Hydration of Proteins: Excess Partial Volumes of Water and Proteins Vladimir A. Sirotkin,* Igor A. Komissarov, and Aigul V. Khadiullina A.M. Butlerov Institute of Chemistry, Kazan (Volga Region) Federal University, Kremlevskaya street, 18, Kazan, 420008, Russia ABSTRACT: High precision densitometry was applied to study the hydration of proteins. The hydration process was analyzed by the simultaneous monitoring of the excess partial volumes of water and the proteins in the entire range of water content. Five unrelated proteins (lysozyme, chymotrypsinogen A, ovalbumin, human serum albumin, and β-lactoglobulin) were used as models. The obtained data were compared with the excess partial enthalpies of water and the proteins. It was shown that the excess partial quantities are very sensitive to the changes in the state of water and proteins. At the lowest water weight fractions (w 1 ), the changes of the excess functions can mainly be attributed to water addition. A transition from the glassy to the flexible state of the proteins is accompanied by significant changes in the excess partial quantities of water and the proteins. This transition appears at a water weight fraction of 0.06 when charged groups of proteins are covered. Excess partial quantities reach their fully hydrated values at w 1 > 0.5 when coverage of both polar and weakly interacting surface elements is complete. At the highest water contents, water addition has no significant effect on the excess quantities. At w 1 > 0.5, changes in the excess functions can solely be attributed to changes in the state of the proteins. 1. INTRODUCTION The hydration of proteins is a phenomenon of considerable fundamental importance and practical interest. It is well-known that water bound to proteins (hydration or biological water) plays a crucial role in determining their stability, dynamics, and functions. 1-5 On the other hand, there are essential differences between the hydration water surrounding the protein and bulk water. 1-7 This means that a characterization of protein hydration requires elucidating the effects of both the protein on water and water on the protein. Volumetric studies have traditionally been of great importance in ascertaining a better understanding of protein- water interactions. Below, a short review of the available studies on the hydration of proteins is given. Because our paper presents a volumetric study of the water-protein systems, a major focus of this section aims to discuss the corresponding volume changes. More comprehensive reviews have been given in refs 1-4. Volume is an important thermodynamic quantity directly related to the compactness or globularity of the protein molecule and is generally thought to arise from a combination of factors. 8-16 The cavities and internal voids appear to represent a major positive contribution to the value of the volume change. The hydration of charged and polar groups causes a decrease in volume. On the other hand, the volume changes associated with the exposure of hydrophobic groups depend on the model compounds selected and fall into the range from small negative to positive values, and it is not clear whether the volume changes associated with the exposure of hydrophobic groups upon protein unfolding is net negative or positive and if the volume change associated with hydrophobic hydration plays an important role in the total volume change. One of the most effective experimental approaches for studying the hydration of proteins is to evaluate changes in the motion of water molecules using nuclear magnetic resonance (NMR) measurements. 1-4 Fullerton et al. 17 identified four water fractions with different correlation times for water motions in the lysozyme-water systems: “superbound” (water molecules bonded to charged sites; w 1 , (water weight fraction) ≈ 0-0.05), “polar bound” (water molecules directly hydrogen bonded to polar sites on the protein macromolecule; w 1 ≈ 0.05-0.2), “structured” (water molecules that are motionally perturbed by a protein but not bonded to it; w 1 ≈ 0.2-0.58), and bulk. Lioutas et al. 18 performed similar experiments and also found three fractions of water with motional properties different from bulk water. This division into four steps is consistent with classifications derived from thermodynamic measurements. For example, Yang and Rupley 19 studied the apparent heat capacity of lysozyme as a function of water content. They identified four stages in the hydration process. Stage I (w 1 = 0-0.06) corresponds to hydration of charged groups. Stage II (w 1 = 0.06-0.2) corresponds to the saturation of the remaining polar sites probably associated with formation of clusters of water molecules. Stage III (w 1 = 0.2-0.28) represents the condensation of water over weakly interacting surface elements. Stage IV (w 1 = 0.28 to dilute solution) corresponds to the addition of water to the fully hydrated protein. Similar division Received: January 21, 2012 Published: March 1, 2012 Article pubs.acs.org/JPCB © 2012 American Chemical Society 4098 dx.doi.org/10.1021/jp300726p | J. Phys. Chem. B 2012, 116, 4098-4105