Skip to main content
SpringerLink
Log in

Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme

  • Published:
Journal of Protein Chemistry Aims and scope Submit manuscript

Abstract

A three-disulfide form of hen egg white lysozyme with Cys6 and Cys127 blocked by carboxymethyl groups was prepared, purified, and characterized for eventual use in protein folding experiments. Trypsin digestion followed by proline-specific endopeptidase digestion facilitated the unambiguous assignment of the disulfide bond pairings and the modified residues in this derivative. 3SS-lysozyme demonstrated nearly full enzymatic activity at itspH optimum,pH 5.5. The 3SS-lysozyme derivative and unmodified lysozyme were shown to be identical by CD spectroscopy atpH 3.6. Immunochemical binding assays demonstrated that the conformation of lysozyme was perturbed predominantly only locally by breaking and blocking the disulfide bond between Cys6 and Cys127. Both 3SS-lysozyme and unmodified lysozyme exhibited reversible thermally induced transitions atpH 2.0 but theT m of 3SS-lysozyme, 18.9°C, was found to be 34° lower than that of native lysozyme under the same conditions. The conformational chemical potential of the denatured form of unmodified lysozyme was determined from the transition curves to be approximately 6.7 kcal/mol higher than that of the denatured form of 3SS-lysozyme, atpH 2.0 and 35°C, if the conformational chemical potential for the folded forms ofboth 3SS-lysozyme and unmodified lysozyme is arbitrarily assumed to be 0.0 kcal/mol. A calculation of the increase in the theoretical loop entropy of denatured 3SS-lysozyme resulting from the cleavage of the Cys6-Cys127 disulfide bond, however, yielded a value of only 5.4 kcal/mol for the difference in conformational chemical potential. This suggests that, in addition to the entropic component, there is also an enthalpic contribution to the difference in the conformational chemical potential corresponding to approximately 1.3 kcal/mol. Thus, it is concluded that the reduction and blocking of the disulfide bond between Cys6 and Cys127 destabilizes 3SS-lysozyme relative to unmodified lysozyme predominantly by stabilizing the denatured conformation by increasing its chain entropy.

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

Similar content being viewed by others

References

  • Acharya, A. S., and Taniuchi, H. (1978).Biochemistry 17, 3064–3070.

    CAS  PubMed  Google Scholar 

  • Acharya, A. S., and Taniuchi, H. (1980).Int. J. Peptide Protein Res. 15, 503–509.

    CAS  Google Scholar 

  • Adler, M., and Scheraga, H. A. (1988).Biochemistry 27, 2471–2480.

    CAS  PubMed  Google Scholar 

  • Amit, A. G., Mariuzza, R. A., Phillips, S. E. V., and Poljak, R. J. (1986).Science 233, 747–753.

    CAS  PubMed  Google Scholar 

  • Anfinsen, C. B., and Scheraga, H. A. (1975).Adv. Protein Chem. 29, 205–300.

    CAS  PubMed  Google Scholar 

  • Atassi, M. Z., Habeeb, A. F. S. A., and Ando, K. (1973).Biochim. Biophys. Acta 303, 203–209.

    CAS  PubMed  Google Scholar 

  • Blake, C. C. F., Mair, G. A., North, A. C. T., Phillips, D. C., and Sarma, V. R. (1967).Proc. R. Soc. London 167, 365–377.

    CAS  Google Scholar 

  • Canfied, R. E. (1963).J. Biol. Chem. 238, 2691–2697.

    Google Scholar 

  • Chang, K. Y., and Carr, C. W. (1971).Biochim. Biophys. Acta 229, 496–503.

    CAS  PubMed  Google Scholar 

  • Chard, T. (1980).Meth. in Enzymol. 70, 280–291.

    CAS  Google Scholar 

  • Chavez, L. G., Jr., and Scheraga, H. A. (1980).Biochemistry 19, 1005–1012.

    CAS  PubMed  Google Scholar 

  • Colman, P. M., Laver, W. G., Varghese, J. N., Baker, A. T., Tulloch, P. A., Air, G. M., and Webster, R. G. (1987).Nature 326, 358–363.

    CAS  PubMed  Google Scholar 

  • David, G. S. (1972).Biochem. Biophys. Res. Commun. 48, 464–471.

    CAS  PubMed  Google Scholar 

  • David, G. S., and Reisfeld, R. A. (1974).Biochemistry 13, 1014–1021.

    CAS  PubMed  Google Scholar 

  • Davies, D. R., Sheriff, S., Padlan, E. A., Silverton, E. W., Cohen, G. H., and Smith-Gill, S. J. (1989). InThe Immune Response to Structurally Defined Proteins: The Lysozyme Model (Smith-Gill, S. J., and Sercarz, E. E., eds.), Adenine Press, Guilderland, New York, pp. 125–132.

    Google Scholar 

  • Davies, R. C., Neuberger, A., and Wilson, B. M. (1969).Biochim. Biophys. Acta 178, 294–305.

