19036
J. Phys. Chem. B 2004, 108, 19036-19042
Lysozyme-Water Interactions Studied by Sorption Calorimetry Vitaly Kocherbitov,*,† Thomas Arnebrant,† and Olle So1 derman‡ Health and Society, Malmo¨ UniVersity, SE-205 06 Malmo¨, Sweden, and Physical Chemistry 1, Center for Chemistry and Chemical Engineering, P.O. Box 124, Lund UniVersity, SE-221 00 Lund, Sweden ReceiVed: June 1, 2004; In Final Form: September 10, 2004
Hydration of hen egg white lysozyme was studied by using the method of sorption calorimetry at 25, 40, and 50 °C. Desorption calorimetric measurements were performed at 25 and 40 °C. The activity of water and partial molar enthalpy of mixing of water were determined as functions of water content. Hydration of lysozyme occurs in four steps: slow penetration of water into the protein-protein interface; gradual glass transition, which occurs in every protein molecule independently of other molecules; further water uptake with disaggregation of protein aggregates and formation of a monolayer of water; and accumulation of free water. The amount of bound water found in desorption experiments is 420 water molecules per lysozyme molecule. Two hysteresis loops were found in the sorption isotherm of lysozyme. The small loop is caused by the slow penetration of water molecules into the protein-protein interface at very low water contents, while the large loop is due to the slow kinetics of aggregation of protein molecules upon desorption. The phase diagram of the lysozyme-water system is presented.
Introduction Protein-water interactions have been attracting the attention of scientists for many decades because of their crucial importance for protein functions and stability.1,2 Most proteins are inactive in the absence of water. Monitoring of changes that occur in proteins upon hydration (addition of water) from the dry state to aqueous solutions can provide information about the properties of proteins both in the dry state and in the solution. One of the most important properties of proteins that changes upon hydration is their dynamics. Glasslike dynamic transitions in proteins were studied by different methods but the exact mechanism of the transition is not fully understood. All methods used for studies of hydration of proteins can be divided in two groups: methods providing thermodynamic information (e.g., water activity, enthalpy of mixing or sorption, heat capacity) and methods providing structural, molecular level information. Since proteins are very complex biochemical objects, there is no single method that alone would provide enough experimental data to explain the behavior of protein systems. Only the combination of several structural and thermodynamic methods will allow complete understanding of the processes of hydration of proteins. Hen egg while lysozyme is a model protein often used to study general features of protein hydration. It is stable and relatively cheap and its structure is well-known.3 Below a short review of the studies of hydration of lysozyme is given. Since the present paper presents a calorimetric study of the hydration of lysozyme, we focused mostly on thermodynamic methods. More comprehensive reviews have been given by Rupley and Careri2 and Gregory.1 In 1959 Hnojewyj and Reyerson4 studied sorption of H2O and D2O by lyophilized lysozyme at 17 and 27 °C. The same * Address correspondence to this author. Phone: +4640 6657946. Fax: +4640 6658100. E-mail:
[email protected]. † Malmo ¨ University. ‡ Center for Chemistry and Chemical Engineering.
