CAS  Google Scholar 

  25. Greenfield NJ, Fasman GD. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry. 1969;8:4108–16.

    Article  CAS  Google Scholar 

  26. Brahms S, Brahms J. Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism. J Mol Biol. 1980;138:149–78.

    Article  CAS  Google Scholar 

  27. Reed J, Reed TA. A set of constructed type spectra for the practical estimation of peptide secondary structure from circular dichroism. Anal Biochem. 1997;254:36–40.

    Article  CAS  Google Scholar 

  28. Mao D, Wachter E, Wallace BA. Folding of the mitochondrial proton adenosine triphosphatase proteolipid channel in phospholipid vesicles. Biochemistry. 1982;21:4960–8.

    Article  CAS  Google Scholar 

  29. Freer ST, Kraut J, Robertus JD, Wright HT. Chymotrypsinogen: 2,5-Å crystal structure, comparison with α-chymotrypsin, and implications for zymogen activation. Biochemistry. 1970;9:1997–2009.

    Article  CAS  Google Scholar 

  30. Wang D, Bode W, Huber R. Bovine chymotrypsinogen A X-ray crystal structure analysis and refinement of a new crystal form at 1.8 A resolution. J Mol Biol. 1985;185:595–624.

    Article  CAS  Google Scholar 

  31. Khan F, Khan RH, Muzammil S. Alcohol-induced versus anion-induced states of alpha-chymotrypsinogen A at low pH. Biochim Biophys Acta. 2000;1481:229–36.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by Universidad Nacional de Colombia and COLCIENCIAS and by Grants BFU2010-16297, BFU2010-19451, PI078/08 and CTPR02/09 (Spain).

Author information

Authors and Affiliations

  1. Departamento de Química, Universidad Nacional de Colombia, Bogotá, Colombia

    Carmen M. Romero & Juan S. Abella

  2. Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Zaragoza, Spain

    Javier Sancho

  3. Instituto de Biocomputación y física de sistemas complejos (BIFI), Universidad de Zaragoza, Zaragoza, Spain

    Adrian Velázquez & Javier Sancho

Authors
  1. Carmen M. Romero
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Juan S. Abella
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Adrian Velázquez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Javier Sancho
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carmen M. Romero.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Romero, C.M., Abella, J.S., Velázquez, A. et al. Thermal denaturation of α-chymotrypsinogen A in presence of polyols at pH 2.0 and pH 3.0. J Therm Anal Calorim 120, 489–499 (2015). https://doi.org/10.1007/s10973-014-4374-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10973-014-4374-2

Keywords

Search

Navigation