Skip to main content
SpringerLink
Log in

Spectroscopic and differential scanning calorimetric studies on the unfolding of Trichosanthes dioica seed lectin. Similar modes of thermal and chemical denaturation

  • Published:
Glycoconjugate Journal Aims and scope Submit manuscript

Abstract

Physico-chemical and unfolding studies have been carried out on Trichosanthes dioica seed lectin (TDSL). The lectin exhibited maximum activity between pH 7.0 and 10.0, which decreased steeply at lower pH. The hemagglutination activity of TDSL was unaffected in the temperature range 4–50°C, but decreased rapidly at higher temperatures. Differential scanning calorimetric studies indicate that thermal unfolding of TDSL is an irreversible process, which could be described by a three-state model. The calorimetric scan recorded at pH 7.0 consists of two transitions, occurring at around 338.6 K, and 342.8 K. In the presence of carbohydrate ligands both these transitions shifted to higher temperatures, suggesting that ligand binding stabilizes the native conformation of the protein. The unfolding temperature was highest at pH 5.0 indicating that TDSL is more stable at acidic pH. Gdn.HCl induced unfolding, monitored by following changes in the intrinsic fluorescence properties of the protein, was also observed to be a three-state process involving an intermediate. CD spectroscopy indicates that the secondary and tertiary structures of TDSL are rather similar at different pH values, indicating that the lectin structure remains essentially unchanged over a wide range of pH.

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

Similar content being viewed by others

Abbreviations

TDSL:

Trichosanthes dioica seed lectin

CD:

Circular dichroism

ΔC p :

Change in excess heat capacity

DSC:

Differential scanning calorimetry

GalNAc:

N-acetyl-D-galactosamine

Gdn.HCl:

Guanidine hydrochloride

ΔH c :

Change in calorimetric enthalpy

ΔH v :

Change in van’t Hoff enthalpy

MeαGal:

Methyl-α-D-galactopyranoside

MeβGal:

Methyl-β-D-galactopyranoside

T m :

Transition temperature

References

  1. Sharon, N., Lis, H.: Lectins, p. 454pp. Kluwer Academic, Dordrecht, The Netherlands (2003)

    Google Scholar 

  2. Lis, H., Sharon, N.: Lectins: Carbohydrate-specific proteins that mediate cellular recognition. Chem. Rev. 98, 637 (1998). doi:10.1021/cr940413g

    Article  CAS  PubMed  Google Scholar 

  3. Van Damme, E.J.M., Peumans, W.J., Barre, A., Rougé, P.: Plant lectins: A composite of several distinct families of structurally and evolutionarily related proteins with diverse biological roles. Crit. Rev. Plant Sci. 17, 575–692 (1998). doi:10.1016/S0735-2689(98)00365-7

    Article  Google Scholar 

  4. Sharon, N., Lis, H.: How proteins bind carbohydrates: Lessons from legume lectins. J. Agric. Food Chem. 50, 6586–6591 (2002). doi:10.1021/jf020190s

    Article  CAS  PubMed  Google Scholar 

  5. Sinha, S., Gupta, G., Vijayan, M., Surolia, A.: Subunit assembly of plant lectins. Curr. Opin. Struct. Biol. 17, 498–505 (2007). doi:10.1016/j.sbi.2007.06.007

    Article  CAS  PubMed  Google Scholar 

  6. Loris, L., Hamelryck, T., Bouckaert, J., Wyns, L.: Legume lectin structure. Biochim. Biophys. Acta 1383, 9–36 (1998)

    CAS  PubMed  Google Scholar 

  7. Brinda, K.V., Surolia, A., Vishveshwara, S.: Insights into the quaternary association of proteins through structure graphs: A case study of lectins. Biochem. J. 391, 1–15 (2005). doi:10.1042/BJ20050434

    Article  CAS  PubMed  Google Scholar 

  8. Srinivas, V.R., Reddy, G.B., Ahmad, N., Swaminathan, C.P., Mitra, N., Surolia, A.: Legume lectin family, the ‘natural mutants of the quaternary state’, provide insights into the relationship between protein stability and oligomerization. Biochim. Biophys. Acta 1527, 102–111 (2001)

    CAS  PubMed  Google Scholar 

  9. Reddy, G.B., Bharadwaj, S., Surolia, A.: Thermal stability and mode of oligomerization of the tetrameric peanut agglutinin: A differential scanning calorimetry study. Biochemistry 38, 4464–4470 (1999). doi:10.1021/bi982828s

    Article  CAS  PubMed  Google Scholar 

  10. Srinivas, V.R., Singha, N.C., Schwarz, F.P., Surolia, A.: Differential scanning calorimetric studies of the glycoprotein, winged bean acidic lectin, isolated from the seeds of Psophocarpus tetragonolobus. FEBS Lett. 425, 57–60 (1998). doi:10.1016/S0014-5793(98)00197-5

    Article  CAS  PubMed  Google Scholar 

  11. Schwarz, F.P., Puri, K., Surolia, A.: Thermodynamics of the binding of galactopyranoside derivatives to the basic lectin from winged bean (Psophocarpus tetragonolobus). J. Biol. Chem. 266, 24344–24350 (1991)

    CAS  PubMed  Google Scholar 

  12. Schwarz, F.P., Puri, K., Bhat, R.G., Surolia, A.: Thermodynamics of monosaccharide binding to concanavalin A, pea (Pisum sativum) lectin and lentil (Lens culinaris) lectin. J. Biol. Chem. 268, 7668–7677 (1993)

    CAS  PubMed  Google Scholar 

  13. Komath, S.S., Kavitha, M., Swamy, M.J.: Beyond carbohydrate binding: New directions in plant lectin research. Org. Biomol. Chem. 4, 973–988 (2006). doi:10.1039/b515446d

    Article  CAS  PubMed  Google Scholar 

  14. Sultan, N.A.M., Swamy, M.J.: Energetics of carbohydrate binding to Momordica charantia lectin: An isothermal titration calorimetric study. Arch. Biochem. Biophys. 437, 115–125 (2005). doi:10.1016/j.abb.2005.03.005

    Article  CAS  PubMed  Google Scholar 

  15. Kenoth, R., Komath, S.S., Swamy, M.J.: Physicochemical and saccharide binding studies on the galactose-specific seed lectin from Trichosanthes cucumerina. Arch. Biochem. Biophys. 413, 131–138 (2003). doi:10.1016/S0003-9861(03)00094-8

    Article  CAS  PubMed  Google Scholar 

  16. Kenoth, R., Reddy, D.R., Maiya, B.G., Swamy, M.J.: Thermodynamic and kinetic analysis of porphyrin binding to Trichosanthes cucumerina seed lectin. Eur. J. Biochem. 268, 5541–5549 (2001). doi:10.1046/j.1432-1033.2001.02491.x

    Article  CAS  PubMed  Google Scholar 

  17. Komath, S.S., Kenoth, R., Swamy, M.J.: Thermodynamic analysis of saccharide binding to snake gourd (Trichosanthes anguina) seed lectin. Fluorescence and absorption spectroscopic studies. Eur. J. Biochem. 268, 111–119 (2001). doi:10.1046/j.1432-1327.2001.01852.x

    Article  CAS  PubMed  Google Scholar 

  18. Sultan, N.A.M., Kenoth, R., Swamy, M.J.: Purification, physicochemical characterization, saccharide specificity, and chemical modification of a Gal/GalNAc specific lectin from the seeds of Trichosanthes dioica. Arch. Biochem. Biophys. 432, 212–221 (2004). doi:10.1016/j.abb.2004.09.016

    Article  CAS  PubMed  Google Scholar 

  19. Sultan, N.A.M., Swamy, M.J.: Fluorescence quenching and time-resolved fluorescence studies on Trichosanthes dioica seed lectin. J. Photochem. Photobiol. B Biol. 80, 93–100 (2005). doi:10.1016/j.jphotobiol.2005.03.003

