Atomic-scale analysis of the solvation thermodynamics of hydrophobic hydration.

Molecular dynamics simulations are used to model the transfer thermodynamics of krypton from the gas phase into water. Extra long, nanosecond simulations are required to reduce the statistical uncertainty of the calculated "solvation" enthalpy to an acceptable level. Thermodynamic integration is used to calculate the "solvation" free energy, which together with the enthalpy is used to calculate the "solvation" entropy. A comparison series of simulations are conducted using a single Lennard-Jones sphere model of water to identify the contribution of hydrogen bonding to the thermodynamic quantities. In contrast to the classical "iceberg" model of hydrophobic hydration, the favorable enthalpy change for the transfer process at room temperature is found to be due primarily to the strong van der Waals interaction between the solute and solvent. Although some stabilization of hydrogen bonding does occur in the solvation shell, this is overshadowed by a destabilization due to packing constraints. Similarly, whereas some of the unfavorable change in entropy is attributed to the reduced rotational motion of the solvation shell waters, the major component is due to a decrease in the number of positional arrangements associated with the translational motions.     

Loss of translational entropy in molecular associations

Molecular associations in solution are opposed by the loss of entropy (DeltaS) that results from the restriction of motion of each component in the complex. Theoretical estimates of DeltaS are essential for rationalizing binding affinities, as well as for calculating entropic contribution to enzyme catalysis. Recently a statistical-mechanical framework has been proposed for estimating efficiently the translational entropy loss (DeltaS(trsl)), while taking explicitly into account the complex intermolecular interactions between the solute and the solvent. This framework relates the translational entropy of a solute in solution to its "free volume," defined as the volume accessible to the center of mass of the solute in the presence of the solvent and calculated by using an extension of the cell model (CM) for condensed phases. The translational entropy of pure water, estimated with the CM algorithm, shows good agreement with the experimental information. The free volume of various solutes in water, calculated within the CM by using molecular dynamics simulations with explicit solvent, displays a strong correlation with the solutes' polar and total surface areas. This correlation is used to propose a parameterization that can be used to calculate routinely the translational entropy of a solute in water. We also applied the CM formalism to calculate the free volume and translational entropy loss (DeltaS(trsl)) on binding of benzene to a cavity in a mutant T4-lysozyme. Our results agree with previously published estimates of the binding of benzene to this mutant T4-lysozyme. These and other considerations suggest that the cell model is a simple yet efficient theoretical framework to evaluate the translational entropy loss on molecular association in solution.     

Thermodynamics of binding water and solute to myosin

From the isopiestic measurements of the extents of adsorption of water vapour by fish myosin at various values of water activities at three different temperatures, the changes in free energy, enthalpy and entropy of dehydration of the protein have been calculated. Extents of excess binding of solvent and solute to myosin have also been determined from isopiestic experiments in the presence of different inorganic salts, sucrose and urea respectively. Mols of water and solute respectively bound in absolute amounts to myosin have been evaluated from these data in limited range of solute concentrations. Free energy changes at different concentrations of these solutes have also been evaluated and their relations with ‘salting-in’ and ‘salting-out’ phenomena have been discussed. The order of the values of the standard free energy change for excess binding calculated with respect to an unified thermodynamic scale are found to be consistent with relative reactivity of binding water to myosin in the presence of inorganic salts, sucrose and urea. Part of this work was presented at the 20th Annual Convention of Chemists of the Indian Chemical Society, Cuttack, 26th-30th December 1983.     

Driving force of water entry into hydrophobic channels of carbon nanotubes: entropy or energy?

Spontaneous entry of water molecules inside single-wall carbon nanotubes (SWCNTs) has been confirmed by both simulations and experiments. Using molecular dynamics simulations, we have studied the thermodynamics of filling of a (6,6) carbon nanotube in a temperature range from 273 to 353 K and with different strengths of the nanotube–water interaction. From explicit energy and entropy calculations using the two-phase thermodynamics method, we have presented a thermodynamic understanding of the filling behaviour of a nanotube. We show that both the energy and the entropy of transfer decrease with increasing temperature. On the other hand, scaling down the attractive part of the carbon–oxygen interaction results in increased energy of transfer while the entropy of transfer increases slowly with decreasing the interaction strength. Our results indicate that both energy and entropy favour water entry into (6,6) SWCNTs. Our results are compared with those of several recent studies of water entry into carbon nanotubes.     

