Hydration of a glycoprotein: relative water affinity of peptide and glycan moieties

Glycosylation, the most prevalent post-translational modification of proteins, affects a number of physical properties including the interactions with the surrounding aqueous solvent. Such glycan-water interactions have been discussed with respect to the increased solubility generally observed for glycoproteins, but experimental support of this correlation remains sparse. We have applied a two-channel calorimetric method to measure the free energy and enthalpy of hydration at 25 degrees C for the glycoprotein phytase (Phy) and a deglycosylated form (dgPhy) of the same protein. Comparisons of results for Phy and dgPhy show that the polypeptide moiety has a higher affinity for water than the glycans. In fact, at moderate hydration levels (approximately 0.3 g water/g macromolecule) the water uptake appears to be entirely governed by adsorption to the peptide groups. We conclude that strengthened interaction with the solvent is unlikely to be the mechanism underlying the increased solubility and lowered propensity of aggregation often reported to result from the glycosylation of proteins.     

Interrelationship of Steric Stabilization and Self-Crowding of a Glycosylated Protein

In the eukaryotic cell, protein glycosylation takes place in the crowded environment of the endoplasmatic reticulum. With the purpose of elucidating the impact of high concentration on the interactions of glycoproteins, we have conducted a series of small-angle x-ray scattering experiments on the heavily glycosylated enzyme Peniophora lycii phytase (Phy) and its deglycosylated counterpart (dgPhy). The small-angle x-ray scattering data were analyzed using an individual numerical form factor for each of the two glycoforms combined with two structure factors, a hard sphere and a screened coulomb potential structure factor, respectively, as determined by ab initio analysis. Based on this data analysis, three main conclusions could be drawn. First, at comparable protein concentrations (mg/ml), the relative excluded volume of Phy was ∼75% higher than that of dgPhy, showing that the glycans significantly increase excluded-volume interactions. Second, the relative excluded volume of dgPhy increased with concentration, as expected; however, the opposite effect was observed for Phy, where the relative excluded volume decreased in response to increasing protein concentration. Third, a clear difference in the effect of salinity on the excluded-volume interactions was observed between the two glycol forms. Although the relative excluded volume of dgPhy decreased with increasing ionic strength, the relative excluded volume of Phy was basically insensitive to increased salinity. We suggest that protrusion forces from the glycans contribute to steric stabilization of the protein, and that glycosylation helps to sustain repulsive electrostatic interactions under crowded conditions. In combination, this aids in stabilizing high concentrations of glycosylated proteins.     

The effect of glycosylation on interparticle interactions and dimensions of native and denatured phytase

Glycosylation affects the physical properties of proteins in a number of ways including solubility and aggregation behavior. To elucidate the mechanism underlying these effects, we have measured second virial coefficients (A₂) of the heavily glycosylated pheniophora lycii phytase (Phy) and its enzymatically deglycosylated counterpart (dgPhy) in native and in denatured form by means of small angle x-ray scattering. The measured A₂-values show that the native forms of Phy and dgPhy are equally repulsive at the studied pH 8 where A₂ equals 10.9 ± 0.1 × 10⁴ mL mol g⁻². However, when thermally denatured, the A₂ of dgPhy decreases to 9.0 ± 0.2 × 10⁴ mL mol g⁻² whereas it remained unchanged for Phy. In accord with earlier investigations, the p(r)-function measured here suggested that the glycans did not affect the peptide structure of the native protein. Conversely, glycosylation markedly changed the structure of thermally denatured protein. This was evident from the radius of gyration, which increased by 32% for Phy and only 11% for dgPhy on denaturation. We suggest that this expanding effect of the glycans on the denatured protein conformation relies on steric hindrance that limits the range of torsion angles available to the polypeptide.     