authors later reported the sorption isotherm (as amount of adsorbed water vs vapor pressure) of lysozyme at 37, 47, and 57 °C.5 From the temperature dependence of the isotherms they calculated differential heats of sorption. Leeder and Watt6 studied the sorption isotherm (water content as function of water activity) of lysozyme in the composition range 0-27 wt % of water at 35 °C using a sorption balance. The experimental sorption isotherm was compared with the results of calculations based on the D’Arcy and Watt model.7 Luscher-Mattli and Ruegg8,9 calculated enthalpy and entropy of water sorption on lysozyme at water contents up to 17 wt %. Calculations were done by using the temperature dependence of the water vapor pressure in the range 25-40 °C. Lioutas et al.10 measured the sorption isotherm of lysozyme at 20 °C by equilibrating lysozyme samples with water vapor from salt solutions and sucrose solutions. A modification of the D’ArcyWatt theory was used for the treatment of the results. Bone11 studied the sorption isotherm of lysozyme in the composition range 1.5-19 wt % of water at nine temperatures in the range 6-46 °C using a vacuum microbalance. From the temperature dependence of the water vapor pressure the enthalpy and entropy of hydration were calculated. Smith et al.12 used a quartz crystal microbalance/heat conduction calorimeter to study the hydration of lysozyme. They obtained both the sorption isotherm and the enthalpy of hydration of the protein in the composition range 0-18 wt % of water at 25 °C. The observed discontinuity in the slope of enthalpy of sorption at 10 wt % was attributed to the binding of water to highly polar groups of lysozyme below this water content.12 Most of the authors evaluated the experimental results on water sorption by proteins using the D’Arcy-Watt model.7 This model takes into account the heterogeneity of a protein molecule: sorption sites with two different heats of sorption were assumed. When all the sites of these two groups are occupied, further adsorption leads to the formation of a multilayer on top of the primarily formed monolayers.7 The
10.1021/jp0476388 CCC: $27.50 © 2004 American Chemical Society Published on Web 11/03/2004
Lysozyme-Water Interactions division of the sorption isotherm of proteins into three regions12,13 apparently arises from the assumptions of the D’ArcyWatt model. From these assumptions an equation that describes the amount of adsorbed water as a function of water vapor pressure was derived7 and widely used for treatment of experimental sorption isotherms of different types of proteins (see, for example, ref 14). Nonetheless, since the D’Arcy-Watt equation contains fitting parameters, its ability to describe experimental sorption isotherms does not necessarily mean that the assumptions on which the equation is based are equally correct for all types of proteins. A very important feature of protein-water systems not taken into account by the D’ArcyWatt model is the change of the dynamics of proteins on hydration, and in particular, the presence of glass transition. Glasslike dynamic transitions in proteins were observed by several experimental techniques (for a review see ref 1). The thermodynamic property commonly used for determination of glass transition is the heat capacity Cp. In synthetic polymers a well-pronounced step in the heat capacity curve is observed at a glass transition. In the case of proteins the appearance of the Cp curve near the glass transition depends on the properties of a particular protein system. In protein systems at high water contents, the glass transition is observed at subzero temperatures and is highly dependent on the dynamic properties of water itself. For several proteins in the hydrated form it was determined that they exhibit a glass transition at about 200 K.1 Sartor et al.15 observed a glass transition in noncrystalline lysozyme at about 168 K at a water content of 0.35 h (g of water/g of protein). For hydrated lysozyme crystals a broad glass transition was found at about 150 K and a tendency of increasing glass transition temperature Tg with dehydration of the crystals was observed.16 Unlike proteins in the native state, showing very broad and therefore not always pronounced changes in the heat capacity curves corresponding to glass transition, denatured proteins exhibit a well-pronounced step in the Cp curves.17,18 The glass transition temperature of denatured proteins increases with dehydration of proteins.18 Several studies of heat capacities of proteins in the dry state have been reported (see, for example, refs 19-21) but the results have not been discussed in terms of glass transitions. Of a particular interest for the present study is the heat capacity of the lysozyme-water system at the composition region of hydration (below 50 wt % of water). Yang and Rupley21 determined the heat capacity of the lysozyme-water system with respect to water content at 25 °C. They divided the composition range of 0-0.5 g of water/g of protein into four