Skip to main content
SpringerLink
Log in

Evidence for a Molten Globule State in Cicer α-Galactosidase Induced by pH, Temperature, and Guanidine Hydrochloride

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Physiologically as well as industrially, α-galactosidases are very important enzymes, but very little is known about the stability and folding aspect of enzyme. In the present study, we have investigated the temperature, pH, and guanidine hydrochloride (GuHCl) induced unfolding of Cicer α-galactosidase using circular dichroism and fluorescence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the presence of both α and β structures in Cicer α-galactosidase and showed that its secondary structure belongs to α + β class of proteins with 31 % α-helicity. For Cicer α-galactosidase the emission maximum was found to be 345 nm which suggests that tryptophan residues are less exposed to solvent. However, at pH 2.0, protein showed blue-shift. This state of protein lacked activity but it retained significant secondary structure. Enhanced binding of ANS at pH 2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded state of Cicer α-galactosidase showed a red-shift of 15 nm with a concomitant decrease in the fluorescence intensity. The enzyme maintained its native structure and full activity up to 40 °C; however, above this temperature, denaturation was observed.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

GuHCl:

Guanidine hydrochloride

CD:

Circular dichroism

MG:

Molten globule

RFOs:

Raffinose family oligosaccharides

pNPGal:

p-Nitrophenylα-d-galactopyranoside

ANS:

8-Anilino-1-napthalenesulfonic acid

References

  1. Haq, S. K., Rasheedi, S., & Khan, R. H. (2002). European Journal of Biochemistry, 269, 47–52.

    Article  CAS  Google Scholar 

  2. Ptitsyn, O. B., & Uversky, V. N. (1994). FEBS Letters, 341, 15–18.

    Article  CAS  Google Scholar 

  3. Ptitsyn, O. B. (1995). Advances in Protein Chemistry, 47, 83–229.

    Article  CAS  Google Scholar 

  4. Naumoff, D. G. (2004). Molecular Biology, 38, 388–399.

    Article  CAS  Google Scholar 

  5. Gitzelmann, R., & Auricchio, S. (1965). Pediatrics, 36, 231–235.

    CAS  Google Scholar 

  6. Steggerda, F. R. (1968). Annals of the New York Academy of Sciences, 150, 57–66.

    Article  CAS  Google Scholar 

  7. Kobayashi, H., & Suzuki, H. (1972). Journal of Fermentation Technology, 50, 625–632.

    CAS  Google Scholar 

  8. Ratto, M., Siika-aho, M., Buchert, J., Valkeajarvi, A., & Viikari, L. (1993). Applied Microbiology and Biotechnology, 40, 449–454.

    Article  CAS  Google Scholar 

  9. Eng, C. M., Guffon, N., Wilcox, W. R., Germain, D. P., Lee, P., Waldek, S., et al. (2001). The New England Journal of Medicine, 345, 9–16.

    Article  CAS  Google Scholar 

  10. Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., et al. (2007). Nature, 25, 454–463.

    Article  CAS  Google Scholar 

  11. Singh, N., & Kayastha, A. M. (2012). Journal of Agricultural and Food Chemistry, 60, 3253–3259.

    Article  CAS  Google Scholar 

  12. Bulpin, P. V., Gidley, M. J., Jeffcoat, R., & Underwood, D. J. (1990). Carbohydrate Polymers, 12, 155–168.

    Article  CAS  Google Scholar 

  13. Eneyskaya, E. V., Golubev, A. M., Kachurin, A. M., Savel’ev, A. N., & Neustroev, K. N. (1998). Carbohydrate Research, 303, 83–91.

    Google Scholar 

  14. Singh, N., & Kayastha, A. M. (2012). Carbohydrate Research, 358, 61–66.

    Article  CAS  Google Scholar 

  15. Malhotra, O. P., & Dey, P. M. (1967). The Biochemical Journal, 103, 508–513.

    CAS  Google Scholar 

  16. Bradford, M. M. (1976). Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  17. Balasubramanian, D., & Kumar, C. (1976). Applied Spectroscopy Reviews, 11, 223–286.

    Article  CAS  Google Scholar 

  18. Perez-Iratxeta, C., & Andrade-Navarro, M. A. (2008). BMC Structures O Biologicos, 8, 25.

    Article  Google Scholar 

  19. Semisotnov, G. V., Rodionova, N. A., Razgulyaev, O. I., Uverskii, V. N., Gripas, F., & Gilmanshin, R. I. (1991). Biopolymer, 31, 119–128.

    Article  CAS  Google Scholar 

  20. Khurana, R., & Udgaonkar, J. B. (1994). Biochemistry, 33, 106–115.

    Article  CAS  Google Scholar 

  21. Fujimoto, Z., Kaneko, S., Momma, M., Kobayashi, H., & Mizuno, H. (2003). The Journal of Biological Chemistry, 278, 20313–20318.

    Article  CAS  Google Scholar 

  22. Golubev, A. M., Nagem, R. A. P., Neto, J. R. B., Neustroev, K. N., Eneyskaya, E. V., Kulminskaya, A. A., et al. (2004). Journal of Molecular Biology, 339, 413–422.

    Article  CAS  Google Scholar 

  23. Guce, A. I., Clark, N. E., Salgado, E. N., Ivanen, D. R., Kulminskaya, A. A., & Brummer, H. (2010). The Journal of Biological Chemistry, 285, 3625–3632.

    Article  CAS  Google Scholar 

  24. Pawar, S. A., & Deshpande, V. V. (2000). European Journal of Biochemistry, 29, 6331–6338.

    Article  Google Scholar 

  25. Tripathi, P., Hofmann, H., Kayastha, A. M., & Hofmann, R. U. (2008). Biophysical Chemistry, 137, 95–99.

    Article  CAS  Google Scholar 

  26. Tomar, R., Dubey, V. K., & Jagannadham, M. V. (2009). The Protein Journal, 28, 213–223.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

One of us (N.S.) would like to thank the University Grants Commission (UGC), New Delhi, India, for financial support in the form of junior and senior research fellowships.

Author information

Authors and Affiliations

  1. School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi, 221005, India

    Neelesh Singh & Arvind M. Kayastha

  2. Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India

    Reetesh Kumar & M. V. Jagannadham

Authors
  1. Neelesh Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Reetesh Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. V. Jagannadham
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Arvind M. Kayastha
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Arvind M. Kayastha.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Singh, N., Kumar, R., Jagannadham, M.V. et al. Evidence for a Molten Globule State in Cicer α-Galactosidase Induced by pH, Temperature, and Guanidine Hydrochloride. Appl Biochem Biotechnol 169, 2315–2325 (2013). https://doi.org/10.1007/s12010-013-0163-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-013-0163-9

Keywords

Search

Navigation