Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2001 Dec;81(6):3489–3502. doi: 10.1016/S0006-3495(01)75980-2

pH corrections and protein ionization in water/guanidinium chloride.

M M Garcia-Mira 1, J M Sanchez-Ruiz 1
PMCID: PMC1301804  PMID: 11721010

Abstract

More than 30 years ago, Nozaki and Tanford reported that the pK values for several amino acids and simple substances in 6 M guanidinium chloride differed little from the corresponding values in low salt (Nozaki, Y., and C. Tanford. 1967. J. Am. Chem. Soc. 89:736-742). This puzzling and counter-intuitive result hinders attempts to understand and predict the proton uptake/release behavior of proteins in guanidinium chloride solutions, behavior which may determine whether the DeltaG(N-D) values obtained from guanidinium chloride-induced denaturation data can actually be interpreted as the Gibbs energy difference between the native and denatured states (Bolen, D. W., and M. Yang. 2000. Biochemistry. 39:15208-15216). We show in this work that the Nozaki-Tanford result can be traced back to the fact that glass-electrode pH meter readings in water/guanidinium chloride do not equal true pH values. We determine the correction factors required to convert pH meter readings in water/guanidinium chloride into true pH values and show that, when these corrections are applied, the effect of guanidinium chloride on the pK values of simple substances is found to be significant and similar to that of NaCl. The results reported here allow us to propose plausible guanidinium chloride concentration dependencies for the pK values of carboxylic acids in proteins and, on their basis, to reproduce qualitatively the proton uptake/release behavior for the native and denatured states of several proteins (ribonuclease A, alpha-chymotrypsin, staphylococcal nuclease) in guanidinium chloride solutions. Finally, the implications of the pH correction for the experimental characterization of protein folding energetics are briefly discussed.

Full Text

The Full Text of this article is available as a PDF (270.8 KB).

Selected References

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

  1. Becktel W. J., Schellman J. A. Protein stability curves. Biopolymers. 1987 Nov;26(11):1859–1877. doi: 10.1002/bip.360261104. [DOI] [PubMed] [Google Scholar]
  2. Bolen D. W., Yang M. Effects of guanidine hydrochloride on the proton inventory of proteins: implications on interpretations of protein stability. Biochemistry. 2000 Dec 12;39(49):15208–15216. doi: 10.1021/bi001071d. [DOI] [PubMed] [Google Scholar]
  3. Cortijo M., Llor J., Sanchez-Ruiz J. M. Thermodynamic constants for tautomerism, hydration, and ionization of vitamin B6 compounds in water/dioxane. J Biol Chem. 1988 Dec 5;263(34):17960–17969. [PubMed] [Google Scholar]
  4. Courtenay E. S., Capp M. W., Saecker R. M., Record M. T., Jr Thermodynamic analysis of interactions between denaturants and protein surface exposed on unfolding: interpretation of urea and guanidinium chloride m-values and their correlation with changes in accessible surface area (ASA) using preferential interaction coefficients and the local-bulk domain model. Proteins. 2000;Suppl 4:72–85. doi: 10.1002/1097-0134(2000)41:4+<72::aid-prot70>3.0.co;2-7. [DOI] [PubMed] [Google Scholar]
  5. Greene R. F., Jr, Pace C. N. Urea and guanidine hydrochloride denaturation of ribonuclease, lysozyme, alpha-chymotrypsin, and beta-lactoglobulin. J Biol Chem. 1974 Sep 10;249(17):5388–5393. [PubMed] [Google Scholar]
  6. Hagihara Y., Aimoto S., Fink A. L., Goto Y. Guanidine hydrochloride-induced folding of proteins. J Mol Biol. 1993 May 20;231(2):180–184. doi: 10.1006/jmbi.1993.1272. [DOI] [PubMed] [Google Scholar]
  7. Ibarra-Molero B., Loladze V. V., Makhatadze G. I., Sanchez-Ruiz J. M. Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability. Biochemistry. 1999 Jun 22;38(25):8138–8149. doi: 10.1021/bi9905819. [DOI] [PubMed] [Google Scholar]
  8. Ibarra-Molero B., Sanchez-Ruiz J. M. A model-independent, nonlinear extrapolation procedure for the characterization of protein folding energetics from solvent-denaturation data. Biochemistry. 1996 Nov 26;35(47):14689–14702. doi: 10.1021/bi961836a. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Mayr L. M., Schmid F. X. Stabilization of a protein by guanidinium chloride. Biochemistry. 1993 Aug 10;32(31):7994–7998. doi: 10.1021/bi00082a021. [DOI] [PubMed] [Google Scholar]
  11. Monera O. D., Kay C. M., Hodges R. S. Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions. Protein Sci. 1994 Nov;3(11):1984–1991. doi: 10.1002/pro.5560031110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Nozaki Y., Tanford C. Acid-base titrations in concentrated guanidine hydrochloride. Dissociation constants of the guamidinium ion and of some amino acids. J Am Chem Soc. 1967 Feb 15;89(4):736–742. doi: 10.1021/ja00980a002. [DOI] [PubMed] [Google Scholar]
  13. Nozaki Y., Tanford C. Proteins as random coils. II. Hydrogen ion titration curve of ribonuclease in 6 M guanidine hydrochloride. J Am Chem Soc. 1967 Feb 15;89(4):742–749. doi: 10.1021/ja00980a003. [DOI] [PubMed] [Google Scholar]
  14. Pace C. N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986;131:266–280. doi: 10.1016/0076-6879(86)31045-0. [DOI] [PubMed] [Google Scholar]
  15. Pace C. N., Laurents D. V., Thomson J. A. pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1. Biochemistry. 1990 Mar 13;29(10):2564–2572. doi: 10.1021/bi00462a019. [DOI] [PubMed] [Google Scholar]
  16. Santoro M. M., Bolen D. W. A test of the linear extrapolation of unfolding free energy changes over an extended denaturant concentration range. Biochemistry. 1992 May 26;31(20):4901–4907. doi: 10.1021/bi00135a022. [DOI] [PubMed] [Google Scholar]
  17. Santoro M. M., Bolen D. W. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry. 1988 Oct 18;27(21):8063–8068. doi: 10.1021/bi00421a014. [DOI] [PubMed] [Google Scholar]
  18. Whitten S. T., García-Moreno E B. pH dependence of stability of staphylococcal nuclease: evidence of substantial electrostatic interactions in the denatured state. Biochemistry. 2000 Nov 21;39(46):14292–14304. doi: 10.1021/bi001015c. [DOI] [PubMed] [Google Scholar]
  19. Yao M., Bolen D. W. How valid are denaturant-induced unfolding free energy measurements? Level of conformance to common assumptions over an extended range of ribonuclease A stability. Biochemistry. 1995 Mar 21;34(11):3771–3781. doi: 10.1021/bi00011a035. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES