Analysis of Bulk and Hydration Water During Thermal Lysozyme Denaturation Using Raman Scattering
- 383 Downloads
We describe a method for analyzing protein hydration by Raman spectroscopy on the model protein lysozyme. The analysis of the protein hydration shell is made possible by dissolving the protein in D2O, providing via isotopic exchange the uncoupled O – H stretching spectrum of water molecules early bound to the protein, which are thereafter spread into the solvent. The spectrum of the hydration water can be obtained by subtracting the spectrum of the contribution of D2O from that of the aqueous lysozyme solution in the intramolecular O – D stretching vibrations region (2,200–2,800 cm−1). Raman investigations were simultaneously carried out in the amide I region (1,500–1,800 cm−1) and in the O – D/H stretching spectrum (3,200–3,800 cm−1) during thermal denaturation of lysozyme, to analyze structural changes of the protein in relation to the physical properties of hydration water. It was found that the H-bond network of hydration water is slightly distorted compared to the bulk water at room temperature, with a loss of the tetrahedral local order. The difference between hydration and bulk water is significantly enhanced at T = 90 °C in the denaturated state of the protein. The quantification of water molecules in direct interaction with the protein provides the temperature dependence of the solvent-accessible surface area during the denaturation process. Both kinds of information on hydration water and protein structure lead to a detailed description and overall understanding of the mechanism of protein denaturation.
KeywordsRaman spectroscopy Hydration water Uncoupled OH stretch Dilute DHO solution Thermal denaturation Lysozyme
This work was supported by the ANR (Agence Nationale de la Recherche) through the BIOSTAB project (“Physique Chimie du Vivant” program), by FEDER and Nord-Pas de Calais region.
- 2.G. Careri, Collective Effects in Hydrated Proteins, in Hydration Processes in Biology: Theoretical and Experimental Approaches, ed. by M.-C. Bellissent-Funel (Ios Press, Amsterdam, 1999)Google Scholar
- 9.C. Schroder, T. Rudas, S. Boresch, O.J. Steinhauser, Chem. Phys. 124, 234907 (2006)Google Scholar
- 20.R. Ionov, A. Hédoux, Y. Guinet, P. Bordat, A. Lerbret, F. Affouard, D. Prevost, M. Descamps, J. Non-Cryst. Solids. 2006.Google Scholar
- 24.J.-A. Seo, A. Hedoux, Y. Guinet, L. Paccou, F. Affouard, A. Lerbret, M. Descamps, J. Phys. Chem. B114, 6675 (2010)Google Scholar
- 29.G. D’Arrigo, G. Maisano, F. Mallamace, P. Migliardo, F.J. Wanderlingh, Chem. Phys. 75, 4264 (1981)Google Scholar
- 30.G.E.J. Walrafen, Chem. Phys. 47, 114 (1967)Google Scholar
- 32.T.T. Wall, D.F.J. Hornig, Chem. Phys. 43, 2079 (1965)Google Scholar
- 34.A. Hedoux, F. Affouard, M. Descamps, Y. Guinet, L. Paccou, Phys.: Condens. Matter 19 (2007). 8 pGoogle Scholar