Advertisement

Journal of Protein Chemistry

, Volume 12, Issue 5, pp 525–531 | Cite as

Cysteine 17 of recombinant human granulocyte-colony stimulating factor is partially solvent-exposed

  • Tsutomu Arakawa
  • Steven J. Prestrelski
  • Linda O. Narhi
  • Thomas C. Boone
  • William C. Kenney
Article

Abstract

Oh-edaet al. have shown instability of granulocyte-colony stimulating factor (G-CSF) upon storage abovepH 7.0 [J. Biol. Chem. (1990)265, 11,432–11,435]. To clarify the mechanism of this instability, the accessibility of a free cysteinyl residue at position 17 for disulfide exchange reaction was examined using a sulfhydryl reagent. The results show that the cysteine is partially solvent-exposed in both glycosylated and nonglycosylated forms, suggesting that the exposure of the cysteine plays a critical role in the instability of the protein. This is supported by the facts that at lowpH where the cysteine is protonated, both proteins have much greater stability and that a Cys17 → Ser analog is extremely stable at neutralpH and 37°C. It was observed that the rate of sulfhydryl titration is slower for the glycosylated form than for the nonglycosylated form, suggesting that the cysteine residue is less solvent-exposed for the former protein or that the pK a is somewhat more basic. In either case, the carbohydrate appears to affect the reactivity of the sulfhydryl group through steric hindrance or alteration in local conformation. Both the glycosylated and nonglycosylated proteins showed essentially identical conformation as determined by circular dichroism, fluorescence, and infrared spectroscopy. Unfolding of these two proteins, induced either by guanidine hydrochloride or bypH, showed an identical course, indicating comparable conformational stability. Contribution of conformational changes to the observed instability at higherpH is unlikely, since little difference in fluorescence spectrum occurs betweenpH 6.0 and 8.0. Based on these observations, G-CSF, whether glycosylated or not, should not be stored above pH 7.0 in solution. On the other hand, G-CSF is extremely stable in acidic solution as expected from the proposed mechanism.

Key words

Circular dichroism FTIR disulfide exchange G-CSF sulfhydryl titration 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Byler, D. M., and Susi, H. (1986).Biopolymers 25, 469–487.Google Scholar
  2. Habeeb, A. F. S. A. (1972).Methods Enzymol. 25, 457–465.Google Scholar
  3. Kauppinen, J. R., Moffat, D. J., Mantsch, H. H., and Cameron, D. C. (1981).Appl. Spectrosc. 25, 271–277.Google Scholar
  4. Kitagawa, S., Yuo, A., Souza, L. M., Saito, M., Miura, Y., and Takaku, F. (1987).Biochem. Biophys. Res. Commun. 144, 1143–1146.Google Scholar
  5. Kolvenbach, C. G., Langley, K. E., Strickland, T. W., Kenney W. C., and Arakawa, T. (1991).J. Biochem. Biophys. Meth. 23, 295–300.Google Scholar
  6. Kubota, N., Orita, T., Hattori, K., Oh-eda, M., Ochi, N., and Yamazaki, T. (1990).J. Biochem. 107, 486–492.Google Scholar
  7. Lu, H.-S., Boone, T. C., Souza, L. M., and Lai, P.-H. (1989).Arch. Biochem. Biophys. 268, 81–92.Google Scholar
  8. Metcalf, D. (1984).The Haemopoietic Colony Stimulating Factors. Elsevier, Amsterdam.Google Scholar
  9. Metcalf, D. (1985).Science 229, 16–22.Google Scholar
  10. Narhi, L. O., Kenney, W. C., and Arakawa, T. (1991a)J. Protein Chem. 10, 359–367.Google Scholar
  11. Narhi, L. O., Arakawa, T., Aoki, K. H., Elmore, R., Rohde, M. F., Boone, T., and Strickland, T. W. (1991b).J. Biol. Chem. 266, 23,022–23,026.Google Scholar
  12. Oh-eda, M., Hasegawa, M., Hattari, K., Kuboniwa, H., Kojima, T., Orita, T., Tomonou, K., Yamazaki, T., and Ochi, N. (1990).J. Biol. Chem. 265, 11,432–11,435.Google Scholar
  13. Pace, N. (1970).Trends Biochem. Sci. 15, 14–17.Google Scholar
  14. Prestrelski, S. J., Byler, D. M., and Liebman, M. N. (1991).Biochemistry 30, 133–143.Google Scholar
  15. Prestrelski, S. J., Byler, D. M., and Thompson, M. P. (1991).Int. J. Peptide Prot. Res. 37, 508–512.Google Scholar
  16. Privalov, P. L., and Khechinashvili, N. N. (1974).J. Mol. Biol. 86, 665–684.Google Scholar
  17. Purcell, J. M., and Susi, H. (1984).J. Biochem. Biophys. Meth. 9, 193–199.Google Scholar
  18. Souza, L. M., Boone, T. C., Gabrilove, J., Lai, P.-H., Zsebo, K. M., Murdock, D. C., Chazin, V. R., Bruszewski, J., Lu, H., Chen, K. K., Barendt, J., Platzer, E., Moore, M. A. S., Mertelsmann, R., and Welte, K. (1986).Science 232, 61–65.Google Scholar
  19. Surewicz, W. R., Leddy, J. J., and Mantsch, H. H. (1990).Biochemistry 29, 8106–8111.Google Scholar
  20. Surewicz, W. K., and Mantsch, H. H. (1988).Biochim. Biophys. Acta 952, 115–130.Google Scholar
  21. Susi, H., and Byler, D. M. (1983).Biochem. Biophys, Res. Commun. 115, 391–397.Google Scholar
  22. You, A., Kitagawa, S., Okabe, T., Urabe, A., Komatsu, Y., Itoh, S., and Takaku, F. (1987).Blood 70, 404–411.Google Scholar
  23. Zsebo, K. M., Cohen, A. M., Murdock, D. C., Boone, T. C., Inoue, H., Chazin, V. R., Hines, D., and Souza, L. M. (1986).Immunobiology 172, 175–184.Google Scholar

Copyright information

© Plenum Publishing Corporation 1993

Authors and Affiliations

  • Tsutomu Arakawa
    • 1
  • Steven J. Prestrelski
    • 1
  • Linda O. Narhi
    • 1
  • Thomas C. Boone
    • 1
  • William C. Kenney
    • 1
  1. 1.Amgen CenterAmgen, Inc.Thousand Oaks

Personalised recommendations