Stabilization of firefly luciferase against thermal stress by osmolytes

The effects of osmolytes, including sucrose, sorbitol and proline on the remaining activity of firefly luciferase were measured. Heat inactivation studies showed that these osmolytes maintain the remaining activity of enzyme and increase activation energy of thermal unfolding reaction. Fluorescence and circular dichroism (CD) experiments showed changes in secondary and tertiary structure of firefly luciferase, in the presence of sucrose, sorbitol and proline. The unfolding curves of luciferase (obtained by far-UV CD spectra), indicated an irreversible thermal denaturation and raising of the midpoint of the unfolding transition temperature (T(m)) in the presence of osmolytes.     

Mechanism of solvent induced thermal stabilization of papain

In the present study an attempt is made to elucidate the effects of various cosolvents, such as sorbitol, sucrose, xylose and glycerol, on papain. The stabilizing effects of these cosolvents on the structure and function of papain is determined by the activity measurements, fluorescence spectroscopy and differential scanning calorimetry (DSC). The enzyme activity measurements indicate several fold increase in the thermal stability of the enzyme in all the cosolvents used. The thermal denaturation studies of papain in presence of various concentrations of cosolvents indicated a shift in the apparent thermal denaturation temperature (app Tm) suggesting increased thermal stability of papain in presence of cosolvents. The app Tm shifted from a control value of 83+/-1 degrees C to a value of >90+/-1 degrees C in presence of 40% sorbitol. The DSC thermogram for native papain can be clearly deconvoluted into two transitions corresponding to left and right domain and in presence of cosolvents both transitions A and B shift to higher temperature. Maximum stabilization was seen in case of 30% sorbitol where the thermal transition temperatures increased compared to control. The results from partial specific volume measurements of papain in presence of cosolvents suggest that the preferential interaction parameter (xi3) was negative in all cosolvents and maximum hydration was observed in the case of glycerol where the preferential interaction parameter was 0.165g/g. These above results suggest that there is a considerable increase in the thermal stability of papain in presence of these cosolvents as a result of preferential hydration.     

Sorbitol counteracts temperature- and chemical-induced denaturation of a recombinant α-amylase from alkaliphilic Bacillus sp. TS-23

Enzymes are highly complex systems with a substantial degree of structural variability in their folded state. In the presence of cosolvents, fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways; alternatively, certain conformations can be stabilized or destabilized. To understand the contribution of osmolytes to the stabilization of structural changes and enzymatic activity of a truncated Bacillus sp. TS-23 α-amylase (BACΔNC), we monitored amylolytic activity, circular dichroism, and fluorescence as a function of osmolytes. In the presence of trimethylamine N-oxide (TMAO) and sorbitol, BACΔNC activity was retained significantly at elevated temperatures. As compared to the control, the secondary structures of this enzyme were essentially conserved upon the addition of these two kinds of osmolytes. Fluorescence results revealed that the temperature-induced conformational change of BACΔNC was prevented by TMAO and sorbitol. However, glycerol did not provide profound protection against thermal denaturation of the enzyme. Sorbitol was further found to counteract guanidine hydrochloride- and SDS-induced denaturation of BACΔNC. Thus, some well-known naturally occurring osmolytes make a dominant contribution to the stabilization of BACΔNC.     

Effects of water-miscible solvents and polyhydroxy compounds on the structure and enzymatic activity of thermolysin

The effect of organic solvents (n-propanol, isopropanol, dimethylformamide and dimethylsulfoxide) on the structure, activity and stability of thermolysin was the focus of this investigation. Results show the ability of the solvents to cause mixed inhibition of thermolysin, which was indicated by kinetic and structural studies (near-UV CD spectra and intrinsic fluorescence). Inhibitory effect of the solvents increased with increments in solvents logP. Thermoinactivation of thermolysin was studied at 80 degrees C in 50% of solvents and showed that with the increase in solvent hydrophobicity, thermal stability of the enzyme decreased. For the stabilization of thermolysin at high temperature, additives such as glycerol, sorbitol and trehalose were employed. In the presence of DMF with a relatively low logP, trehalose was shown to be a good stabilizer, whereas glycerol had a marked stabilization effect in the presence of n-propanol and isopropanol with a relatively high logP. Consequently, it was concluded that the stabilizing effect of additives can be correlated with the logP of solvents.     

Thermal stability of <Emphasis Type="Italic">α</Emphasis>-amylase in aqueous cosolvent systems

The activity and thermal stability of α-amylase were studied in the presence of different concentrations of trehalose, sorbitol, sucrose and glycerol. The optimum temperature of the enzyme was found to be 50 ± 2°C. Further increase in temperature resulted in irreversible thermal inactivation of the enzyme. In the presence of cosolvents, the rate of thermal inactivation was found to be significantly reduced. The apparent thermal denaturation temperature (T _m )_app and activation energy (E _a ) of α-amylase were found to be significantly increased in the presence of cosolvents in a concentration-dependent manner. In the presence of 40% trehalose, sorbitol, sucrose and glycerol, increments in the (T _m )_app were 20°C, 14°C, 13°C and 9°C, respectively. The E _a of thermal denaturation of α-amylase in the presence of 20% (w/v) trehalose, sorbitol, sucrose and glycerol was found to be 126, 95, 90 and 43 kcal/mol compared with a control value of 40 kcal/mol. Intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence studies indicated that thermal denaturation of the enzyme was accompanied by exposure of the hydrophobic cluster on the protein surface. Preferential interaction parameters indicated extensive hydration of the enzyme in the presence of cosolvents.     

The effect of some osmolytes on the activity and stability of mushroom tyrosinase

The thermodynamical stability and remained activity of mushroom tyrosinase (MT) fromAgaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40°C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obeye the first order law. Inactivation rate constant (k_inact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of k_inact(0.7×10⁻⁴s⁻¹) in comparison with their absence (2.5×10⁻⁴s⁻¹). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodyanamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.     

Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions

Osmolytes of the polyol series are known to accumulate in biological systems under stress and stabilize the structures of a wide variety of proteins. While increased surface tension of aqueous solutions has been considered an important factor in protein stabilization effect, glycerol is an exception, lowering the surface tension of water. To clarify this anomalous effect, the effect of a series of polyols on the thermal stability of a highly thermolabile two domain protein yeast hexokinase A has been investigated by differential scanning calorimetry and by monitoring loss in the biological activity of the enzyme as a function of time. A larger increase in the T(m) of domain 1 compared with that of domain 2, varying linearly with the number of hydroxyl groups in polyols, has been observed, sorbitol being the best stabilizer against both thermal as well as urea denaturation. Polyols help retain the activity of the enzyme considerably and a good correlation of the increase in T(m) (DeltaT(m)) and the retention of activity with the increase in the surface tension of polyol solutions, except glycerol, which breaks this trend, has been observed. However, the DeltaT(m) values show a linear correlation with apparent molal heat capacity and volume of aqueous polyol solutions including glycerol. These results suggest that while bulk solution properties contribute significantly to protein stabilization, interfacial properties are not always a good indicator of the stabilizing effect. A subtle balance of various weak binding and exclusion effects of the osmolytes mediated by water further regulates the stabilizing effect. Understanding these aspects is critical in the rational design of stable protein formulations.     

Effects of trehalose and sorbitol on the activity and structure of Pseudomonas cepacia lipase: spectroscopic insight

The stability of enzymes with no reduction in their catalytic activity still remains a critical issue in industrial applications. Naturally occurring osmolytes are commonly used as protein stabilizers. In this study we have investigated the effects of sorbitol and trehalose on the structural stability and activity of Pseudomonas cepacia lipase (PCL), using UV-visible, circular dichroism (CD) and fluorescence spectroscopy. Surface plasmon resonance (SPR) technique was used to trace changes in the refractive index and dielectric constant of the environment. The results revealed that catalytic activity and intrinsic fluorescence intensity of PCL increased in the presence of both osmolytes. Far-UV CD spectra indicated that the protein has undergone some conformational changes upon interacting with these osmolytes. Increasing the concentration of sorbitol led to changes in the refractive index and consequently the dielectric constant of environment; whereas in the case of trehalose, such changes were not significant. Unfavorable interactions of trehalose with protein surface induced higher preferential exclusion from the enzyme-water interface than that of sorbitol. Results of this report could give further insights about the stabilization mechanism of osmolytes.     

