Protein and Water Dynamics in Bovine Serum Albumin-Water Mixtures over Wide Ranges of Composition A. Panagopoulou,* ,† A. Kyritsis, † N. Shinyashiki, ‡ and P. Pissis † † Department of Physics, National Technical University of Athens, Zografou Campus, 157 80 Athens, Greece ‡ Department of Physics, Tokai University, Hiratsuka, Kanagawa, 259-1292 Japan ABSTRACT: Dielectric dynamic behavior of bovine serum albumin (BSA)- water mixtures over wide ranges of water fractions, from dry protein until 40 wt % in water, was studied through dielectric relaxation spectroscopy (DRS). The α relaxation associated with the glass transition of the hydrated system was identified. The evolution of the low temperature dielectric relaxation of small polar groups of the protein surface with hydration level results in the enhancement of dielectric response and the decrease of relaxation times, until a critical water fraction, which corresponds to the percolation threshold for protonic conductivity. For water fractions higher than the critical one, the position of the secondary ν relaxation of water saturates in the Arrhenius diagram, while contributions originating from water molecules in excess (uncrystallized water or ice) follow separate relaxation modes slower than the ν relaxation. 1. INTRODUCTION It is known that the dynamics of water is experimentally inaccessible for the liquid state in a wide temperature range (150-230 K at atmospheric pressure), often called the no man’s land. This is due to the fact that, even after quenching to very low temperatures, bulk supercooled water crystallizes upon heating at approximately 150 K. 1 In order to confront the difficulty in monitoring water dynamics in the no man’s land region, several approaches have been made in the literature, aiming to prevent crystallization of water, either by mixing with hydrophilic glass-forming solutes 2-6 and biopolymers 7,8 or by confinement on the nanometer length scale, 9-13 e.g., within nanopores of silica gels. 9 Homogeneous water solutions of several systems, such as alcohols, ethylene and propylene glycols, sugars, or carbohydrates (mono-, di-, and polysacchar- ides), and some hydrophilic macromolecular systems including biopolymers (from polypeptides to several proteins), 3,4,14,15 in concentrations of water up to 50% in weight, can be easily supercooled down to form glass, while no crystallization occurs when water molecular clusters are reduced down to sizes smaller than the critical size necessary for homogeneous nucleation, in case of confinement. 9 Another effect of confinement is the disorder induced by the interfaces that prevents the water molecules from forming a crystalline lattice. Dielectric studies focused on the dynamics of supercooled water in different host environments in the hydration range 30-50 wt %, all providing one major result concerning the main relaxation of water. Its relaxation time, τ, has an Arrhenius temperature dependence, at least below the glass transition temperature, T g , of the hydrated system, has an almost universal activation energy, E act , of about 0.45-0.55 eV, 4,16 and shows a symmetric or nearly symmetric shape of its response function on a logarithmic frequency scale, and its magnitude increases systematically with increasing water content. 16 These character- istics apply not only in the case of aqueous mixtures but also in the case of water confined in various confining systems. 17 Another interesting feature of the observed main relaxation is a change in the temperature dependence of its characteristic relaxation time from an Arrhenius to a non-Arrhenius one (strong to fragile crossover), typically at about 180 ± 20 K. 4,14,18 In the case of hydrated proteins, this dynamic crossover has been assigned to the saturation of the cooperative ordering of hydrogen bonds, by combining experimental results and molecular simulations. 19 However, several experimental studies on hydrated proteins by dielectric relaxation spectroscopy (DRS) and nuclear magnetic resonance (NMR) show no sign of such a crossover, 20,21 which has also been suggested to be an artifact of the data analysis method. 22 More recent studies propose the presence of more than one dynamical crossover, one at about 250 K and one at about 180 K, for protein hydration water. 23 The crossover at about 180 K has been also observed in the case of protein hydration water and water confined in silica gel nanopores. 24 The interpretation of the main relaxation of water and its association to the viscosity related α relaxation of bulk water has been highly debated in the literature. Unlike simple liquids, there is a difficulty to extrapolate the time scales of the dielectric response of water above the homogeneous nucleation temperature (235 K) to the one in the deeply supercooled regime. The T g of water is suggested to be in the range of 136 K, 25-28 while other studies suggest that the actual glass transition temperature of water lies in the range 160-180 Received: November 3, 2011 Revised: March 7, 2012 Published: April 2, 2012 Article pubs.acs.org/JPCB © 2012 American Chemical Society 4593 dx.doi.org/10.1021/jp2105727 | J. Phys. Chem. B 2012, 116, 4593-4602