Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 Jan;8(1):222–233. doi: 10.1110/ps.8.1.222

A mechanistic analysis of the increase in the thermal stability of proteins in aqueous carboxylic acid salt solutions.

J K Kaushik 1, R Bhat 1
PMCID: PMC2144102  PMID: 10210200

Abstract

The stability of proteins is known to be affected significantly in the presence of high concentration of salts and is highly pH dependent. Extensive studies have been carried out on the stability of proteins in the presence of simple electrolytes and evaluated in terms of preferential interactions and increase in the surface tension of the medium. We have carried out an in-depth study of the effects of a series of carboxylic acid salts: ethylene diamine tetra acetate, butane tetra carboxylate, propane tricarballylate, citrate, succinate, tartarate, malonate, and gluconate on the thermal stability of five different proteins that vary in their physico-chemical properties: RNase A, cytochrome c, trypsin inhibitor, myoglobin, and lysozyme. Surface tension measurements of aqueous solutions of the salts indicate an increase in the surface tension of the medium that is very strongly correlated with the increase in the thermal stability of proteins. There is also a linear correlation of the increase in thermal stability with the number of carboxylic groups in the salt. Thermal stability has been found to increase by as much as 22 C at 1 M concentration of salt. Such a high thermal stability at identical concentrations has not been reported before. The differences in the heat capacities of denaturation, deltaCp for RNase A, deduced from the transition curves obtained in the presence of varying concentrations of GdmCl and that of carboxylic acid salts as a function of pH, indicate that the nature of the solvent medium and its interactions with the two end states of the protein control the thermodynamics of protein denaturation. Among the physico-chemical properties of proteins, there seems to be an interplay of the hydrophobic and electrostatic interactions that lead to an overall stabilizing effect. Increase in surface free energy of the solvent medium upon addition of the carboxylic acid salts appears to be the dominant factor in governing the thermal stability of proteins.