Molecular-size corrections to the strength of the hydrophobic effect: a critical review

Large increases in the strength of the hydrophobic effect and, consequently, in the estimates of the hydrophobic contribution to the thermodynamics of protein folding (and other biologically-relevant processes), have been recently advocated on the basis of the application, to model transfer thermodynamic data, of corrections for the solute/solvent size disparity. In this work we first examine the effect of molecular-size corrections on the values calculated from several types of model transfer data. For the transfer of a solute from an organic solvent to water, the above increase is exclusively associated with the application of a solute/water molecular-size correction. Secondly, we critically review and assess the several theoretical arguments that lead to these corrections. In particular, we show that, contrary to previous claims in the literature, the analysis of dissolution processes in terms of ideal-gas, intermediate states does not lead to the molecular-size correction term, but only to expressions equivalent (although not strictly identical) to those derived from the well-known Ben-Naim's statistical-mechanical approach. In general, the several analyses offered or discussed in this work disfavor the application of the solute/water molecular-size corrections.     

153 Wonderful roles of the entropy in protein dynamics,binding and folding

The entropy, which is central to the second law of thermodynamics, determines that the thermal energy always flows spontaneously from regions of higher temperature to regions of lower temperature. In the protein–solvent thermodynamic system, the entropy is defined as a measure of how evenly the thermal energy would distribute over the entire system (Liu et al., 2012). Such tendency to distribute energy as evenly as possible will reduce the state of order of the initial system, and hence, the entropy can be regarded as an expression of the disorder, or randomness of the system (Yang et al., 2012). For a protein–solvent system under a constant solvent condition, the origin of entropy is the thermal energy stored in atoms, which makes atoms jostle around and bump onto one another, thus leading to vibrations of the covalent bonds connecting two atoms (occurring on the fs timescale) and the rotational and translational motions of amino acid side chain groups (occurring on ps timescale) and water molecules. These motions break the noncovalent bonds around structural regions that are weakly constrained thereby triggering the competitive interactions among residues or between residues and water molecules leading ultimately to the loop motions (occurring on ns timescale) around the protein surface. The loop motions can further transmit either through the water network around the protein surface or via specific structural components (such as the hinge-bending regions) over the entire protein molecule leading to large concerted motions (occurring on μs to s timescales) that are most relevant to protein functions (Amadei, Linssen & Berendsen, 1993; Tao, Rao & Liu, 2010). Thus, the multiple hierarchies of the protein dynamics on distinct timescales (Henzler-Wildman & Kern, 2007) are a consequence of the cascade amplification of the microscopic motions of atoms and groups for which the entropy originating from atomic thermal energy is most fundamental. In the case of protein–ligand binding, the importance of the entropy is embodied in the following aspects. (i) The release of the water molecule kinetic energy (which is a process of the solvent entropy maximization) will cause Brownian motions of individual water molecules which result in strong Brownian bombardments to solute molecules causing molecule wanders/diffusions and subsequent accident contacts/collisions between proteins and ligands. (ii) Such collisions will inevitably cause water molecule displacement and, if the contact interfaces are properly complementary, the requirement to increase the solvent entropy would further displace the water network around the binding interfaces thus leading to the formation the initial protein-ligand complex. (iii) In the initial complex, the loose association of the two partners provide the opportunity for protein to increase conformational entropy, thus triggering the conformational adjustments through competitive interaction between protein residues and ligand, leading ultimately to the formation of tightly associated complex (Liu et al., 2012). In the protein folding process, the first stage, i.e. the rapid hydrophobic collapse (Agashe, Shastry & Udgaonkar, 1995; Dill, 1985), is in fact driven by the effect of the solvent entropy maximization. Specifically, the requirement to maintain as many as possible the dynamic hydrogen bonds among the water molecules will squeeze/sequestrate the hydrophobic amino acid side chains into the interior of the folding intermediates and expose the polar/charged side chains onto the intermediate surface. This will minimize the solvent accessible surface area of the folding intermediates and as thus maximize the entropy of the solvent. The resulting molten globule states (Ohgushi & Wada, 1983) may contain a few secondary structural components and native tertiary contacts, while many native contacts, or close residue–residue interactions present in the native state have not yet formed. However, the nature to increase the protein conformational entropy can trigger a further conformational adjustment process, i.e. the conformational entropy increase breaks the transient secondary or tertiary contacts and triggers the competitive interactions among protein residues and between residues and water. This process may repeat many rounds until the negative enthalpy change resulting from the noncovalent formations can overcompensate for protein conformational entropy loss. In summary, we consider that the tendency to maximize the entropy of the protein–solvent system, which originates from the atomic thermal energy, is the most fundamental driving factor for protein folding, binding, and dynamics, whereas the enthalpy reduction, an opposing factor that tends to make the system become ordered, can compensate for the effect of entropy loss to ultimately allow the system to reach equilibrium at the free energy minima, either global or local.     