regions according to the appearance of the curve of the apparent specific heat capacity of lysozyme. The most interesting feature of these data is a linear increase of the apparent heat capacity of lysozyme in the composition range 0.07-0.27 h. We shall use these results in combination with the enthalpy data obtained in the present study for analysis of the processes of hydration of lysozyme. Although the literature on the studies of dynamics of proteins is very extensive, the exact mechanism underlying glass transitions in proteins is still unclear. For example, Careri et al.2,22 discussed changes that occur in proteins during hydration mostly in terms of percolation theory focusing on properties of water molecules adsorbed on the surface of proteins, while Gregory1 described glass transition in proteins as the transition from rigid protein molecules to rigid “knots” and soft “matrixes”. Another interesting property of water sorption on proteins is hysteresis in the sorption isotherm. Several studies showed that
J. Phys. Chem. B, Vol. 108, No. 49, 2004 19037 the sorption isotherms obtained during sorption and desorption of water differ significantly. From the data of Hnojewyj and Reyerson4 it follows that the activity of water in the mixture with lysozyme was higher during adsorption than during desorption. The same authors5 showed that the activity of water during the first run was higher than that during following runs. On the other hand, the heat capacity data of Yang and Rupley21 did not show any hysteresis. Bryan23 presented four thermodynamic models of hysteresis of protein-water interactions. Cerofolini and Cerofolini24 proposed a model for adsorption of water by proteins, which takes into account hysteresis, but this model apparently does not take into account the glass transition and specific aggregation of protein molecules. Aggregation of proteins in solution is a subject of very extensive studies (see, for example, refs 25-30). Although these studies deal with proteins in solution (at low concentrations of proteins), some results can be relevant for the concentration region of hydration. While aggregation of lysozyme at high water content does not change its tertiary structure, protein-protein interactions at low water contents can strongly affect not only tertiary but also secondary structure. Griebenow and Klibanov31 studied several proteins and showed that lyophilization increases β-sheet content and lowers R-helix content of the proteins. Costatino et al.32 using FTIR spectroscopy found that the β-sheet content in lysozyme in the lyophilized form was about 44%, while in the aqueous solution it was about 18%. The decrease of the R-helix content upon lyophilization was lower than the increase of β-sheet content. The reported changes were essentially reversible. Here we present a study of hydration of lysozyme using a method of sorption calorimetry.33,34 This method is based on the use of the double twin microcalorimeter33 and was never applied for protein studies before. The method of sorption calorimetry allows simultaneous monitoring of water activity (sorption isotherm) and partial molar enthalpy of mixing of water Hm w . The most important reason to use this technique for protein studies is that it provides direct high-resolution measurements of the enthalpy of hydration of a studied substance at isothermal conditions (no analysis of the temperature dependence of the sorption isotherm for calculation of enthalpy is required). Since there are two modifications of the method, sorption and desorption calorimetry, hysteresis of the sorption isotherm can be studied. The method has previously proved to be very efficient for accurate measurements of phase transitions that occur at tiny changes of water contents in surfactant systems.35,36 A glass transition (from glassy crystals to liquid crystals) at isothermal conditions upon hydration of a surfactant37 was also studied with this method. One should also mention the studies of hydration of phospholipids38 and DNA39 performed with the method of sorption calorimetry. Materials and Methods Hen egg white lysozyme was purchased from Sigma (catalog no. L6876). This preparation contains up to 5% buffer salts, such as sodium acetate and sodium chloride. The sample dialyzed against water gave similar results to that observed without dialysis. The results presented here are obtained with nondialyzed samples. All lysozyme samples were dried in a vacuum at room temperature in contact with 4Å molecular sieves for at least 20 h before use. To check the impact of conditions of drying on the sorption results, in some cases further drying at 100 °C in a vacuum for 1 h was applied. The results obtained with lysozyme dried at 100 °C and at room temperature were very similar.
19038 J. Phys. Chem. B, Vol. 108, No. 49, 2004
Kocherbitov et al.
Figure 1. Water activity as a function of water content at 25 °C, solid line - sorption calorimetric data; asterisks - data from experiments with saturated salt solutions.