    CAS  PubMed  Google Scholar 

  • Denton, M. E., and Scheraga, H. A. (1989). InThe Immune Response to Structurally Defined Proteins: The Lysozyme Model (Smith-Gill, S. J., and Sercarz, E. E., eds.), Adenine Press, Guilderland, New York, pp. 39–40.

    Google Scholar 

  • Dobson, C. M., and Evans, P. A. (1984).Biochemistry 23, 4267–4270.

    CAS  Google Scholar 

  • Dubois, T., Guillard, R., Prieels, J.-P., and Perraudin, J.-P. (1982).Biochemistry 21, 6516–6523.

    CAS  PubMed  Google Scholar 

  • Ellman, G. L. (1959).Arch. Biochem. Biophys. 82, 70–77.

    CAS  PubMed  Google Scholar 

  • Farr, R. S. (1958).J. Infect. Dis. 103, 239–262.

    CAS  PubMed  Google Scholar 

  • Flory, P. J. (1956).J. Am. Chem. Soc. 78, 5222–5235.

    CAS  Google Scholar 

  • Friguet, B., Chaffotte, A. F., Djavadi-Ohaniance, L., and Goldberg, M. E. (1985).J. Immunol. Methods 77, 305–319.

    CAS  PubMed  Google Scholar 

  • Furie, B., Schechter, A. N., Sachs, D. H., and Anfinsen, C. B. (1975).J. Mol. Biol. 92, 497–506.

    CAS  PubMed  Google Scholar 

  • Haeffner-Gormley, L., Parente, L., and Wetlaufer, D. B. (1985).Int. J. Peptide Protein Res. 26, 83–91.

    CAS  Google Scholar 

  • Haeffner-Gormley, L., Poludniak, N. H., and Wetlaufer, D. B. (1981).J. Chromatogr. 214, 185–196.

    CAS  Google Scholar 

  • Hayashi, K., Shimoda, T., Imoto, T., and Funatsu, M. (1968).J. Biochem. 64, 365–370.

    CAS  PubMed  Google Scholar 

  • Hubbard, A. L., and Cohn, Z. A. (1972).J. Cell Biol. 55, 390–405.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Imoto, T., Johnson, L. N., North, A. C. T., Phillips, D. C., and Rupley, J. A. (1972).The Enzymes, Vol. 7, Academic Press, New York, pp. 665–868.

    Google Scholar 

  • Johnson, R. E., Adams, P., and Rupley, J. A. (1978).Biochemistry 17, 1479–1485.

    CAS  PubMed  Google Scholar 

  • Kauzmann, W. (1959). InSulfur in Proteins (Benesch, R., Benesch, R. E., Boyer, P. D., Klotz, I. M., Middlebrook, W. R., Szent-Györgyi, A. G., and Schwarz, D. R., eds.), Academic Press, New York, pp. 93–108.

    Google Scholar 

  • Konishi, Y., Ooi, T., and Scheraga, H. A. (1982).Biochemistry 21, 4741–4748.

    CAS  PubMed  Google Scholar 

  • Krohn, K. A., and Welch, M. J. (1974).Int. J. Appl. Radiat. Isot. 25, 315–323.

    CAS  PubMed  Google Scholar 

  • Lavoie, T. B., Kam-Morgan, L. N. W., Hartman, A. B., Mallett, C. P., Sherriff, S., Saroff, D. A., Mainhart, C. R., Hamel, P. A., Kirsch, J. F., Wilson, A. C., and Smith-Gill, S. J. (1989). InThe Immune Response to Structurally Defined Proteins: The Lysozyme Model (Smith-Gill, S. J., and Sercarz, E. E., eds.), Adenine Press, Guilderland, New York, pp. 151–168.

    Google Scholar 

  • Lin, S. H., Konishi, Y., Denton, M. E., and Scheraga, H. A. (1984).Biochemistry 23, 5504–5512.

    CAS  PubMed  Google Scholar 

  • Marchalonis, J. J. (1969).Biochem. J. 113, 299–305.

    CAS  PubMed  PubMed Central  Google Scholar 

  • McWherter, C. A., Thannhauser, T. W., Fredrickson, R. A., Zagotta, M. T., and Scheraga, H. A. (1984).Anal. Biochem. 141, 523–537.

    CAS  PubMed  Google Scholar 

  • Miller, A., Ch'ng, L.-K., Benjamin, C., Sercarz, E., Brodeur, P., and Riblet, R. (1983).Ann. N.Y. Acad. Sci. 418, 140–150.

    CAS  PubMed  Google Scholar 

  • Minden, P., and Farr, R. S. (1978). InHandbook of Experimental Immunology, 3rd ed. (Weir, D. M., ed.), Blackwell Scientific Publications, Oxford, England, pp. 13.1–13.22.

    Google Scholar 

  • O'Reilly, J. M., and Karasz, F. E. (1970).Biopolym. 9, 1429–1435.

    CAS  Google Scholar 

  • Padlan, E. A., Silverton, E. W., Sheriff, S., Cohen, G. H., Smith-Gill, S. J., and Davies, D. R. (1989).Proc. Natl. Acad. Sci. U.S.A. 86, 5938–5942.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Parsons, S. M., and Raftery, M. A. (1972).Biochemistry 11, 1623–1629.