    Article  CAS  Google Scholar 

  20. Agashe, V.R., Udgaonkar, J.B.: Thermodynamics of denaturation of barstar: Evidence for cold denaturation and evaluation of the interaction with guanidine hydrochloride. Biochemistry 34, 3286–3299 (1995). doi:10.1021/bi00010a019

    Article  CAS  PubMed  Google Scholar 

  21. Nicholson, E.M., Scholtz, J.M.: Conformational stability of the Escherichia coli HPr protein: Test of the linear extrapolation method and a thermodynamic characterization of cold denaturation. Biochemistry 35, 11369–11378 (1996). doi:10.1021/bi960863y

    Article  CAS  PubMed  Google Scholar 

  22. Johnson, C.R., Morin, P.E., Arrowsmith, C.H., Freire, E.: Thermodynamic analysis of the structural stability of the tetrameric oligomerization domain of p53 tumor suppressor. Biochemistry 34, 5309–5316 (1995). doi:10.1021/bi00016a002

    Article  CAS  PubMed  Google Scholar 

  23. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 480–485 (1970). doi:10.1038/227680a0

    Article  Google Scholar 

  24. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–273 (1951)

    CAS  PubMed  Google Scholar 

  25. Manly, S.P., Matthews, K.S., Sturtevant, J.M.: Thermal denaturation of the core protein of lac Repressor. Biochemistry 24, 3842–3846 (1985). doi:10.1021/bi00336a004

    Article  CAS  PubMed  Google Scholar 

  26. Hinz, H.J., Schwarz, F.P.: Measurement and analysis of results obtained on biological substances with differential scanning calorimetry. Pure Appl. Chem. 73, 745–759 (2001). doi:10.1351/pac200173040745

    Article  CAS  Google Scholar 

  27. Schellman, J.A.: Macromolecular binding. Biopolymers 14, 999–1018 (1975). doi:10.1002/bip.1975.360140509

    Article  CAS  Google Scholar 

  28. Fukada, H., Sturtevant, J.M., Quiocho, F.A.: Thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli. J. Biol. Chem. 258, 13163–13198 (1983)

    Google Scholar 

  29. Lackowicz, J.R.: Principles of fluorescence spectroscopy. Plenum, New York (1989)

    Google Scholar 

  30. Khan, F., Ahmad, A., Khan, M.I.: Chemical, thermal and pH-induced equilibrium unfolding studies of Fusarium solani lectin. IUBMB Life 59, 34–43 (2007). doi:10.1080/15216540601178075

    Article  CAS  PubMed  Google Scholar 

  31. Krupakar, J., Swaminathan, C.P., Das, P.K., Surolia, A., Podder, S.K.: Calorimetric studies on the stability of the ribosome-inactivating protein abrin II: Effects of pH and ligand binding. Biochem. J. 338, 273–279 (1999). doi:10.1042/0264-6021:3380273

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by a research grant from the Department of Biotechnology (India) to MJS. MK is supported by a Senior Research Fellowship from CSIR (India). The Central Instrumentation Laboratory, University of Hyderabad is gratefully acknowledged for the use of the Jasco J-810 CD spectropolarimeter. We acknowledge the University Grants Commission (India) for their support through the UPE and CAS programs, to the University of Hyderabad and School of Chemistry, respectively.

Author information

Authors and Affiliations

  1. School of Chemistry, University of Hyderabad, Hyderabad, 500 046, India

    M. Kavitha & Musti J. Swamy

Authors
  1. M. Kavitha
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Musti J. Swamy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Musti J. Swamy.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kavitha, M., Swamy, M.J. Spectroscopic and differential scanning calorimetric studies on the unfolding of Trichosanthes dioica seed lectin. Similar modes of thermal and chemical denaturation. Glycoconj J 26, 1075–1084 (2009). https://doi.org/10.1007/s10719-009-9228-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10719-009-9228-y

Keywords

Search

Navigation