The method of sorption calorimetry was used to monitor water activity aw and partial molar enthalpy of mixing of water Hm w. A two-chamber calorimetric cell with the sample chamber on top was used. The calorimetric cell was inserted into the doubletwin calorimeter.33 Sorption and desorption calorimetric experiments were conducted following the procedures described in detail elsewhere.33,34,40,41 The raw data corresponding to the process of sorption of water vapor on lysozyme were used for the calculation of enthalpy of mixing of liquid water with the protein. Masses of lysozyme samples were in the range 12-60 mg. A typical time of a calorimetric experiment was about 5 days. Sorption experiments were performed at 25, 40, and 50 °C, desorption at 25 and 40 °C. In the desorption experiments40 a nonsaturated solution of Mg(NO3)2 was used as a water vapor sink. To compare sorption calorimetric measurements with static measurements on sorption of water on lysozyme, an isopiestic method was used. Lysozyme samples were equilibrated with water vapor from saturated salt solutions at 25 (0.3 °C over 5-7 days. The following salts were used: LiCl, KCH3COO, NaI, Mg(NO3)2, NaBr, NaCl, (NH4)2SO4, and KNO3. The masses of lysozyme samples used in the equilibration were in the range 3-10 mg. The obtained protein-water mixtures were weighed to obtain the sorption isotherm and were then used in DSC experiments (the DSC results will be presented in a separate paper). Results and Discussion Sorption Results. We performed sorption calorimetric experiments on lysozyme at 25, 40, and 50 °C. The sorption isotherm of lysozyme (water activity as a function of water content) at 25 °C is presented in Figure 1. Since sorption of water in the calorimetric experiment is a dynamic process (water content changes during the experiments), the obtained data can be influenced by kinetic effects. To check this, we also performed static sorption experiments. Lysozyme samples were equilibrated with water vapor from saturated salt solutions with known water activities (the isopiestic method). These results are also presented in Figure 1. The sorption isotherms obtained by the two different methods are in good agreement at high water contents. At low water contents (up to 8 wt %) the activities obtained calorimetrically are higher than the activities obtained by equilibration with water vapor produced by salt solutions. In other words, at the same water activity, the mass of absorbed water was lower in the calorimetric experiments
Figure 2. Partial molar enthalpy of mixing of water measured in a sorption calorimetric experiment at 40 °C. The numbers 1, 2, 3 denote different regimes of sorption (see text for details).
than at the equilibrium. The reason for this is a smaller characteristic time for the water sorption in the calorimetric experiments (hours) compared to days for the isopiestic experiments. This indicates that the kinetics of sorption is slow in the region of 0-8 wt % of water. In the calorimetric experiments performed at 40 °C, the sorption isotherms obtained at different rates of water sorption also differed from each other in a similar composition range, while at higher water content they were in agreement. The partial molar enthalpy of mixing of water Hm w was measured simultaneously with the measurements of water activity in sorption calorimetric experiments. The results of the measurements at 40 °C are presented in Figure 2. The values of Hm w are very exothermic in the beginning of sorption (about 15 kJ/mol H2O) while at the end the enthalpy becomes endothermic. One should stress that these values correspond to the mixing of protein with liquid water, not to the sorption of water from vapor, which is exothermic in the whole range of concentrations because of the highly negative value of enthalpy of condensation of pure water Hcond w : cond Hsorp + Hmix w ) Hw w
(1)
The enthalpy data obtained by sorption calorimetry have better resolution and cover a wider composition and temperature range than the data from previous studies.9,11,12 This allowed us to use the present data for deeper analysis of the processes that occur during hydration of lysozyme. To give the complete thermodynamic characteristic of the processes corresponding to the different regimes of sorption, we shall also use the data on heat capacity of the lysozyme-water system.21 The enthalpy curve presented in Figure 2 can be divided into three parts corresponding to three regimes of sorption. Although the changes on the enthalpy curves at the changes of the regimes are not very pronounced, they are well reproducible in different experiments and at different conditions. The processes of hydration of proteins were divided into several regimes before12,13,21 but the basis for the division was mostly the diversity of the energies of interaction of water with sorption sites. Our model also includes aggregation properties of lysozyme and the glass transition. In the discussion below the composition ranges of the regimes of sorption are given for 40 °C (as in Figure 2);
Lysozyme-Water Interactions the temperature dependence of the transitions is discussed below in the phase diagram section. Regime 1: 0-8 wt % of Water. The main features of regime 1 are the following: highly exothermic Hm w , slow sorption of water, and very low heat capacity.21 To explain these features one needs to take into account not only protein-water but also protein-protein interactions. At higher water contents the voids between protein molecules are filled with water. At low water contents, corresponding to regime 1, the amount of water present in the system is not high enough to fill these voids. As a consequence, protein molecules should specifically adjust their positions and probably their shapes to reduce the volume of the system. The protein-protein interactions can include association of hydrophobic elements, formation of hydrogen bonds between polar side chain and backbone atoms, and ionic interactions (salt bridges) between oppositely charged amino acid side chains.30 These interactions can cause reversible changes not only in the tertiary but also in the secondary structure of proteins.31 In particular, the β-sheet content in lysozyme increases from about 18% in aqueous solution to about 44% in solid lyophilized form.32 The increase of β-sheet content is probably due to a better steric match between β-sheets compared to R-helixes. These facts show that interactions between protein molecules are very strong at low water contents. To penetrate between the protein-protein interface, water molecules should break the strong ionic and hydrogen bounds. As a result the structures of protein molecules can undergo some changes. Since the content of water (which can act as a plasticizer1) is very low, the mobility of the lysozyme molecules is low and hence all rearrangements of the structural units are slow. This explains the slow kinetics of sorption of water in regime 1. The fact that lysozyme is in the glassy (rigid) state at the low water content explains other features of regime 1, notably the low heat capacity21 and exothermic enthalpy of sorption (Figure 2). In a previous study we found that during sorption of water on a glassy material (the glassy lamellar structure formed by octyl maltoside), the enthalpy of mixing was highly exothermic.37 The particular value of Hm w was about 10 kJ/mol in the whole range of existence of the glassy state. This value was rather constant due to the fact that water molecules came in contact only with OH groups of the maltoside during the whole process of sorption. For lysozyme, a diversity of charged and hydrophilic groups are present, and therefore Hm w changes during sorption of water in regime 1. Regime 2: 8-19% of Water. The main features of regime 2 are the following: Hm w increases from negative to positive values, aw increases strongly, and Cp increases gradually from very low values in the beginning of the regime to very high values at the end.21 The strong increase of the heat capacity may be caused by an increase of mobility of the structural units of lysozyme, in other words it can be interpreted in terms of a transition of lysozyme molecules from a rigid (glassy) state to a flexible (elastic) state. Indeed, a glasslike dynamic transition in lysozyme was observed with several techniques. Gregory1 summarized results of several experimental studies and came to the conclusion that lysozyme undergoes a glasslike transition at a water content of 0.12h (about 11 wt %) at 25 °C. This water content is within regime 2 in the present study. A glass transition is a typical feature of both synthetic polymers and biopolymers. It is also observed in some nonpolymeric materials.37 In a DSC curve a glass transition is usually seen as a step, indicating an increase of heat capacity. During isothermal sorption of water a glass transition results in 37 a step on the Hm w curve. In the case of lysozyme, both Cp and
J. Phys. Chem. B, Vol. 108, No. 49, 2004 19039 Hm w change not in a stepwise fashion but gradually over the wide range of compositions corresponding to regime 2. One should stress that the wide range of the glass transition is not a kinetic effect caused by a deviation from equilibrium behavior, but an immanent property of the lysozyme-water system. Yang and Rupley21 conducted their isothermal experiments at static conditions but obtained a nonstepwise change of the heat capacity. In the present sorption experiments in regime 2, the calorimetric results were in agreement with static isopiestic measurements (Figure 1). One way to explain the difference in the glass transition behavior between lysozyme and synthetic polymers would be by the fact that different groups having different properties are present in proteins. Therefore the mobility of different groups can change at different water contents. On the other hand, there are data in the literature showing that the glass transition in denatured proteins gives a well-pronounced step of Cp.17,18 This indicates that the main reason for the nonstepwise change of the properties of proteins upon glass transition lies not in the heterogeneity of the proteins but in their globular structure. In the denatured state the chains belonging to different protein molecules are in close contact with each other and therefore the increase of mobility occurs cooperatively. In the native state the protein chains are folded into a tertiary structure, which decreases protein-protein contacts compared to the denatured state. After the first regime of sorption, the ionic and very hydrophilic sites on the surface of protein molecules are occupied by water molecules, which makes interactions between protein molecules weaker, while the strength of the intramolecular interactions does not change much. This can lead to a situation where the protein molecules undergo glass transition independently of each other. In conjunction, we propose the following mechanism of the glass transition in lysozyme: a molecule of the protein in a glassy state (rigid molecule) adsorbs a certain number of water molecules n and turns into a flexible (elastic) molecule:
Rigid + nH2O ) Flexible
(2)
For one lysozyme molecule this chemical reaction-like transition occurs cooperatively, while for the whole system of molecules the transition occurs not simultaneously but gradually, reflecting gradual changes of temperature or water content. During the transition both rigid and flexible molecules are present and the amount of flexible molecules increases with the increase of water content. The change of β-sheet content in lysozyme molecules may also occur during transition 2, but the amount of available experimental data does not allow us to make a definite conclusion about this. One should also note that transition 2 cannot be considered as a phase transition since the water activity is not constant during the transition. The mixture of rigid and flexible molecules can be considered as a solid solution, and therefore the properties of the system (aw and Hm w ) are changing during water sorption although the same process occurs over the whole composition range of regime 2. Regime 3: Water Content AboVe 19 wt %. The main features of regime 3 are the following: endothermic values of Hm w , high values of aw, and high values of Cp. All protein molecules are in the flexible state, and further hydration is driven by entropy (Sm w is positive). At high water activity (above 0.95) all processes in the sorption experiments become very slow and the obtained values of enthalpy less accurate, therefore to study hydration at these water activity values a special modification of the method, desorption calorimetry,40 should be used. Desorption Results. Desorption experiments were performed at 25 and 40 °C and started from about 50 wt % mixture of
19040 J. Phys. Chem. B, Vol. 108, No. 49, 2004
Figure 3. Activity of water in desorption (solid line) and sorption (dashed line) experiments versus water content at 25 °C.