Role of osmolytes as chemical chaperones during the refolding of aminoacylase.

The refolding and reactivation of aminoacylase is particularly difficult because of serious off-pathway aggregation. The effects of 4 osmolytes--dimethylsulphoxide, glycerol, proline, and sucrose--on the refolding and reactivation of guanidine-denatured aminoacylase were studied by measuring aggregation, enzyme activity, intrinsic fluorescence spectra, 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra, and circular dichroism (CD) spectra. The results show that all the osmolytes not only inhibit aggregation but also recover the activity of aminoacylase during refolding in a concentration-dependent manner. In particularly, a 40% glycerol concentration and a 1.5 mol/L sucrose concentration almost completely suppressed the aminoacylase aggregation. The enzyme activity measurements revealed that the influence of glycerol is more significant than that of any other osmolyte. The intrinsic fluorescence results showed that glycerol, proline, and sucrose stabilized the aminoacylase conformation effectively, with glycerol being the most effective. All 4 kinds of osmolytes reduced the exposure of the hydrophobic surface, indicating that osmolytes facilitate the formation of protein hydrophobic collapse. The CD results indicate that glycerol and sucrose facilitate the return of aminoacylase to its native secondary structure. The results of this study suggest that the ability of the various osmolytes to facilitate the refolding and renaturation of aminoacylase is not the same. A survey of the results in the literature, as well as those presented here, suggests that although the protective effect of osmolytes on protein activity and structure is equal for different osmolytes, the ability of osmolytes to facilitate the refolding of various proteins differs from case to case. In all cases, glycerol was found to be the best stabilizer and a folding aid.     

Co-solvent mediated thermal stabilization of chondroitinase ABC I form Proteus vulgaris

Chondroitinase ABC I (cABC I) from Proteus vulgaris cleaves glycosaminoglycan chains which are responsible for most of the inhibition of axon regrowth in spinal cord injury. The clinical utilization of this enzyme is mainly limited by its thermal instability. This study has been undertaken to determine the effects of glycerol, sorbitol and trehalose on cABC I activity and thermal stability. The results indicated that the enzyme catalytic activity and intrinsic fluorescence intensity increased in the presence of these cosolvents whereas no considerable conformational changes observed in far-UV CD spectra. Thermal CD experiment revealed an increase in T(m) of cABC I in the presence of cosolvents which was significant for trehalose. Our results support the idea that cABC I has stabilized in the presence of glycerol, sorbitol and trehalose. Therefore, the use of these cosolvents seems to be promising for improvement in shelf-life and clinical applications of this drug enzyme.     

Molar concentrations of sorbitol and polyethylene glycol inhibit the Plasmodium aquaglyceroporin but not that of E. coli: Involvement of the channel vestibules

The aquaglyceroporins of Escherichia coli, EcGlpF, and of Plasmodium falciparum, PfAQP, are probably the best characterized members of the solute-conducting aquaporin (AQP) subfamily. Their crystal structures have been elucidated and numerous experimental and theoretical analyses have been conducted. However, opposing reports on their rates of water permeability require clarification. Hence, we expressed EcGlpF and PfAQP in yeast, prepared protoplasts, and compared water and glycerol permeability of both aquaglyceroporins in the presence of different osmolytes, i.e. sucrose, sorbitol, PEG300, and glycerol. We found that water permeability of PfAQP strongly depends on the external osmolyte, with full inhibition by sorbitol, and increasing water permeability when glycerol, PEG300, and sucrose were used. EcGlpF expression did not enhance water permeability over that of non-expressing control protoplasts regardless of the osmolyte. Glycerol permeability of PfAQP was also inhibited by sorbitol, but to a smaller extent, whereas EcGlpF conducted glycerol independently of the osmolyte. Mixtures of glycerol and urea passed PfAQP equally well under isosmotic conditions, whereas under hypertonic conditions in a countercurrent with water, glycerol was clearly preferred over urea. We conclude that PfAQP has high and EcGlpF low water permeability, and explain the inhibiting effect of sorbitol on PfAQP by its binding to the extracellular vestibule. The preference for glycerol under hypertonic conditions implies that in a physiological setting, PfAQP mainly acts as a water/glycerol channel rather than a urea facilitator.     