An effective solvent theory connecting the underlying mechanisms of osmolytes and denaturants for protein stability

An all-atom Gō model of Trp-cage protein is simulated using discontinuous molecular dynamics in an explicit minimal solvent, using a single, contact-based interaction energy between protein and solvent particles. An effective denaturant or osmolyte solution can be constructed by making the interaction energy attractive or repulsive. A statistical mechanical equivalence is demonstrated between this effective solvent model and models in which proteins are immersed in solutions consisting of water and osmolytes or denaturants. Analysis of these studies yields the following conclusions: 1), Osmolytes impart extra stability to the protein by reducing the entropy of the unfolded state. 2), Unfolded states in the presence of osmolyte are more collapsed than in water. 3), The folding transition in osmolyte solutions tends to be less cooperative than in water, as determined by the ratio of van 't Hoff to calorimetric enthalpy changes. The decrease in cooperativity arises from an increase in native structure in the unfolded state, and thus a lower thermodynamic barrier at the transition midpoint. 4), Weak denaturants were observed to destabilize small proteins not by lowering the unfolded enthalpy, but primarily by swelling the unfolded state and raising its entropy. However, adding a strong denaturant destabilizes proteins enthalpically. 5), The folding transition in denaturant-containing solutions is more cooperative than in water. 6), Transfer to a concentrated osmolyte solution with purely hard-sphere steric repulsion significantly stabilizes the protein, due to excluded volume interactions not present in the canonical Tanford transfer model. 7), Although a solution with hard-sphere interactions adds a solvation barrier to native contacts, the folding is nevertheless less cooperative for reasons 1–3 above, because a hard-sphere solvent acts as a protecting osmolyte.     

Non-polar solutes in water and in aqueous solutions of protein denaturants. Modeling of solution and transfer processes

A simple molecular model for the thermodynamic behavior of non-polar solutes in water and in aqueous solutions of protein denaturants is presented. Three contributions are considered: (i) combinatorial arising from the mixing process, (ii) interactional characterizing the molecular interactions occurring in the mixture and (iii) a contribution originating from the structural changes occurring in the first shell of water molecules around the solute. The latter is modeled assuming that water molecules in contact with the solute are involved in a chemical equilibrium between two states. The model describes well the temperature and denaturant concentration dependences of the Gibbs energies of solution and transfer for benzene, toluene and alkanes in water and aqueous solutions of urea and guanidine hydrochloride. Model parameters are physically meaningful, allowing a discussion of the molecular interactions involved. A preferential solvation of the solute by the denaturant is found. However, the non-polar solute-denaturant interaction is not specific, i.e. leading to a distinct chemical entity. Urea and guanidine hydrochloride are non-polar solubilizing agents because their interactions with the solute are less unfavorable than those between water and the solute.     

Two different proteins that compete for binding to thrombin have opposite kinetic and thermodynamic profiles

Thrombin binds thrombomodulin (TM) at anion binding exosite 1, an allosteric site far from the thrombin active site. A monoclonal antibody (mAb) has been isolated that competes with TM for binding to thrombin. Complete binding kinetic and thermodynamic profiles for these two protein-protein interactions have been generated. Binding kinetics were measured by Biacore. Although both interactions have similar K(D)s, TM binding is rapid and reversible while binding of the mAb is slow and nearly irreversible. The enthalpic contribution to the DeltaG(bind) was measured by isothermal titration calorimetry and van't Hoff analysis. The contribution to the DeltaG(bind) from electrostatic steering was assessed from the dependence of the k(a) on ionic strength. Release of solvent H(2)O molecules from the interface was assessed by monitoring the decrease in amide solvent accessibility at the interface upon protein-protein binding. The mAb binding is enthalpy driven and has a slow k(d). TM binding appears to be entropy driven and has a fast k(a). The favorable entropy of the thrombin-TM interaction seems to be derived from electrostatic steering and a contribution from solvent release. The two interactions have remarkably different thermodynamic driving forces for competing reactions. The possibility that optimization of binding kinetics for a particular function may be reflected in different thermodynamic driving forces is discussed.     

Enhanced crystal packing due to solvent reorganization through reductive methylation of lysine residues in oxidoreductase from <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Protein crystallization is in part driven by the changes in the entropy of the system, but opinions differ as to whether the solute (protein) or solvent (water) molecules make more of a contribution to the overall entropic change. Methylation of lysine residues in proteins has been used to enhance protein crystallization. We investigated using molecular dynamics simulations with explicit solvent molecules, the behavior of several native proteins and their methylated counterparts chosen from an earlier large-scale study. Methylated lysines are capable of making a variety of interactions including H-bonds with protein residues and solvent. We demonstrate that methylation on the lysine slightly increases its side chain conformational entropy by about 3.5 J mol⁻¹ K⁻¹. Analysis of the radial and spatial distributions of the water molecules around the methylated lysine surface in oxidoreductase from Streptococcus pneumoniae revealed a larger sphere of water molecules with low entropy, as compared with solvent associated with unmethylated lysine. If methylated lysine were to make interactions at the protein–protein interface, the low-entropy water molecules associated with methylated lysines would be released, resulting in a gain of entropy. We show that this gain more than compensates for the loss of protein entropy. Therefore, we propose that lysine methylation favors the formation of crystals through solvent entropic gain.     

Solvent reorganization contribution to the transfer thermodynamics of small nonpolar molecules.

The experimental thermodynamic data for the dissolution of five simple hydrocarbon molecules in water were combined with the solute-solvent interaction energy from a computer simulation study to yield data on the enthalpy change of solvent reorganization. Similar data were generated for dissolving these same solute molecules in their respective neat solvents using the equilibrium vapor pressure and the heat of vaporization data for the pure liquid. The enthalpy and the free energy changes upon cavity formation were also estimated using the temperature dependence of the solute-solvent interaction energy. Both the enthalpy and T delta S for cavity formation rapidly increase with temperature in both solvent types, and the free energy of cavity formation can be reproduced accurately by the scaled particle theory over the entire temperature range in all cases. These results indicate that the characteristic structure formation around an inert solute molecule in water produces compensating changes in enthalpy and entropy, and that the hydrophobicity arises mainly from the difference in the excluded volume effect.     

Practical limitations on the use of thermodynamic data from isothermal processes

Enthalpy, entropy and volume data obtained for processes studied in aqueous solvents generally have been assumed to apply to the solute process without consideration of the coupling between the process and the two-state equilibrium of water. Walrafen's confirmation of the latter in 1983 shows that long-debated model to be correct so the enthalpy and entropy contributions to a free-energy change to give unambiguous information must be corrected for the water contribution. The situation is further complicated by differential chemical interaction of amphiphilic solutes with the two water species since experimental complications make correction difficult or impossible. A more general source of error in isothermal experiments is the linkage to the thermal-equilibrium device. That thermal problem discovered only in 1967 is not yet treated in textbooks although it is always a complication in isothermal processes and responsible for a hierarchy of thermodynamic quantities with different levels of reliability. Major consequences for several familiar thermodynamic and extra-thermodynamic methods are examined in terms of relative reliability. In most cases the thermal corrections are restricted by changes in phase state on cooling.     

Effect of temperature on the modification of spectral properties of tryptophan aqueous solutions induced by water previously treated with laser radiation

It was shown that preliminary exposure of a solvent (water) to low-intensity laser radiation reduces the tryptophan fluorescence intensity, and this fluorescence quenching effect is retained throughout the temperature range explored (from 8 up to 50 degrees C). The effects found are interpreted as resulting from changes in solvent properties induced by the action of electromagnetic radiation on interaction of water molecules with solute.     

Nature of alcohol and anesthetic action on cooperative membrane equilibria.

A generalized, colligative thermodynamic framework is used to treat the action of solutes on cooperative membrane equilibria. Configurational entropy, the randomness imparted by solutes through the partitioning or mixing process, is implicated as the energetic driving force for the action of anesthetics on cooperative membrane equilibria. The equilibria predicted to be most sensitive to solute action--in which the dilute solute causes a perturbation equivalent to a large change in temperature--are (1) low-enthalpy processes that coincide with (2) large partitioning differences between states. The model stresses that solutes do not act at a single site, but on both states in an equilibrium, and that the perturbation is determined by the difference in entropy. Evidence for the thermodynamic framework is obtained from the partitioning behavior of the general anesthetic 1-hexanol into a model lecithin (DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine) membrane as a function of temperature and alcohol concentration. The low-enthalpy equilibrium between the gel (L beta') and ripple states (P beta') (pretransition) is more sensitive to 1-hexanol than the high-enthalpy equilibrium between the ripple (P beta') and fluid bilayer states (L alpha) (main transition). The perturbations of both equilibria are accurately described by the colligative thermodynamic framework. The results suggest that alcohols and anesthetics act through entropy to upset the natural thermal balance that maintains native membrane architecture.     

Water relations in the malonamide-induced haemolysis of mammalian erythrocytes

A revised procedure is described for deriving enthalpy-entropy relations in the haemolysis kinetics of mammalian erythrocytes and its application demonstrates that previously reported linear enthalpy-entropy correlations are statistical artefacts with no real physical basis.Physically valid linear enthalpy-entropy relations do exist between species at constant osmotic concentration, but these are the result of the mutual dependence of the activation parameters on erythrocyte solvent volume. Non-linear enthalpy-entropy dependence on osmotic concentration, which is also physically valid and occurs within species, is attributed to erythrocyte solvent volume variation due to the osmotic properties of haemoglobin.Further development of the data indicates that malonamide-induced haemolysis is essentially an osmotic phenomenon and that the water permeability of all those cells is probably the same.From a consideration of the process in relation to the molecular dynamics of water it appears that the activation enthalpy, entropy and internal energy of haemolysis may refer to the molecular mobility of water during osmosis.     

Structure and solvation of melittin in hexafluoroacetone/water

Intermolecular (1)H[(19)F] and (1)H[(1)H] nuclear Overhauser effects have been used to explore interaction of solvent components with melittin dissolved in 50% hexafluoroacetone trihydrate (HFA)/water. Standard nuclear Overhauser effect experiments and an analysis of C(alpha)H proton chemical shifts confirm that the conformation of the peptide in this solvent is alpha-helical from residues Ala4 to Thr11 and from Leu13 to Arg24. The two helical regions are not collinear; the interhelix angle (144 +/- 20 degrees ) found in this work is near that observed in the solid state and previous NMR studies. Intermolecular NOEs arising from interactions between spins of the solvent and the solute indicate that both fluoroalcohol and water molecules are strongly enough bound to the peptide that solvent-solute complexes persist for > or =2 ns. Preferential interactions of HFA with many hydrophobic side chains of the peptide are apparent while water molecules appear to be localized near hydrophilic side chains. These results indicate that interactions of both HFA and water are qualitatively different from those present when the peptide is dissolved in 35% hexafluoro-2-propanol/water, a chemically similar helix-supporting solvent system.     

Enthalpy–entropy compensation phenomena in water solutions of proteins and small molecules: A ubiquitous properly of water

This article presents evidence for the existence of a specific linear relationship between the entropy change and the enthalpy change in a variety of processes of small solutes in water solution. The processes include solvation of ions and nonelectrolytes, hydrolysis, oxidation–reduction, ionization of weak electrolytes, and quenching of indole fluorescence among others. The values of the proportionality constant, called the compensation temperature, lie in a relatively narrow range, from about 250 to 315 °K, for all these processes. Such behavior can be a consequence of experimental errors but for a number of the processes the precision of the data is sufficient to show that the enthalpy–entropy compensation pattern is real. It is tentatively concluded that the pattern is real, very common and a consequence of the properties of liquid water as a solvent regardless of the solutes and the solute processes studied. As such the phenomenon requires that theoretical treatments of solute processes in water be expanded by inclusion of a specific treatment of the characteristic of water responsible for compensation behavior. The possible bases of the effect are proposed to be temperature-independent heat-capacity changes and/or shifts in concentrations of the two phenomenologically significant species of water. The relationship of these alternatives to the two-state process of water suggested by spectroscopic and relaxation studies is examined. The existence of a similar and probably identical relationship between enthalpy and entropy change in a variety of protein reactions suggests that liquid water plays a direct role in many protein processes and may be a common participant in the physiological function of proteins. It is proposed that the linear enthalpy–entropy relationship be used as a diagnostic test for the participation of water in protein processes. On this basis the catalytic processes of chymotrypsin and acetylcholinesterase are dominated by the properties of bulk water. The binding of oxygen by hemoglobin may fall in the same category. Similarities and differences in the behavior of small-solute and protein processes are examined to show how they may be related. No positive conclusions are established, but it is possible that protein processes are coupled to water via expansions and contractions of the protein and that in general the special pattern of enthalpy–entropy compensation is a consequent of the properties of water which require that expansions and contractions of solutes effect changes in the free volume of the nearby liquid water. It is shown that proteins can be expected to respond to changes in nearby water and interfacial free energy by expansions and contractions. Such responses may explain a variety of currently unexplained characteristics of protein solutions. More generally, the enthalpy–entropy compensation pattern appears to be the thermodynamic manifestation of “structure making” and “structure breaking,” operationally defined terms much used in discussions of water solutions. If so, the compensation pattern is ubiquitous and requires re-examination of a large body of molecular interpretations derived from quantitative studies of processes in water. Theories of processes in water may have to be expanded to accommodate this aspect of water behavior.     

