KEY WORDS: dipole moment, enzymic activity, hydration, hydrogen bonds, orientational relaxation time, proton percolation
INTRODUCTIONIn one of the earliest reports of the physicochemical properties of a protein, Sorensen (1) asked "Does crystallised egg-albumin contain water?" Fol lowing a detailed compositional analysis, he found that such samples contain about 0.22 g water per g water-free egg-albumin. In later work, he gives the water content of crystallized hemoglobin as 0.35 gjg (2). We now know that protein molecules can have from 0.20 to 0.70 g strongly associated (bound) water per g protein, and through modern x-ray and neutron diffraction studies, we have for some proteins a detailed knowledge of the location and bonding of much of this water (3-5). By using the latest high-resolution NMR techniques, we can even identify individual molecules of hydration water and characterize their binding sites on the protein molecule (6). Thus, in general and as a convenient method of classification, two kinds of water molecules associated with proteins have been identified: internal water and peripheral water. The internal water molecules, which form an integral part of a protein structure, diminish local charge-charge inter actions and reduce destabilizing effects that arise from otherwise unbonded proton donors and acceptors (7,8). These molecules exchange with bulk water in time scales ranging from tens of seconds to months (9, 10). As a rough gauge of the upper limit of internal water, we can consider the small 177 0066-426Xj92jll01-0l77$ 02.00 Annu. Rev. Phys. Chem. 1992.43:177-205. Downloaded from www.annualreviews.org by University of Sydney on 09/17/13. For personal use only.Quick links to online content Further ANNUAL REVIEWS 178 PETHIG enzyme pancreatic trypsin inhibitor (mol wt 6700), which contains fo ur internal waters (8). As this represents a hydration content of 1.07 wt%, we can conclude that most of the water bound to proteins is associated with the protein surface. Such hydration shells of peripheral water do not freeze upon cooling (11). And, although distinct nonrandom distributions of water molecules occur at the polar side-chains, consistent with the expected stereochemistry of the potential hydrogen-bonding sites (5), there is no evidence fo r the existence of extensive ice-like fo rmations of water molecules around protein molecules. Klotz (12) proposed that water might form clathrate-type structures around hydrophobic groups at protein sur faces. Evidence for this is found for the small, hydrophobic plant protein crambin, in which a cluster of pentagonal arrays, made up of 16 hydrogen bonded water molecules, sits at a hydrophobic intermolecular cleft (13).Of the approximately 90 water molecules per crambin molecule, 73 have been located (14), and all of them contribute to an ordered hydrogen bonded network around the protein surface. From NMR studies (6, and references cited therein), it appears that protein hydration is more dis ordered in solution than has been indicated from single crystal...