Thermal Destabilization of Stem Bromelain by Trehalose

Trehalose, a naturally occurring osmolyte, is considered as a universal protein stabilizer. We investigated the effect of the disaccharides, trehalose and sucrose, on the thermal stability and conformation of bromelain. To our surprise, bromelain in the presence of 1 M trehalose/sucrose was destabilized under thermal stress. The average Tm values as determined by UV spectroscopy and CD spectropolarimetry decreased by 5° and 7°C for bromelain in 1 M sucrose or trehalose solutions, respectively. The enzyme was also found to inactivate faster at 60°C in the presence of these osmolytes. The tertiary and secondary structure of bromelain undergoes small changes in the presence of sucrose/trehalose. Studies on the binding of these osmolytes with the native and the heat denatured enzyme revealed that sucrose/trehalose lead to preferential hydration of the denatured bromelain as compared to the native one, hence stabilizing more the denatured conformation. This is perhaps the first report on the destabilization of a protein by trehalose.     

Interaction of selected cosolvents with bovine alpha-lactalbumin

Alpha-lactalbumin constitutes about 3% of bovine milk proteins. The preferential solvent interactions between selected cosolvents (sorbitol, sucrose and glycerol) and alpha-lactalbumin at pH 7.5 was determined using precision densitimetry. The preferential interaction parameter (xi(3)) and other thermodynamic parameters were calculated at different solvent concentrations. The xi(3) parameter was maximum at 30%, 45% and 40% (w/v) concentrations with the values of -0.282g/g, -0.171g/g and -0.299g/g for sorbitol, sucrose and glycerol, respectively. Thus the principal driving energy in the system being preferential hydration and mutual exclusion of bulk solvent. There was only a marginal change in the CD spectra of the protein with these cosolvents indicating the integrity of secondary structures. The results of thermal denaturation measurements indicated an increase in thermal stability of alpha-lactalbumin with these cosolvents. In the presence of 30% sorbitol there was an increase in the apparent thermal transition temperature (apparent T(m)) from 65 to 71 degrees C. These results indicate that the selected cosolvents in this study stabilizes alpha-lactalbumin without altering the structure of the protein.     

Co-solvent effects on structure and function properties of savinase: solvent-induced thermal stabilization

The industrial utilization of savinase is mainly constrained by its stability limitations. In the present study, the irreversible thermoinactivation of savinase has been evaluated at 70 degrees C, and various possible mechanisms for irreversible thermoinactivation of savinase were examined. The main process seemed to be autodigestion of savinase at higher temperatures. To improve the thermal stability of the enzyme, the effect of two co-solvents (sorbitol and trehalose) on the enzyme's activity and stability was investigated. Both osmolytes prevented the autolysis of savinase at 70 degrees C without inactivating the enzyme; furthermore, the structural and kinetic stabilities of the enzyme increased in the presence of additives.     

Enthalpic and entropic effects of salt and polyol osmolytes on site-specific protein-DNA association: the integrase Tn916-DNA complex