Thermal stability of collagen fibers in ethylene glycol

The mechanism that renders collagen molecules more stable when precipitated as fibers than the same molecules in solution is controversial. According to the polymer-melting mechanism the presence of a solvent depresses the melting point of the polymer due to a thermodynamic mechanism resembling the depression of the freezing point of a solvent due to the presence of a solute. On the other hand, according to the polymer-in-a-box mechanism, the change in configurational entropy of the collagen molecule on denaturation is reduced by its confinement by surrounding molecules in the fiber. Both mechanisms predict an approximately linear increase in the reciprocal of the denaturation temperature with the volume fraction (epsilon) of solvent, but the polymer-melting mechanism predicts that the slope is inversely proportional to the molecular mass of the solvent (M), whereas the polymer-in-a-box mechanism predicts a slope that is independent of M. Differential scanning calorimetry was used to measure the denaturation temperature of collagen in different concentrations of ethylene glycol (M = 62) and the slope found to be (7.29 +/- 0.37) x 10(-4) K(-1), compared with (7.31 +/- 0.42) x 10(-4) K(-1) for water (M = 18). This behavior was consistent with the polymer-in-a-box mechanism but conflicts with the polymer-melting mechanism. Calorimetry showed that the enthalpy of denaturation of collagen fibers in ethylene glycol was high, varied only slowly within the glycol volume fraction range 0.2 to 1, and fell rapidly at low epsilon. That this was caused by the disruption of a network of hydrogen-bonded glycol molecules surrounding the collagen is the most likely explanation.     

Computer simulation of hydration networks around amino acids

The networks of solvent hydrogen bonds around polar and apolar amino acids have been studied by computer simulation techniques using a non-pair additive model for the water molecules interactions. Analysis of the simulated aqueous solutions has shown the presence of water molecules which (a) form a bridge around individual polar solute atoms (self-bridging loops) and (b) form chains between different polar solute atoms (polar bridging chains). Some of these networks associated with polar solute atoms from pentagons but 4, 6 and 7 sided polygons are also seen. The water molecule close to apolar solute atoms (<4.0 Å) also form irregular networks with polygons of 4, 5, 6 and 7 sides. These networks are compared with those found experimentally in ice, clathrates and crystal hydrates of macromolecules.     

High-affinity binding of water by proteins is similar in air and in organic solvents

Published data for water adsorption by proteins suspended in organic solvents (of interest as enzyme reaction mixtures) have been converted to a basis of thermodynamic water activity (aw). The resulting adsorption isotherms have been compared with those known for proteins equilibrated with water from a gas phase. This comparison can show any effects of the solvent on the interaction between the protein and water at the molecular level. At lower water contents (aw less than about 0.4), similar adsorption isotherms are found in each solvent and in the gas phase; differences are probably less than the likely errors. Hence, it may be concluded that the presence of an organic solvent has little effect on the interaction between proteins and tightly bound water; on a molecular scale there is probably little penetration of the primary hydration layer by solvent molecules, even fairly polar ones such as EtOH. At higher aw values, there are differences between the isotherms which probably are significant. Nonpolar solvents increase the amount of water bound by the enzyme (at fixed aw), while polar solvents (mainly EtOH) may reduce the amount of water bound by the enzyme, presumably by occupying part of the secondary hydration layers in place of water.