Full Text

The Full Text of this article is available as a PDF (595.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abe Y., Ueda T., Iwashita H., Hashimoto Y., Motoshima H., Tanaka Y., Imoto T. Effect of salt concentration on the pKa of acidic residues in lysozyme. J Biochem. 1995 Nov;118(5):946–952. doi: 10.1093/jb/118.5.946. [DOI] [PubMed] [Google Scholar]
  2. Arakawa T., Bhat R., Timasheff S. N. Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system. Biochemistry. 1990 Feb 20;29(7):1914–1923. doi: 10.1021/bi00459a036. [DOI] [PubMed] [Google Scholar]
  3. Arakawa T., Bhat R., Timasheff S. N. Why preferential hydration does not always stabilize the native structure of globular proteins. Biochemistry. 1990 Feb 20;29(7):1924–1931. doi: 10.1021/bi00459a037. [DOI] [PubMed] [Google Scholar]
  4. Arakawa T., Timasheff S. N. Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding. Biochemistry. 1984 Dec 4;23(25):5912–5923. doi: 10.1021/bi00320a004. [DOI] [PubMed] [Google Scholar]
  5. Arakawa T., Timasheff S. N. Preferential interactions of proteins with salts in concentrated solutions. Biochemistry. 1982 Dec 7;21(25):6545–6552. doi: 10.1021/bi00268a034. [DOI] [PubMed] [Google Scholar]
  6. Arakawa T., Timasheff S. N. Preferential interactions of proteins with solvent components in aqueous amino acid solutions. Arch Biochem Biophys. 1983 Jul 1;224(1):169–177. doi: 10.1016/0003-9861(83)90201-1. [DOI] [PubMed] [Google Scholar]
  7. Arakawa T., Timasheff S. N. The stabilization of proteins by osmolytes. Biophys J. 1985 Mar;47(3):411–414. doi: 10.1016/S0006-3495(85)83932-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Baldwin R. L. How Hofmeister ion interactions affect protein stability. Biophys J. 1996 Oct;71(4):2056–2063. doi: 10.1016/S0006-3495(96)79404-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bonneté F., Madern D., Zaccaï G. Stability against denaturation mechanisms in halophilic malate dehydrogenase "adapt" to solvent conditions. J Mol Biol. 1994 Dec 9;244(4):436–447. doi: 10.1006/jmbi.1994.1741. [DOI] [PubMed] [Google Scholar]
  10. Breslow R., Guo T. Surface tension measurements show that chaotropic salting-in denaturants are not just water-structure breakers. Proc Natl Acad Sci U S A. 1990 Jan;87(1):167–169. doi: 10.1073/pnas.87.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Busby T. F., Atha D. H., Ingham K. C. Thermal denaturation of antithrombin III. Stabilization by heparin and lyotropic anions. J Biol Chem. 1981 Dec 10;256(23):12140–12147. [PubMed] [Google Scholar]
  12. Busby T. F., Ingham K. C. Thermal stabilization of antithrombin III by sugars and sugar derivatives and the effects of nonenzymatic glycosylation. Biochim Biophys Acta. 1984 May 25;799(1):80–89. doi: 10.1016/0304-4165(84)90329-5. [DOI] [PubMed] [Google Scholar]
  13. Carbonnaux C., Ries-Kautt M., Ducruix A. Relative effectiveness of various anions on the solubility of acidic Hypoderma lineatum collagenase at pH 7.2. Protein Sci. 1995 Oct;4(10):2123–2128. doi: 10.1002/pro.5560041018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Edge V., Allewell N. M., Sturtevant J. M. Differential scanning calorimetric study of the thermal denaturation of aspartate transcarbamoylase of Escherichia coli. Biochemistry. 1988 Oct 18;27(21):8081–8087. doi: 10.1021/bi00421a017. [DOI] [PubMed] [Google Scholar]
  15. Eftink M. R., Anusiem A. C., Biltonen R. L. 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. 1983 Aug 2;22(16):3884–3896. doi: 10.1021/bi00285a025. [DOI] [PubMed] [Google Scholar]
  16. Gekko K., Morikawa T. Preferential hydration of bovine serum albumin in polyhydric alcohol-water mixtures. J Biochem. 1981 Jul;90(1):39–50. doi: 10.1093/oxfordjournals.jbchem.a133468. [DOI] [PubMed] [Google Scholar]
  17. Hernández-Arana A., Rojo-Domínguez A., Altamirano M. M., Calcagno M. L. Differential scanning calorimetry of the irreversible denaturation of Escherichia coli glucosamine-6-phosphate deaminase. Biochemistry. 1993 Apr 13;32(14):3644–3648. doi: 10.1021/bi00065a017. [DOI] [PubMed] [Google Scholar]
  18. Jensen W. A., Armstrong J. M., De Giorgio J., Hearn M. T. Stability studies on maize leaf phosphoenolpyruvate carboxylase: the effect of salts. Biochemistry. 1995 Jan 17;34(2):472–480. doi: 10.1021/bi00002a011. [DOI] [PubMed] [Google Scholar]
  19. Kita Y., Arakawa T., Lin T. Y., Timasheff S. N. Contribution of the surface free energy perturbation to protein-solvent interactions. Biochemistry. 1994 Dec 20;33(50):15178–15189. doi: 10.1021/bi00254a029. [DOI] [PubMed] [Google Scholar]