The effect of low molecular-weight compounds on the equilibrium constant K(A) can be used to explore the energetics and molecular mechanism of protein-DNA interactions. Here we use the complex composed of the integrase Tn916 DNA-binding domain and its target DNA duplex to investigate the effects of salt and the nonionic osmolytes glycerol and sorbitol on sequence-specific protein-DNA association. Increasing Na(+) concentration from 0.12 to 0.32 M weakens the binding affinity by a factor of 20. The decrease of affinity is dominated by a large loss of binding enthalpy but only a small loss of binding entropy. This contrasts the concept that the salt-induced weakening of protein-DNA binding is mainly entropic. The large enthalpy loss is discussed in the light of recent views about the nature of the general salt effect. Addition of up to 2.5 M sorbitol and up to 3.3 M glycerol causes a slight increase of the binding affinity. However, both osmolytes lead to a large enthalpy gain and a similarly large entropy loss. This intriguing enthalpy-entropy compensation can be explained in part by an enthalpic chelate effect: The osmolyte tightens the structure of the protein-DNA complex whereby the formation of enthalpically favorable noncovalent interactions is promoted at the entropic cost of a more rigid complex. The results were obtained by isothermal titration calorimetry. They are supported by kinetic experiments showing that the rate of formation of the complex is reduced by salt, but the rate of complex dissociation is not. Glycerol and sorbitol reduce both rates in line with an only small effect on complex stability. This work clarifies the thermodynamic and kinetic response of a novel protein-DNA complex to increased salt and the presence of two common, nonionic osmolytes.     

Osmolyte effect on the stability and folding of a hyperthermophilic protein

Proteins are known to be stabilized by naturally occurring osmolytes such as amino acids, sugars, and methylamines. Here, we examine the effect of trimethylamine-N-oxide (TMAO) on the conformational stability of ribonuclease HII from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII), which inherently possesses high conformational stability. Heat- and guanidine hydrochloride-induced unfolding experiments demonstrated that the conformational stability of Tk-RNase HII in the presence of 0.5M TMAO was higher than that in the absence of TMAO at all examined temperatures. TMAO affected the unfolding and refolding kinetics of Tk-RNase HII to a similar extent. These results indicate that proteins are universally stabilized by osmolytes, regardless of their robustness, and suggest a stabilization mechanism by osmolytes, caused by the unfavorable interaction of osmolytes with protein backbones in the denatured state. Our results also imply that the basic protein folding principle is not dependent on protein stability and evolution.     

Effects of glycerol on the compaction and stability of the wild type and mutated rabbit muscle creatine kinase

All organisms have developed detect, repair, regulation, and stabilization mechanisms to survive from cellular and molecular damage induced by diverse stresses. Among them, the accumulation of osmolytes is a common mechanism evolved by cells to maintain cell volume and stabilize macromolecules against various environmental stresses. The molecular mechanisms by which osmolytes stabilize proteins and prevent aggregation have been well-established. However, little is known about the effects of osmolytes on mutated or damaged proteins. In this research, we investigated the effects of glycerol on the activity, structure, and stability of the wild type (WT) and D54G CK under normal and extreme (high temperature) conditions. It was found that glycerol had similar effects on the suppression of the aggregation during the refolding of both proteins. Under native conditions, the effect of glycerol on the mutated protein was more obvious than on the WT protein. Glycerol could efficiently force the mutated protein to fold to a state close to the WT protein, and thus stabilize the native state of the mutated protein. Glycerol could also protect both the WT and mutated proteins against heat-induced denaturation. However, the change in the transition free energy of heat-induced inactivation of the WT protein was larger than that of the mutated protein. These results suggested that glycerol might have differential effects on the changes of the chemical potential and the transition free energy of the WT and mutated proteins.     

Polyol osmolytes stabilize native-like cooperative intermediate state of yeast hexokinase A at low pH

Osmolytes produced under stress in animal and plant systems have been shown to increase thermal stability of the native state of a number of proteins as well as induce the formation of molten globule (MG) in acid denatured states and compact conformations in natively unfolded proteins. However, it is not clear whether these solutes stabilize native state relative to the MG state under partially denaturing conditions. Yeast hexokinase A exists as a MG state at pH 2.5 that does not show any cooperative transition upon heating. Does the presence of some of these osmolytes at pH 2.5 help in the retention of structure that is typical of native state? To answer this question, the effect of ethylene glycol (EG), glycerol, xylitol, sorbitol, trehalose and glucose at pH 2.5 on the structure and stability of yeast hexokinase A was investigated using spectroscopy and calorimetry. In presence of the above osmolytes, except EG, yeast hexokinase at pH 2.5 retains native secondary structure and hydrophobic core and unfolds with excessive heat absorption upon thermal denaturation. However, the cooperative structure binds to ANS suggesting that it is an intermediate between MG and the native state. Further, we show that at high concentration of polyols at pH 2.5, except EG, which populates a non-native ensemble, ΔH_cal/ΔH_van approaches unity indicative of two-state unfolding. The results suggest that osmolytes stabilize cooperative protein structure relative to non-cooperative ensemble. These findings have implications toward the structure formation, folding and stability of proteins produced under stress in cellular systems.     

Increased thermal stability of proteins in the presence of naturally occurring osmolytes.

Organisms and cellular systems which have adapted to stresses such as high temperature, desiccation, and urea-concentrating environments have responded by concentrating particular organic solutes known as osmolytes. These osmolytes are believed to confer protection to enzyme and other macromolecular systems against such denaturing stresses. Differential scanning calorimetric (DSC) experiments were performed on ribonuclease A and hen egg white lysozyme in the presence of varying concentrations of the osmolytes glycine, sarcosine, N,N-dimethylglycine, and betaine. Solutions containing up to several molar concentrations of these solutes were found to result in considerable increases in the thermal unfolding transition temperature (Tm) for these proteins. DSC scans of ribonuclease A in the presence of up to 8.2 M sarcosine resulted in reversible two-state unfolding transitions with Tm increases of up to 22 degrees C and unfolding enthalpy changes which were independent of Tm. On the basis of the thermodynamic parameters observed, 8.2 M sarcosine results in a stabilization free energy increase of 7.2 kcal/mol for ribonuclease A at 65 degrees C. This translates into more than a 45,000-fold increase in stability of the native form of ribonuclease A over that in the absence of sarcosine at this temperature. Catalytic activity measurements in the presence of 4 M sarcosine give kcat and Km values that are largely unchanged from those in the absence of sarcosine. DSC of lysozyme unfolding in the presence of these osmolytes also results in Tm increases of up to 23 degrees C; however, significant irreversibly occurs with this protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of high hydrostatic pressure for increasing activity and stability of enzymes

Elevated hydrostatic pressure has been used to increase catalytic activity and thermal stability of alpha-chymotrypsin (CT). For an anilide substrate, characterized by a negative value of the reaction activation volume (DeltaV( not equal)), an increase in pressure at 20 degrees C results in an exponential acceleration of the hydrolysis rate catalyzed by CT reaching a 6.5-fold increase in activity at 4700 atm (4.7 kbar). Due to a strong temperature dependence of DeltaV( not equal), the acceleration effect of high pressure becomes more pronounced at high temperatures. For example, at 50 degrees C, under a pressure of 3.6 kbar, CT shows activity which is more than 30 times higher than the activity at normal conditions (20 degrees C, 1 atm). At pressures of higher than 3.6 kbar, the enzymatic activity is decreased due to a pressure-induced denaturation.Elevated hydrostatic pressure is also efficient for increasing stability of CT against thermal denaturation. For example, at 55 degrees C, CT is almost instantaneously inactivated at atmospheric pressure, whereas under a pressure of 1.8 kbar CT retains its anilide-hydrolyzing activity during several dozen minutes. Additional stabilization can be achieved in the presence of glycerol, which is most effective for protection of CT at an intermediate concentration of 40% (v/v). There has been observed an additivity in stabilization effects of high pressure and glycerol: thermal inactivation of pressure-stabilized CT can be decelerated in a supplementary manner by addition of 40% (v/v) glycerol. The protection effect of glycerol on the catalytic activity and stability of CT becomes especially pronounced when both extreme factors of temperature and pressure reach critical values. For example, at approximately 55 degrees C and 4.7 kbar, enzymatic activity of CT in the presence of 40% (v/v) glycerol is severalfold higher than in aqueous buffer.The results of this study are discussed in terms of the hypotheses which explain the action of external and medium effects on protein structure, such as preferential hydration and osmotic pressure. (c) 1996 John Wiley & Sons, Inc.