Interrelationships of glycosylation and aggregation kinetics for Peniophora lycii phytase

The kinetics of thermally induced aggregation of the glycoprotein Peniophora lycii phytase (Phy) and a deglycosylated form (dgPhy) was studied by dynamic (DLS) and static (SLS) light scattering. This provided a detailed insight into the time course of the formation of small aggregates ( approximately 10-100 molecules) of the enzyme. The thermodynamic stability of the two forms was also investigated using scanning calorimetry (DSC). It was found that the glycans strongly promoted kinetic stability (i.e., reduced the rate of irreversible denaturation) while leaving the equilibrium denaturation temperature, T(d), defined by DSC, largely unaltered. At pH 4.5-5.0, for example, dgPhy aggregated approximately 200 times faster than Phy, even though the difference in T(d) was only 1-3 degrees C. To elucidate the mechanism by which the glycans promote kinetic stability, we measured the effect of ionic strength and temperature on the aggregation rate. Also, the second virial coefficients (B(22)) for the two forms were measured by SLS. These results showed that the aggregation rate of Phy scaled with the concentration of thermally denatured protein. This suggested first-order kinetics with respect to the concentration of the thermally denatured state. A similar but less pronounced correlation was found for dgPhy, and it was suggested that while the aggregation process for the deglycosylated form is dominated by denatured protein, it also involves a smaller contribution from associating molecules in the native state. The measurements of B(22) revealed that dgPhy had slightly higher values than Phy. This suggests that dgPhy interacts more favorably with the buffer than Phy and hence rules out strong hydration of the glycans as the origin of their effect on the kinetic stability. On the basis of this and the effects of pH and ionic strength, we suggest that the inhibition of aggregation is more likely to depend on steric hindrance of the glycans in the aggregated form of the protein.     

Glycoprotein-surfactant interactions: a calorimetric and spectroscopic investigation of the phytase-SDS system

The interactions of sodium dodecyl sulfate (SDS) and two glyco-variants of the enzyme phytase from Peniophora lycii were investigated. One variant (Phy) was heavily glycosylated while the other (dgPhy) was enzymatically deglycosylated. Effects at 24 degrees C of titrating SDS to Phy and dgPhy were studied by Isothermal Titration Calorimetry (ITC) and Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. Comparisons of results for the two variants were used to elucidate glycan-surfactant interrelationships. The CD spectra suggested that both the native and the SDS-denatured states of the two variants were mutually similar, and hence that the denaturation process was structurally equivalent for the two glyco-variants. The denatured state was far from fully unfolded and probably retained a substantial content of native-like structure. Furthermore, it was found that the glycans brought about only a small increase in the resistance towards SDS induced denaturation. The SDS concentration required to denature half of the protein molecules differed less than 1 mM for the two variants. The affinity for SDS of both variants was unusually low. The amount of bound SDS (w/w) at different stages of the binding isotherm was 3-10 times lower than that reported for the most previously investigated globular proteins. Analysis of the relative affinity of the glycan and peptide moieties suggested that the carbohydrates bind much less surfactant. At saturation, glycans adsorbed about half as much SDS (in g/g) as the peptide moiety of Phy and about five times less than average proteins.     

Influence of Polyols on the Stability and Kinetic Parameters of Invertase from Candida utilis: Correlation with the Conformational Stability and Activity

Invertase (β-d-fructofuranoside fructohydrolase-E.C. 3.2.1.26) is a sucrose hydrolyzing enzyme found in microbial, plant and animal sources. Invertase from Candida utilis is a dimeric glycoprotein composed of two identical monomer subunits with an apparent molecular mass of 150 kDa. We investigated the mechanism of stabilization of invertase with polyols (glycerol, xylitol, and sorbitol). Activity, thermodynamic and kinetic measurements of invertase were performed as a function of polyol concentration and showed that polyols act as very effective stabilizing agents. The result from the solvent-invertase interaction shows preferential exclusion of the polyols from the protein domain leading to preferential hydration of protein. Apparent thermal denaturation temperature of the protein (T _m ) rose from 75 °C to a maximum of 85 °C in 30% glycerol. The stabilization has been attributed to the preferential hydration of the enzyme.     

Thermostability Engineering of Fungal Phytases Using Low-Mr Additives and Chemical Crosslinking

With the ultimate goal to develop preparations of phytase (myo-inositol hexakisphosphate phosphohydrolase) with improved thermal resistance for inclusion in animal feed, several thermostabilization approaches were investigated with a set of fungal (Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, and Aspergillus niger phytase) and consensus phytases. Screening of different low-M_r additives revealed that polyethylene glycols increase the thermostability of all phytases in a molecular weight-dependent fashion. The polyols ribitol, xylitol (C₅ sugars) and sorbitol (C₆ sugar) also improved their thermostability, whereas polyols containing more or less carbon atoms, such as glycerol, erythritol and mannoheptulose, showed only minor effects. The stabilizing effects of PEGs and polyols were concentration dependent. In a second series of experiments, crosslinking of the carbohydrate chains of A. fumigatus and consensus phytase using sodium periodate and adipic acid dihydrazide resulted in the formation of oligomeric forms, which may explain the observed thermostability enhancement of 10–15d`C.     

Exclusion and retention of compensatory kosmotropes by HPLC columns

With water as the elution solvent, zwitterionic solutes and polyols were retained on HPLC columns, more than was water, by totally hydrophobic packing materials. Relative retentions were systematically affected by oxygen functional groups in the packing material, explicable as specific retention of water. Reproducible elution sequences of 20 solutes at a variety of hydrophobic surfaces (aromatic and both long- and short-alkyl aliphatic surfaces) showed there is a general process, consistent with interactions with hydration water at the surface having solvent properties distinct from bulk water. Early eluting solutes included glycine, sarcosine and taurine. Glycine betaine followed both these and N,N-dimethylglycine. The natural betaines propionobetaine and dimethylsulfoniopropionate also preceded glycine betaine. Dimethylsulfoxide was strongly retained, as (to a lesser extent) was proline betaine. Polyols eluted in the sequence sorbitol, trehalose, glycerol. Changes in the chemical nature of the surface or base material affected relative retentions of water and solutes. The presence of hydrogen-bonding functions increased retention of polyols, as well as water, relative to zwitterionic solutes. Specific effects with some solutes may be related to inconsistencies seen in biological systems. Pressures up to 8 MPa did not affect relative retention, constraining models based on the formation of low-density water.     

Polyol-induced molten globule of cytochrome c: an evidence for stabilization by hydrophobic interaction.

To address the contribution of hydrophobic interaction to the stability of molten globule (MG) of proteins, the effects of various polyols (ethylene glycol, glycerol, erythritol, xylitol, sorbitol, and inositol) on the structure of acid-unfolded horse cytochrome c were examined at pH 2, by means of circular dichroism (CD), partial specific volume, adiabatic compressibility, and differential scanning calorimetry (DSC). Addition of polyols induced the characteristic CD spectra of MG, the effect being enhanced with an increase in their concentration and chain length (the number of OH groups) of polyols except for ethylene glycol. The free energy change of MG formation by sorbitol was comparable with those for the salt-induced MG formation but the heat capacity change was negligibly small. The partial specific volume did not change within the experimental error but the adiabatic compressibility largely increased by MG formation. The sorbitol-induced MG showed a highly cooperative DSC thermogram with a large heat capacity change in comparison with the salt-induced one. These results demonstrate that polyols can stabilize the MG state of this protein through the enhanced hydrophobic interaction overcoming the electrostatic repulsion between charged residues. The stabilizing mechanism and structure of MG state induced by polyols were discussed in terms of the preferential solvent interactions and osmotic pressure of the medium, in comparison with the salt-induced one.     

The Effect of Sugars and Polyols on the Heat Resistance of Salmonellae

S ummary . The heat resistance at 65° of 3 strains of salmonellae in solutions of sugars or polyols was enhanced as the concentration of the solutes increased. There was no linear relationship between heat resistance and water activity ( a_w ), but for all solutes except glycerol there was a linear relationship between log D ₆₅ and concentration (% w/w) of solute. Comparison of D ₆₅ at a particular a_w or percentage (w/w) solute concentration showed that the value decreased in the order: sucrose > glucose > sorbitol > fructose > glycerol. In glycerol, D ₆₅ values were always very much lower than in any other solute. With sucrose–glycerol or sucrose–glucose mixtures, heat resistance depended both on the total concentration (% w/w) of solutes present and also on the a_w of the solution.     

Unilamellar DMPC vesicles in aqueous glycerol: preferential interactions and thermochemistry

Glycerol is accumulated in response to environmental stresses in a diverse range of organisms. Understanding of favorable in vivo effects of this solute requires insight into its interactions with biological macromolecules, and one access to this information is the quantification of so-called preferential interactions in glycerol-biopolymer solutions. For model membrane systems, preferential interactions have been discussed, but not directly measured. Hence, we have applied a new differential vapor pressure equipment to quantify the isoosmotic preferential binding parameter, Gamma( micro 1), for systems of unilamellar vesicles of DMPC in aqueous glycerol. It is found that Gamma( micro 1) decreases linearly with the glycerol concentration with a slope of -0.14 +/- 0.014 per molal. This implies that glycerol is preferentially excluded from the membrane-solvent interface. Calorimetric investigations of the same systems showed that the glycerol-DMPC interactions are weakly endothermic, and the temperature of the main phase transition increases slightly (0.16 degrees C per molal) with the glycerol concentration. The results are discussed with respect to a molecular picture which takes into account both the partitioning of glycerol into the membrane and the preferential exclusion from the hydration layer, and it is concluded that the latter effect contributes about four times stronger than the former to the net interaction.     

Critical importance of phytase for yeast growth and alcohol fermentation in Japanese sake brewing

The high phytase producing mutant of Aspergillus oryzae (KL-38) previously isolated was employed for koji making, and the produced koji rice then supplied for sake brewing. The alcohol fermentation was improved compared to that with the parent strain (A. oryzae BP-1). The effects of two phytase isozymes (Phy I and Phy II) produced by A. oryzae on yeast growth and inorganic phosphate liberation were investigated using a synthetic medium containing phytic acid as a sole phosphate source. Yeast growth and the liberation of inorganic phosphate were both enhanced by the combination of Phy I and Phy II at a ratio of 1 to 3, which was compatible with the production ratio in KL-38. Based on these results, phytase plays important role in sake brewing, and that the maximum inorganic phosphate liberation from phytic acid can be obtained by a suitable combination of Phy I and Phy II.     

Crowding alone cannot account for cosolute effect on amyloid aggregation

Amyloid fiber formation is a specific form of protein aggregation, often resulting from the misfolding of native proteins. Aimed at modeling the crowded environment of the cell, recent experiments showed a reduction in fibrillation halftimes for amyloid-forming peptides in the presence of cosolutes that are preferentially excluded from proteins and peptides. The effect of excluded cosolutes has previously been attributed to the large volume excluded by such inert cellular solutes, sometimes termed "macromolecular crowding". Here, we studied a model peptide that can fold to a stable monomeric β-hairpin conformation, but under certain solution conditions aggregates in the form of amyloid fibrils. Using Circular Dichroism spectroscopy (CD), we found that, in the presence of polyols and polyethylene glycols acting as excluded cosolutes, the monomeric β-hairpin conformation was stabilized with respect to the unfolded state. Stabilization free energy was linear with cosolute concentration, and grew with molecular volume, as would also be predicted by crowding models. After initiating the aggregation process with a pH jump, fibrillation in the presence and absence of cosolutes was followed by ThT fluorescence, transmission electron microscopy, and CD spectroscopy. Polyols (glycerol and sorbitol) increased the lag time for fibril formation and elevated the amount of aggregated peptide at equilibrium, in a cosolute size and concentration dependent manner. However, fibrillation rates remained almost unaffected by a wide range of molecular weights of soluble polyethylene glycols. Our results highlight the importance of other forces beyond the excluded volume interactions responsible for crowding that may contribute to the cosolute effects acting on amyloid formation.     

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.     

Competing solvent effects of polyols and guanidine hydrochloride on protein stability

The denaturation of lysozyme and ribonuclease A by guanidine hydrochloride was followed in the presence and absence of glycerol and sorbitol by means of circular dichroism measurements at 25 degrees C. The protein-solvent interactions in the presence of these polyols were also studied by means of density measurements, for discussion of the mechanism of protein stabilization by polyols in terms of the multicomponent thermodynamic theory. The free energy of denaturation depends linearly on the molarity of guanidine hydrochloride at a given polyol concentration, without modification of the cooperativity of the transition. The free energy of denaturation at an infinite dilution of guanidine hydrochloride increases in proportion to the polyol concentration. These results indicate the competing solvent effects of polyols and guanidine hydrochloride on the structures of proteins. In water-protein-polyol systems, protein is preferentially hydrated to elevate its chemical potential, predominantly due to the unfavorable interaction of polyols with the exposed nonpolar amino acid residues. By linkage with the free energy of denaturation, it was quantitatively determined that the chemical potential of denatured protein is more extensively elevated by addition of polyols than that of native protein. These results demonstrate that polyols stabilize the protein structure through strengthening of the hydrophobic interaction, competing with the effect of guanidine hydrochloride.     

Solute stresses affect growth patterns, endogenous water potentials and accumulation of sugars and sugar alcohols in cells of the biocontrol yeast Candida sake

AIM: To evaluate the effect of modifications of water activity (aw 0. 996-0.92) of a molasses medium with different solutes (glycerol, glucose, NaCl, proline or sorbitol) on growth, intracellular water potentials (psi(c)) and endogenous accumulation of polyols/sugars in the biocontrol yeast Candida sake. METHODS AND RESULTS: Modification of solute stress significantly influenced growth, psi(c) and accumulation of sugars (glucose/trehalose) and polyols (glycerol, erythritol, arabitol and mannitol) in the yeast cells. Regardless of the solute used to modify aw, growth was always decreased as water stress increased. Candida sake cells grew better in glycerol- and proline-amended media, but were sensitive to NaCl. The psi(c) measured using psychrometry showed a significant effect of solutes, aw and time. Cells from the 0.96 aw NaCl treatment presented the lowest psic value (- 5.20 MPa) while cells from unmodified media (aw = 0. 996) had the highest value (- 0.30 MPa). In unmodified medium, glycerol was the predominant reserve accumulated. Glycerol and arabitol were the major compounds accumulated in media modified with glucose or NaCl. In proline media, the concentration of arabitol increased. In glycerol- and sorbitol-amended media, the concentration of glycerol rose. Some correlations were obtained between compatible solutes and psi(c). CONCLUSIONS AND SIGNIFICANCE: This study demonstrates that subtle changes in physiological parameters significantly affect the endogenous contents of C. sake cells. It may be possible to utilize such physiological information to develop biocontrol inocula with improved quality.     

Chemical chaperone-mediated protein folding: stabilization of P22 tailspike folding intermediates by glycerol

Polyol co-solvents such as glycerol increase the thermal stability of proteins. This has been explained by preferential hydration favoring the more compact native over the denatured state. Although polyols are also expected to favor aggregation by the same mechanism, they have been found to increase the folding yields of some large, aggregation-prone proteins. We have used the homotrimeric phage P22 tailspike protein to investigate the origin of this effect. The folding of this protein is temperature-sensitive and limited by the stability of monomeric folding intermediates. At non-permissive temperature (>or=35 degrees C), tailspike refolding yields were increased significantly in the presence of 1-4 m glycerol. At low temperature, tailspike refolding is prevented when folding intermediates are destabilized by the addition of urea. Glycerol could offset the urea effect, suggesting that the polyol acts by stabilizing crucial folding intermediates and not by increasing solvent viscosity. The stabilization effect of glycerol on tailspike folding intermediates was confirmed in experiments using a temperature-sensitive folding mutant protein, by fluorescence measurements of subunit folding kinetics, and by temperature up-shift experiments. Our results suggest that the chemical chaperone effect of polyols observed in the folding of large proteins is due to preferential hydration favoring structure formation in folding intermediates.     

Molecular level probing of preferential hydration and its modulation by osmolytes through the use of pyranine complexed to hemoglobin

Two spectroscopic probes are used to expose molecular level changes in hydration shell water interactions that directly relate to such issues as preferential hydration and protein stability. The major focus of the present study is on the use of pyranine (HPT) fluorescence to probe as a function of added osmolytes (PEG, urea, trehalose, and magnesium), the extent to which glycerol is preferentially excluded from the hydration shell of free HPT and HPT localized in the diphosphoglycerate (DPG) binding site of hemoglobin in both solution and in Sol-Gel matrices. The pyranine study is complemented by the use of vibronic side band luminescence from the gadolinium cation that directly exposes the changes in hydrogen bonding between first and second shell waters as a function of added osmolytes. Together the results form the basis for a water partitioning model that can account for both preferential hydration and water/osmolyte-mediated conformational changes in protein structure.     

In vitro inhibition of the actions of basic FGF by a novel 16 amino acid peptide

We have examined the temperature-dependent effects of several organic compounds on the activity of the purified Ca²⁺-ATPase of erythrocytes. The monomeric enzyme was activated either by interaction with calmodulin or by oligomerization in the absence of calmodulin. Of the four homologous solute series studied including polyols, alkanols, aprotic solvents, and N-methyl derivatives of formamide and acetamide only polyols stabilized the enzyme over a broad range of concentration and temperature. Similarity of Ca²⁺-ATPase activity patterns at 25 and 37°C and in the presence of glycerol is in agreement with indirect, stabilizing interactions. Glycerol also protected the Ca²⁺-ATPase from thermal denaturation at 45°C. Within each homologous series, inhibitory effects increased with increasing solute concentration and with increasing structural similarity to detergents, indicating that direct destabilizing interactions are responsible for the observed inhibition. These were comparable to the destabilizing effect of urea. Oligomers were more resistant to all inhibitory solutes as compared to calmodulin-activated monomers suggesting that the nonpolar patches of the oligomerized enzyme are less accessible to solutes.     

Vapor pressure osmometry studies of osmolyte-protein interactions: implications for the action of osmoprotectants in vivo and for the interpretation of "osmotic stress" experiments in vitro

To interpret or to predict the responses of biopolymer processes in vivo and in vitro to changes in solute concentration and to coupled changes in water activity (osmotic stress), a quantitative understanding of the thermodynamic consequences of interactions of solutes and water with biopolymer surfaces is required. To this end, we report isoosmolal preferential interaction coefficients (Gamma(mu1) determined by vapor pressure osmometry (VPO) over a wide range of concentrations for interactions between native bovine serum albumin (BSA) and six small solutes. These include Escherichia coli cytoplasmic osmolytes [potassium glutamate (K(+)Glu(-)), trehalose], E. coli osmoprotectants (proline, glycine betaine), and also glycerol and trimethylamine N-oxide (TMAO). For all six solutes, Gamma(mu1) and the corresponding dialysis preferential interaction coefficient Gamma(mu1),(mu3) (both calculated from the VPO data) are negative; Gamma(mu1), (mu3) is proportional to bulk solute molality (m(bulk)3) at least up to 1 m (molal). Negative values of Gamma(mu1),(mu3) indicate preferential exclusion of these solutes from a BSA solution at dialysis equilibrium and correspond to local concentrations of these solutes in the vicinity of BSA which are lower than their bulk concentrations. Of the solutes investigated, betaine is the most excluded (Gamma(mu1),(mu3)/m(bulk)3 = -49 +/- 1 m(-1)); glycerol is the least excluded (Gamma(mu1),(mu3)/m(bulk)3 = -10 +/- 1 m(-1)). Between these extremes, the magnitude of Gamma(mu1),(mu3)/m(bulk)3 decreases in the order glycine betaine > proline >TMAO > trehalose approximately K(+)Glu(-) > glycerol. The order of exclusion of E. coli osmolytes from BSA surface correlates with their effectiveness as osmoprotectants, which increase the growth rate of E. coli at high external osmolality. For the most excluded solute (betaine), Gamma(mu1),(mu3) provides a minimum estimate of the hydration of native BSA of approximately 2.8 x 10(3) H(2)O/BSA, which corresponds to slightly less than a monolayer (estimated to be approximately 3.2 x 10(3) H(2)O). Consequently, of the solutes investigated here, only betaine might be suitable for use in osmotic stress experiments in vitro as a direct probe to quantify changes in hydration of protein surface in biopolymer processes. More generally, however, our results and analysis lead to the proposal that any of these solutes can be used to quantify changes in water-accessible surface area (ASA) in biopolymer processes once preferential interactions of the solute with biopolymer surface are properly taken into account.