SPECIAL ISSUE ARTICLE Glass Transition and Dynamics in Lysozyme–Water Mixtures Over Wide Ranges of Composition Anna Panagopoulou & Apostolos Kyritsis & Anna-Maria Aravantinou & Dionysios Nanopoulos & Roser Sabater i Serra & Jose Luis Gómez Ribelles & Naoki Shinyashiki & Polycarpos Pissis Received: 6 August 2010 / Accepted: 28 December 2010 / Published online: 13 January 2011 # Springer Science+Business Media, LLC 2011 Abstract Differential scanning calorimetry (DSC) and two dielectric techniques, broadband dielectric relaxation spec- troscopy and thermally stimulated depolarization currents (TSDC), were employed to study glass transition and water and protein dynamics in mixtures of water and a globular protein, lysozyme, in wide ranges of water content, both solutions, and hydrated solid samples. In addition, water equilibrium sorption isotherms (ESI) measurements were performed at room temperature. The main objective was to correlate results by different techniques to each other and to determine critical water contents for various processes. From ESI measurements the content of water directly bound to primary hydration sites was determined to 0.088 (grams of water per grams of dry protein), corresponding to 71 water molecules per protein molecule, and that where clustering becomes significant to about 0.25. Crystallization and melting events of water were first observed at water contents 0.270 and 0.218, respectively, and the amount of uncrystallized water was found to increase with increasing water content. Two populations of ice crystals were observed by DSC, primary and bulk ice crystals, which give rise to two separate relaxations in dielectric measurements. In addition, the relaxation of uncrystallized water was observed, superimposed on a local relaxation of polar groups on the protein surface. The glass transition temperature, determined by DSC and TSDC in rather good agreement to each other, was found to decrease significantly with increasing water content and to stabilize at about -90 °C for water contents higher than about 0.25. This is a novel result of this study with potential impact on cryoprotection and pharmaceutics. Keywords Glass transition . Molecular mobility . Hydrated proteins . Uncrystallized water . Dielectric relaxation . Plasticization Introduction The hydration properties of proteins and water and protein dynamics in protein–water mixtures have been studied in the past several decades by a variety of experimental techniques. The results indicated the significant influence of protein–water interactions on the structure, the dynamics and the biological function of proteins. 1–6 A minimum amount of water is necessary for enzymatic activity of a protein and dielectric results indicated a correlation between the onset of enzymatic activity and of a percolation type displacement process of protons on single macromole- cules. 3 Thermal and dynamic studies of fully hydrated A. Panagopoulou : A. Kyritsis : A.-M. Aravantinou : D. Nanopoulos : P. Pissis (*) Department of Physics, National Technical University of Athens, Zografou Campus, 15780 Athens, Greece e-mail: ppissis@central.ntua.gr R. S. i Serra : J. L. Gómez Ribelles Centro de Biomateriales e Ingeniería Tisular, Universidad Politécnica de Valencia, 46022 Valencia, Spain N. Shinyashiki Department of Physics, Tokai University, Hiratsuka, Kanagawa 259-1292, Japan J. L. Gómez Ribelles Centro de Investigación Príncipe Felipe, 46013 Valencia, Spain J. L. Gómez Ribelles CIBER en Bioingeniería, Biomateriales y Nanotecnología, Valencia, Spain Food Biophysics (2011) 6:199–209 DOI 10.1007/s11483-010-9201-0