  20. Lee J. C., Timasheff S. N. The stabilization of proteins by sucrose. J Biol Chem. 1981 Jul 25;256(14):7193–7201. [PubMed] [Google Scholar]
  21. Lin T. Y., Timasheff S. N. On the role of surface tension in the stabilization of globular proteins. Protein Sci. 1996 Feb;5(2):372–381. doi: 10.1002/pro.5560050222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Liu Y., Bolen D. W. The peptide backbone plays a dominant role in protein stabilization by naturally occurring osmolytes. Biochemistry. 1995 Oct 3;34(39):12884–12891. doi: 10.1021/bi00039a051. [DOI] [PubMed] [Google Scholar]
  23. Liu Y., Sturtevant J. M. The observed change in heat capacity accompanying the thermal unfolding of proteins depends on the composition of the solution and on the method employed to change the temperature of unfolding. Biochemistry. 1996 Mar 5;35(9):3059–3062. doi: 10.1021/bi952198j. [DOI] [PubMed] [Google Scholar]
  24. Lumry R., Rajender S. Enthalpy-entropy compensation phenomena in water solutions of proteins and small molecules: a ubiquitous property of water. Biopolymers. 1970;9(10):1125–1227. doi: 10.1002/bip.1970.360091002. [DOI] [PubMed] [Google Scholar]
  25. Makhatadze G. I., Lopez M. M., Richardson J. M., 3rd, Thomas S. T. Anion binding to the ubiquitin molecule. Protein Sci. 1998 Mar;7(3):689–697. doi: 10.1002/pro.5560070318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Makhatadze G. I., Privalov P. L. Energetics of protein structure. Adv Protein Chem. 1995;47:307–425. doi: 10.1016/s0065-3233(08)60548-3. [DOI] [PubMed] [Google Scholar]
  27. Matthew J. B., Richards F. M. Anion binding and pH-dependent electrostatic effects in ribonuclease. Biochemistry. 1982 Sep 28;21(20):4989–4999. doi: 10.1021/bi00263a024. [DOI] [PubMed] [Google Scholar]
  28. Melander W., Horváth C. Salt effect on hydrophobic interactions in precipitation and chromatography of proteins: an interpretation of the lyotropic series. Arch Biochem Biophys. 1977 Sep;183(1):200–215. doi: 10.1016/0003-9861(77)90434-9. [DOI] [PubMed] [Google Scholar]
  29. Myers J. K., Pace C. N., Scholtz J. M. Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 1995 Oct;4(10):2138–2148. doi: 10.1002/pro.5560041020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  31. Pace C. N., Grimsley G. R. Ribonuclease T1 is stabilized by cation and anion binding. Biochemistry. 1988 May 3;27(9):3242–3246. doi: 10.1021/bi00409a018. [DOI] [PubMed] [Google Scholar]
  32. Pace C. N., Laurents D. V. A new method for determining the heat capacity change for protein folding. Biochemistry. 1989 Mar 21;28(6):2520–2525. doi: 10.1021/bi00432a026. [DOI] [PubMed] [Google Scholar]
  33. Santoro M. M., Liu Y., Khan S. M., Hou L. X., Bolen D. W. Increased thermal stability of proteins in the presence of naturally occurring osmolytes. Biochemistry. 1992 Jun 16;31(23):5278–5283. doi: 10.1021/bi00138a006. [DOI] [PubMed] [Google Scholar]
  34. Sharp K. A., Nicholls A., Fine R. F., Honig B. Reconciling the magnitude of the microscopic and macroscopic hydrophobic effects. Science. 1991 Apr 5;252(5002):106–109. doi: 10.1126/science.2011744. [DOI] [PubMed] [Google Scholar]
  35. Tanford C. Interfacial free energy and the hydrophobic effect. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4175–4176. doi: 10.1073/pnas.76.9.4175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Timasheff S. N. The control of protein stability and association by weak interactions with water: how do solvents affect these processes? Annu Rev Biophys Biomol Struct. 1993;22:67–97. doi: 10.1146/annurev.bb.22.060193.000435. [DOI] [PubMed] [Google Scholar]
  37. Timasheff S. N. Water as ligand: preferential binding and exclusion of denaturants in protein unfolding. Biochemistry. 1992 Oct 20;31(41):9857–9864. doi: 10.1021/bi00156a001. [DOI] [PubMed] [Google Scholar]
  38. Wang A., Bolen D. W. A naturally occurring protective system in urea-rich cells: mechanism of osmolyte protection of proteins against urea denaturation. Biochemistry. 1997 Jul 29;36(30):9101–9108. doi: 10.1021/bi970247h. [DOI] [PubMed] [Google Scholar]
  39. Woolfson D. N., Cooper A., Harding M. M., Williams D. H., Evans P. A. Protein folding in the absence of the solvent ordering contribution to the hydrophobic interaction. J Mol Biol. 1993 Jan 20;229(2):502–511. doi: 10.1006/jmbi.1993.1049. [DOI] [PubMed] [Google Scholar]
  40. Zale S. E., Klibanov A. M. Why does ribonuclease irreversibly inactivate at high temperatures? Biochemistry. 1986 Sep 23;25(19):5432–5444. doi: 10.1021/bi00367a014. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES