Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1993 Aug;2(8):1291–1300. doi: 10.1002/pro.5560020812

Effect of pH and denaturants on the folding and stability of murine interleukin-6.

L D Ward 1, J G Zhang 1, G Checkley 1, B Preston 1, R J Simpson 1
PMCID: PMC2142433  PMID: 8401214

Abstract

The conformation and stability of a recombinant mouse interleukin-6 (mIL-6) has been investigated by analytical ultracentrifugation, fluorescence spectroscopy, urea-gradient gel electrophoresis, and near- and far-ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4.0, the tryptophan fluorescence of mIL-6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence quantum yield was not due to unfolding of the molecule because the conformation of mIL-6, as judged by both urea-gradient gel electrophoresis and CD spectroscopy, was stable over the pH range 2.0-10.0. Sedimentation equilibrium experiments indicated that mIL-6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicochemical studies. Quenching of tryptophan fluorescence (20%) also occurred in the presence of 6 M guanidine hydrochloride upon going from pH 7.4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp-36 and Trp-160, may be partially involved in the quenching of endogenous fluorescence. In this regard, similar results were obtained for a 17-residue synthetic peptide, peptide H1, which corresponds to an N-terminal region of mIL-6 (residues Val-27-Lys-43). The pH-dependent acid quenching of endogenous tryptophan fluorescence of peptide H1 was 30% in the random coil conformation and 60% in the presence of alpha-helix-promoting solvents. Replacement of His-33 with Ala-33 in peptide H1 alleviated a significant portion of the pH-dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His-33 with the indole ring of Trp-36 is a major determinant responsible for the quenching of the endogenous protein fluorescence of mIL-6.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Brakenhoff J. P., Hart M., Aarden L. A. Analysis of human IL-6 mutants expressed in Escherichia coli. Biologic activities are not affected by deletion of amino acids 1-28. J Immunol. 1989 Aug 15;143(4):1175–1182. [PubMed] [Google Scholar]
  2. Brakenhoff J. P., Hart M., De Groot E. R., Di Padova F., Aarden L. A. Structure-function analysis of human IL-6. Epitope mapping of neutralizing monoclonal antibodies with amino- and carboxyl-terminal deletion mutants. J Immunol. 1990 Jul 15;145(2):561–568. [PubMed] [Google Scholar]
  3. Brems D. N., Plaisted S. M., Havel H. A., Kauffman E. W., Stodola J. D., Eaton L. C., White R. D. Equilibrium denaturation of pituitary- and recombinant-derived bovine growth hormone. Biochemistry. 1985 Dec 17;24(26):7662–7668. doi: 10.1021/bi00347a025. [DOI] [PubMed] [Google Scholar]
  4. COWGILL R. W. Fluorescence and the structure of proteins. I. Effects of substituents on the fluorescence of indole and phenol compounds. Arch Biochem Biophys. 1963 Jan;100:36–44. doi: 10.1016/0003-9861(63)90031-6. [DOI] [PubMed] [Google Scholar]
  5. Creighton T. E. Kinetic study of protein unfolding and refolding using urea gradient electrophoresis. J Mol Biol. 1980 Feb 15;137(1):61–80. doi: 10.1016/0022-2836(80)90157-6. [DOI] [PubMed] [Google Scholar]
  6. Edelhoch H., Brand L., Wilchek M. Fluorescence studies with tryptophyl peptides. Biochemistry. 1967 Feb;6(2):547–559. doi: 10.1021/bi00854a024. [DOI] [PubMed] [Google Scholar]
  7. Hirano T., Akira S., Taga T., Kishimoto T. Biological and clinical aspects of interleukin 6. Immunol Today. 1990 Dec;11(12):443–449. doi: 10.1016/0167-5699(90)90173-7. [DOI] [PubMed] [Google Scholar]
  8. Hollecker M., Creighton T. E. Effect on protein stability of reversing the charge on amino groups. Biochim Biophys Acta. 1982 Mar 4;701(3):395–404. doi: 10.1016/0167-4838(82)90243-6. [DOI] [PubMed] [Google Scholar]