Figure 4. Partial molar enthalpy of mixing of water measured in a desorption experiment at 25 °C.
lysozyme and water. During experiments the water content decreased until it reached a value at which the water activity of the sample was the same as the water activity in the solution of Mg(NO3)2 used as a water vapor sink.40 The activity of water registered in a desorption experiment at 25 °C is shown in Figure 3. In the beginning of the process of desorption the activity of water is very close to one, which is close to the value observed at the end of the sorption experiment. Then at about 35 wt % of water, aw drops and its value becomes much lower than in the sorption experiment. Upon further desorption the activity of water remains lower than that during sorption (in other words, at the same water activity the water content is higher in the case of desorption). To propose an explanation of this phenomenon, one has to use the data on enthalpy of mixing obtained simultaneously in the same experiment. Although water is removed from the system in the desorption experiments, enthalpy data in Figure 4 are presented as enthalpy of mixing (i.e., enthalpy of addition of water to the system) data in order to consider the same variable Hm w obtained during sorption and desorption. At the composition of the sharp fall in the activity of water, a step is observed in the enthalpy curve (presented in Figure 4). Hm w changes from slightly positive values (close to zero within the uncertainty of the measurement) at higher water contents to negative values at lower water contents. We propose the following explanation of the observed phenomena. Above a water content of 35 wt % in the system there is a certain
Kocherbitov et al. amount of water usually referred to as “free water”. Free water by definition has properties which are close to those of pure water. Water molecules, which constitute free water, are mostly in contact with each other but not with protein molecules. Therefore the observed activity of water is close to one as in pure water (Figure 3) and the partial molar enthalpy of mixing of water is close to 0 since the process of mixing is essentially the transfer of water molecules from pure water to free water, which has similar properties. At water contents lower than 35 wt %, free water disappears and only bound water is present in the system. Bound water interacts strongly with the protein, therefore Hm w is negative (exothermic) and decreases further upon removal of water, because at lower water contents there are more hydrophilic sites not occupied by water molecules. The changes of the properties of hydration at 35 wt % of water reflect the disappearance of free water from the sample upon desorption. During sorption these changes were not observed. We suggest that the reason for this is in the aggregation properties of lysozyme. Molecules of lysozyme in the beginning of the sorption experiments are in a highly aggregated state. Interactions between protein molecules at low water content are probably very specific, which would imply that the positions of the protein molecules should be specifically adjusted to match each other’s surfaces. This takes place only if the experiment starts from a highly aggregated state of proteins, i.e., during sorption of water started with dry protein. The initial state of the sample in a desorption experiment is a solution of lysozyme in water where aggregation is less pronounced (although small aggregates, for example, dimers, may still be present) and hence most of the protein surface is available for contact with water. When water is removed from the system during desorption, protein molecules come into closer contact with each other but the same aggregates as those present in the sorption experiments are not formed. The formation of very large aggregates with very specific orientations of protein molecules probably takes place over a longer time scale than a desorption experiment. As a result of the strong interactions between the surfaces of protein molecules in sorption experiments, some parts of these surfaces are excluded from contact with water. This means that aggregation causes a decrease of protein surface accessible for water. This, in turn, means that at the same water activity an aggregated protein can accommodate fewer water molecules than a disaggregated one, in other words the activity of water in sorption experiments is higher than that in desorption ones (cf. Figure 3). The sorption and desorption isotherms then can be characterized as sorption isotherms of aggregated and disaggregated lysozyme, respectively. Another difference between sorption and desorption isotherms is the absence of the step on the activity curve (which indicates the disappearance of free water in case of desorption) during sorption. The free water should certainly appear at some composition during sorption of water, but this is not clearly observed in the sorption isotherm. The reason for this is probably the gradual disaggregation of lysozyme molecules during the uptake of water. When all sorption sites of aggregated protein are occupied, further absorbed water causes disruption of protein-protein hydrogen bonds and occupies newly appearing sorption sites. The composition at which free water disappears recalculated per mole ratio gives 420 water molecules per lysozyme molecule. In other words, 420 water molecules are bound to a lysozyme molecule. The issue of the amount of bound water is closely related to that of the amount of water sufficient for the
Lysozyme-Water Interactions
Figure 5. Two hysteresis loops in the sorption isotherm in the system lysozyme-water at 25 °C. Arrows denote directions of experiments (sorption or desorption). The dashed line denotes extrapolation of the desorption isotherm to lower water contents (not reached in experiments). The curve between sorption and desorption isotherms is the equilibrium sorption isotherm determined by the isopiestic method.
monolayer coverage of the protein surface and also to the problem of nonfreezing water.1,2 Strictly speaking, the amount of water corresponding to these three types can be different. In this study we determined the amount of bound water, which does not necessarily correspond to monolayer coverage. The amount of nonfreezing water determined by DSC,42 IR spectroscopy,42 and NMR42,43 is much lower than the amount of bound water presented here. This can be explained by the fact that the amount of nonfreezing water depends not only on the interactions of water with proteins but also on interactions between water molecules in crystals of ice. In other words, the amount of nonfreezing water reflects the competition between crystallization of water and binding of water to proteins. Therefore, even some water molecules weakly bound to the protein surface can be removed from the surface of protein and then crystallize. The method of determination of the amount of bound water presented here is based only on monitoring the changes of thermodynamic properties (aw and Hm w ) of water in the system and therefore gives a more correct value of the amount of bound water. Hysteresis. Hysteresis of the sorption isotherm is a typical feature of protein-water systems.1,24 The results presented above suggest that this phenomenon also appears in the mixture of lysozyme with water. Depending on the direction of water sorption, the history of the sample, and the method of investigation, we obtained different values of the activity of water at the same water content. According to stability theory, only one state of the sample (which corresponds to the global minimum of Gibbs energy) should be stable at a given composition, other states should be metastable or nonstable. We believe that the sorption isotherm obtained by the isopiestic method (asterisks in Figure 1) represents stable states since the samples were equilibrated for a long time, while the data obtained by sorption calorimetry may sometimes represent metastable states. For most of the sorption isotherm (above 8% of water) the data obtained by sorption calorimetry are in good agreement with the isopiestic data. These two sorption isotherms below 8 wt % of water form a small hysteresis loop (Figure 5). The reason of the hysteresis at this water content is not the direction of the process of sorption but its rate. The slow penetration of water molecules between rigid protein molecules would affect the sorption/ desorption processes in either of the directions. Another (large)
J. Phys. Chem. B, Vol. 108, No. 49, 2004 19041
Figure 6. Phase diagram of the lysozyme-water system. Rigid and Flex stand for rigid and flexible conformations of lysozyme molecules, respectively. xw is water content in the system (in wt %), h is the mass ratio (g of H2O/g of protein).
