Spectroscopic and differential scanning calorimetric studies on the unfolding of Trichosanthes dioica seed lectin. Similar modes of thermal and chemical denaturation
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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.
KeywordsAgglutinin Carbohydrate binding protein Thermal unfolding Van’t Hoff enthalpy Calorimetric enthalpy Chemical denaturation
Trichosanthes dioica seed lectin
Change in excess heat capacity
Differential scanning calorimetry
Change in calorimetric enthalpy
Change in van’t Hoff enthalpy
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.
- 1.Sharon, N., Lis, H.: Lectins, p. 454pp. Kluwer Academic, Dordrecht, The Netherlands (2003)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)PubMedGoogle 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 CrossRefPubMedGoogle 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