    CAS  PubMed  Google Scholar 

  • Pfeil, W., and Privalov, P. L. (1976).Biophys. Chem. 4, 33–40.

    CAS  PubMed  Google Scholar 

  • Phillips, D. C. (1974). InLysozyme (Osserman, E. F., Canfield, R. E., and Beychok, S., eds.), Academic Press, New York, pp. 9–30.

    Google Scholar 

  • Poland, D. C., and Scheraga, H. A. (1965).Biopolymers 3, 379–399.

    CAS  Google Scholar 

  • Privalov, P. L., and Khechinashvili, N. N. (1974).J. Mol. Biol. 86, 665–684.

    CAS  PubMed  Google Scholar 

  • Radford, S. E., Woolfson, D. N., Martin, S. R., Lowe, G., and Dobson, C. M. (1991).Biochem. J. 273, 211–218.

    CAS  PubMed Central  Google Scholar 

  • Richardson, J. S. (1981).Adv. Protein Chem. 34, 167–339.

    CAS  PubMed  Google Scholar 

  • Rodbard, D., Bridson, W., and Rayford, P. L. (1969).J. Lab. Clin. Med. 74, 770–781.

    CAS  PubMed  Google Scholar 

  • Sachs, D. H., Schechter, A. N., Eastlake, A., and Anfinsen, C. B. (1972).Proc. Natl. Acad. Sci. U.S.A. 69, 3790–3794.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Saxena, V. P., and Wetlaufer, D. B. (1970).Biochemistry 9, 5015–5023.

    CAS  PubMed  Google Scholar 

  • Schellman, J. A. (1955).C.R. Trav. Lab. Carlsberg Ser. Chim. 29, 230–259.

    CAS  Google Scholar 

  • Schwarz, F. P. (1989).Thermochim. Acta 147, 71–91.

    CAS  Google Scholar 

  • Sheriff, S., Silverton, E. W., Padlan, E. A., Cohen, G. H., Smith-Gill, S. J., Finzel, B. C., and Davies, D. R. (1987).Proc. Natl. Acad. Sci. U.S.A. 84, 8075–8079.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Shugar, D. (1952).Biochim. Biophys. Acta 8, 302–309.

    CAS  PubMed  Google Scholar 

  • Smith-Gill, S. J., Laboie, T. B., and Mainhart, C. R. (1984).J. Immunol. 133, 384–393.

    CAS  PubMed  Google Scholar 

  • Smith-Gill, S. J., Wilson, A. C., Potter, M., Prager, E. M., Feldmann, R. J., and Mainhart, C. R. (1982).J. Immunol. 128, 314–322.

    CAS  PubMed  Google Scholar 

  • Sophianopoulos, A. J., and Weiss, B. J. (1964).Biochemistry 3, 1920–1928.

    CAS  PubMed  Google Scholar 

  • Thannhauser, T. W., Konishi, Y., and Scheraga, H. A. (1984).Anal. Biochem. 138, 181–188.

    CAS  PubMed  Google Scholar 

  • Thannhauser, T. W., McWherter, C. A., and Scheraga, H. A. (1985).Anal. Biochem. 149, 322–330.

    CAS  PubMed  Google Scholar 

  • Thorell, J. I., and Johansson, B. G. (1971).Biochim. Biophys. Acta 251, 363–369.

    CAS  PubMed  Google Scholar 

  • Thornton, J. M. (1981).J. Mol. Biol. 151, 261–287.

    CAS  PubMed  Google Scholar 

  • Ueda, T., Yamada, H., Hirata, M., and Imoto, T. (1985).Biochemistry 24, 6316–6322.

    CAS  PubMed  Google Scholar 

  • Walsh, K. A. (1970).Methods Enzymol. 19, 41–63.

    Google Scholar 

  • Wang, M. C., and Uhlenbeck, G. E. (1945).Rev. Mod. Phys. 17, 323–342.

    Google Scholar 

  • Yalow, R. S., and Berson, S. A. (1970). InStatistics in Endocrinology (McArthur, J. W., and Colton, T., eds.), MIT Press, Cambridge, Massuchasetts, pp. 327–344.

    Google Scholar 

  • Yamada, H., Imoto, T., Fujita, K., Okazaki, K., and Motomura, M. (1981).Biochemistry 20, 4836–4842.

    CAS  PubMed  Google Scholar 

  • Yamada, H., Kuroki, R., Hirata, M., and Imoto, T. (1983).Biochemistry 22, 4551–4556.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Baker Laboratory of Chemistry, Cornell University, 14853-1301, Ithaca, New York

    Mary E. Denton & Harold A. Scheraga

Authors
  1. Mary E. Denton
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Harold A. Scheraga
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Cornell Biotechnology Army Research Office Predoctoral Fellow, 1986–1989.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Denton, M.E., Scheraga, H.A. Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme. J Protein Chem 10, 213–232 (1991). https://doi.org/10.1007/BF01024786

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01024786

Key words

Search

Navigation