hysteresis loop is formed by sorption (equilibrium) and desorption isotherms. The difference between the two isotherms is caused by the direction of the process and its history (aggregated or disaggregated sample in the beginning of the experiment). Strictly speaking, the existence of this hysteresis loop also depends on the rate of the experiment (if desorption would be infinitely slow, then the two isotherms would be identical), but the rate dependence is not as pronounced in the case of the large loop as in the case of the small loop. Phase Diagram. We have conducted sorption experiments at three temperatures and desorption experiments at two temperatures. From the temperature dependences of the observed events and transitions we have composed the phase diagram of the lysozyme-water system at low water contents (Figure 6). The transitions associated with changes of rigidity of the protein occur at lower water contents at higher temperatures. This behavior is similar to that of the glass transition in the octyl maltoside-water system.37 The difference is that in the considered temperature range the temperature dependence of the glass transition composition in lysozyme is less pronounced (the direction of the curves is more vertical). It is not clear if this transition can occur in the dry protein at all on account of the presence of a number of charged groups in a protein molecule. During sorption of water the first water molecules are tightly bound to the charged groups and therefore cannot act as a plasticizer. The number of water molecules bound to charged groups changes little with temperature, which gives a weak dependence of the beginning of the glass transition composition with temperature. In desorption experiments we did not observe any temperature dependence of the amount of bound water at all (vertical line in the phase diagram). This phenomenon can be explained in two different ways. The first is based on the assumption that the amount of bound water can be very close to monolayer coverage of protein molecules, which is determined by the geometry of the protein globule, which does not change significantly with temperature. The second explanation is the compensation of two different factors. The amount of water bound to a separate protein molecule can decrease with increasing temperature. On the other hand, some aggregates (dimers) can disaggregate at higher temperature providing more surface for binding of water. The balance between these two processes may result in the apparent absence of change of the amount of bound water upon temperature change.
19042 J. Phys. Chem. B, Vol. 108, No. 49, 2004 Conclusions The methods of sorption and desorption calorimetry were applied to study the hydration and dehydration of lysozyme. The phase diagram of the system lysozyme-water was determined. According to the proposed model, the hydration of lysozyme occurs in four steps: (1) slow penetration of water into the protein-protein interface, (2) gradual glass transition, which occurs in every protein molecule independently of other molecules, (3) further water uptake with disaggregation of protein aggregates and formation of a monolayer of water, and (4) accumulation of free water. The amount of bound water determined by desorption calorimetry is 420 water molecules per lysozyme molecule. Two hysteresis loops are found on the sorption isotherm. The small hysteresis loop is caused by slow penetration of water into the protein- protein interface at very low water content while the large loop is caused by slow aggregation of protein molecules at higher water contents. Acknowledgment. The authors thank Gerd Olofsson and Tommy Nylander for fruitful discussions. The Knowledge Foundation and the Swedish Research Council (VR) are acknowledged for financial support. References and Notes (1) Gregory, R. B. Protein-solVent interactions; Marcel Dekker: New York, 1995. (2) Rupley, J. A.; Careri, G. AdV. Protein Chem. 1991, 41, 37. (3) Biswal, B. K.; Sukumar, N.; Vijayan, M. Acta Crystallogr. Sect. D: Biol. Crystallogr. 2000, 56, 1110. (4) Hnojewyj, W. S.; Reyerson, L. H. J. Phys. Chem. 1959, 63, 1653. (5) Hnojewyj, W. S.; Reyerson, L. H. J. Phys. Chem. 1961, 65, 1694. (6) Leeder, J. D.; Watt, I. C. J. Colloid Interface Sci. 1974, 48, 339. (7) D’Arcy, R. L.; Watt, I. C. Trans. Faraday Soc. 1970, 66, 1236. (8) Luscher-Mattli, M. Thermodynamic parameters of biopolymerwater systems. In Thermodynamic data for biochemistry and biotechnology; Hinz, H.-J., Ed.; Springer-Verlag: Berlin, Germany, 1986; p 276. (9) Luscher-Mattli, M.; Ruegg, M. Biopolymers 1982, 21, 419. (10) Lioutas, T. S.; Baianu, I. C.; Steinberg, M. P. J. Agric. Food Chem. 1987, 35, 133. (11) Bone, S. Phys. Med. Biol. 1996, 41, 1265. (12) Smith, A. L.; Shirazi, H. M.; Mulligan, S. R. Biochim. Biophys. Acta: Protein Struct. Mol. Enzymol. 2002, 1594, 150.
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