  9. Kishimoto T. The biology of interleukin-6. Blood. 1989 Jul;74(1):1–10. [PubMed] [Google Scholar]
  10. Loewenthal R., Sancho J., Fersht A. R. Fluorescence spectrum of barnase: contributions of three tryptophan residues and a histidine-related pH dependence. Biochemistry. 1991 Jul 9;30(27):6775–6779. doi: 10.1021/bi00241a021. [DOI] [PubMed] [Google Scholar]
  11. Mattice W. L., Riser J. M., Clark D. S. Conformational properties of the complexes formed by proteins and sodium dodecyl sulfate. Biochemistry. 1976 Sep 21;15(19):4264–4272. doi: 10.1021/bi00664a020. [DOI] [PubMed] [Google Scholar]
  12. Parry D. A., Minasian E., Leach S. J. Cytokine conformations: predictive studies. J Mol Recognit. 1991 Mar-Jun;4(2-3):63–75. doi: 10.1002/jmr.300040205. [DOI] [PubMed] [Google Scholar]
  13. Reid G. E., Simpson R. J. Automated solid-phase peptide synthesis: use of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate for coupling of tert-butyloxycarbonyl amino acids. Anal Biochem. 1992 Feb 1;200(2):301–309. doi: 10.1016/0003-2697(92)90470-r. [DOI] [PubMed] [Google Scholar]
  14. Shanafelt A. B., Miyajima A., Kitamura T., Kastelein R. A. The amino-terminal helix of GM-CSF and IL-5 governs high affinity binding to their receptors. EMBO J. 1991 Dec;10(13):4105–4112. doi: 10.1002/j.1460-2075.1991.tb04987.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Shinitzky M., Goldman R. Fluorometric detection of histiine-tryptophan complexes in peptides and proteins. Eur J Biochem. 1967 Dec;3(2):139–144. doi: 10.1111/j.1432-1033.1967.tb19508.x. [DOI] [PubMed] [Google Scholar]
  16. Simpson R. J., Moritz R. L., Rubira M. R., Van Snick J. Murine hybridoma/plasmacytoma growth factor. Complete amino-acid sequence and relation to human interleukin-6. Eur J Biochem. 1988 Sep 1;176(1):187–197. doi: 10.1111/j.1432-1033.1988.tb14267.x. [DOI] [PubMed] [Google Scholar]
  17. Strickland E. H. Aromatic contributions to circular dichroism spectra of proteins. CRC Crit Rev Biochem. 1974 Jan;2(1):113–175. doi: 10.3109/10409237409105445. [DOI] [PubMed] [Google Scholar]
  18. Van Snick J., Cayphas S., Szikora J. P., Renauld J. C., Van Roost E., Boon T., Simpson R. J. cDNA cloning of murine interleukin-HP1: homology with human interleukin 6. Eur J Immunol. 1988 Feb;18(2):193–197. doi: 10.1002/eji.1830180202. [DOI] [PubMed] [Google Scholar]
  19. Van Snick J. Interleukin-6: an overview. Annu Rev Immunol. 1990;8:253–278. doi: 10.1146/annurev.iy.08.040190.001345. [DOI] [PubMed] [Google Scholar]
  20. WHITE A. Effect of pH on fluorescence of tryosine, tryptophan and related compounds. Biochem J. 1959 Feb;71(2):217–220. doi: 10.1042/bj0710217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. YPHANTIS D. A. EQUILIBRIUM ULTRACENTRIFUGATION OF DILUTE SOLUTIONS. Biochemistry. 1964 Mar;3:297–317. doi: 10.1021/bi00891a003. [DOI] [PubMed] [Google Scholar]
  22. Yang J. T., Wu C. S., Martinez H. M. Calculation of protein conformation from circular dichroism. Methods Enzymol. 1986;130:208–269. doi: 10.1016/0076-6879(86)30013-2. [DOI] [PubMed] [Google Scholar]
  23. Zhang J. G., Moritz R. L., Reid G. E., Ward L. D., Simpson R. J. Purification and characterization of a recombinant murine interleukin-6. Isolation of N- and C-terminally truncated forms. Eur J Biochem. 1992 Aug 1;207(3):903–913. doi: 10.1111/j.1432-1033.1992.tb17123.x. [DOI] [PubMed] [Google Scholar]
  24. Zhong L., Johnson W. C., Jr Environment affects amino acid preference for secondary structure. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4462–4465. doi: 10.1073/pnas.89.10.4462. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES