Interactions of myoglobin with urea and some alkylureas. I. Solvation in urea and alkylurea solutions

The interactions of myoglobin with urea, methyl-, N,N'-dimethyl- and ethylurea in aqueous solutions were studied by density measurements. From the densities at constant chemical potential and constant molality, the partial specific volumes of myoglobin in these solutions as well as the extent of preferential binding of urea and alkylurea to myoglobin were determined. It has been found that water and not the denaturant is preferentially bound in urea solutions and alkylurea solutions up to 4 M so that the Gibbs free energy of myoglobin, i.e., its chemical potential in a denaturant solution, is larger than in water. This behavior of myoglobin is different from that of other globular proteins for which preferential binding of urea has been found. It appears that preferential hydration of myoglobin is due to its high content of ionic groups.     

Interactions of myoglobin with urea and some alkylureas. II. Calorimetric and circular dichroic studies

The interactions of myoglobin with urea, methyl-, N,N'-dimethyl- and ethylurea were studied by means of calorimetry and circular dichroism (CD). The enthalpies of transfer from water to aqueous denaturant solutions are positive for the alkylureas and negative for urea. The difference is due to the presence of hydrophobic groups in the alkylureas. Gibbs free energies of transfer for urea solutions were obtained from preferential binding data determined previously. An attempt is made to interpret the values of the thermodynamic quantities in terms of various interactions between protein and denaturant. Analysis of the far-ultraviolet CD spectra reveals some differences in the denaturing activity of urea and the alkylureas, the latter being stronger denaturants than urea. Myoglobin displays relatively high stability towards these denaturants since concentrations above 5 M are needed for achieving major conformational changes.     

Salting-in of neopentane in the aqueous solutions of urea and glycine-betaine

The effects of urea and glycine-betaine (GB) osmolytes on the hydrophobic interactions of neopentane in water have been studied using molecular dynamics simulations. From the study of the potentials of mean force, it is observed that both urea and GB decrease the association and solvation of neopentane. The calculated equilibrium constants show that urea and GB decrease the population of solvent-separated minima of neopentane. The hydrophobic association as well as solvation of neopentane molecules are stabilised by entropy and enthalpy in the mixtures. The radial distribution functions (RDFs) and running coordination numbers of water, urea and GB molecules show that neopentane shows salting-in behaviour in aqueous-GB, aqueous-urea and aqueous-urea-GB mixtures. Neopentane is preferentially solvated by GB in aqueous-GB and preferentially solvated by urea in aqueous-urea-GB solutions. The preferential solvation of neopentane by GB suggests that GB decreases the interaction between neopentane molecules i.e. salting-in of neopentane. The calculated solvation free energies and radial density profiles of neopentane also support the salting-in behaviour of neopentane in the mixtures of these osmolytes.     

SANS/SAXS study of the BSA solvation properties in aqueous urea solutions via a global fit approach

We report on the solvation properties and intermolecular interactions of a model protein (bovine serum albumine, BSA) in urea aqueous solutions, as obtained by combining small-angle neutron and X-ray scattering experiments. According to a global fit strategy, all the whole set of scattering curves are analysed by considering a unique model which includes the BSA structure, the protein-protein interactions and the thermodynamic exchange process of water/urea molecules at the protein solvent interface. As a main result, the equilibrium constant that accounts for the difference in composition between the bulk solvent and the protein solvation layer is derived. Results confirm that urea preferentially sticks to the protein surface, inducing a noticeable change in both the repulsive and the attractive interaction potentials.     

Apparent specific volumes of some dipeptides (containing L-valine and L-leucine in aqueous alkylurea solutions).

The apparent molal volumes of nine dipeptides containing glycine, L-valine, and L-leucine have been determined in methyl, N,N'-dimethyl and ethylurea solutions from precise density measurements. Limiting partial molal volumes. V2(0), at various solute concentrations have also been calculated. The experimental values of V2(0) in water agree reasonably well with those calculated as the sum of V2(0) of both acids after accounting for the electrostrictive effect and loss of water. There is no correlation between the values of V2(0) of individual dipeptides in alkylureas which means that the intrinsic volume and the electrostrictive effect make the largest contribution to V2(0). The contribution from other effects is within the limit of experimental error. The volumes of transfer from water to alkylurea solutions are all positive and reflect by and large the electrostrictive effect.     

Partial specific volumes and interactions with solvent components of α-globulin fromSesamum indicum L. in urea and guanidine hydrochloride

The interaction of α-globulin with urea/guanidine hydrochloride was investigated by determining the apparent partial specific volumes of the protein in these solvents. The apparent partial specific volumes were determined both under isomolal and isopotential conditions. The preferential interaction parameter with solvent components calculated were 0.08 and 0.1 g of urea and guanidine hydrochloride respectively per g protein. In both the cases the interaction was not preferential with water. The total binding of denaturant to α-globulin was calculated both for urea and guanidine hydrochloride and the correlation between experimentally determined number of mol of denaturant bound per mol of protein and the total number of peptide bonds and aromatic amino acids were found to be in excellent agreement with each other. The changes in volume upon transferring α-globulin from a salt solution to 8 M urea and 6 M guanidine hydrochloride were also calculated. This work was done at the Biochemistry Department, Brandeis University, Waltham, Massachusetts 02254, USA.     

Calorimetric study of the thermal denaturation of beta-lactoglobulin in the presence of urea and phosphate ions]

The denaturation of beta-lactoglobulin in solution with different content of urea and phosphates has been studied calorimetrically. It has been shown that the increase of phosphate ion concentration in solution leads to an increase of beta-lactoglobulin stability, while increase of urea concentration leads to an opposite effect. The variation of these components in solution practically does not influence the value of the heat capacity increment of beta-lactoglobulin in the considered temperature region. Accordingly the denaturation enthalpy is a linear function of temperature whose slope does not differ for solution with urea concentration less than 4.4 M. However, the absolute value of denaturation enthalpy in these solutions at corresponding temperatures differs significantly due to the heat effect of additional urea solvation during transition to the denatured state. The latter leads to a decrease of the overall denaturation enthalpy and, as a result, a shift of the enthalpy plot to higher temperatures providing conditions for studying the thermodynamic and structural characteristics of the molecule in the cold denatured-state.     

Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: a calorimetric and spectroscopic study

The effect of methylurea, N,N'-dimethylurea, ethylurea, and butylurea as well as guanidine hydrochloride (GuHCl), urea and pH on the thermal stability, structural properties, and preferential solvation changes accompanying the thermal unfolding of ribonuclease A (RNase A) has been investigated by differential scanning calorimetry (DSC), UV, and circular dichroism (CD) spectroscopy. The results show that the thermal stability of RNase A decreases with increasing concentration of denaturants and the size of the hydrophobic group substituted on the urea molecule. From CD measurements in the near- and far-UV range, it has been observed that the tertiary structure of RNase A melts at about 3 degrees C lower temperature than its secondary structure, which means that the hierarchy in structural building blocks exists for RNase A even at conditions at which according to DSC and UV measurements the RNase A unfolding can be interpreted in terms of a two-state approximation. The far-UV CD spectra also show that the final denatured states of RNase A at high temperatures in the presence of different denaturants including 4.5 M GuHCl are similar to each other but different from the one obtained in 4.5 M GuHCl at 25 degrees C. The concentration dependence of the preferential solvation change delta r23, expressed as the number of cosolvent molecules entering or leaving the solvation shell of the protein upon denaturation and calculated from DSC data, shows the same relative denaturation efficiency of alkylureas as other methods.     

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

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

Structural thermodynamics of protein preferential solvation: osmolyte solvation of proteins, aminoacids, and peptides

Protein stability and solubility depend strongly on the presence of osmolytes, because of the protein preference to be solvated by either water or osmolyte. It has traditionally been assumed that only this relative preference can be measured, and that the individual solvation contributions of water and osmolyte are inaccessible. However, it is possible to determine hydration and osmolyte solvation (osmolation) separately using Kirkwood-Buff theory, and this fact has recently been utilized by several researchers. Here, we provide a thermodynamic assessment of how each surface group on proteins contributes to the overall hydration and osmolation. Our analysis is based on transfer free energy measurements with model-compounds that were previously demonstrated to allow for a very successful prediction of osmolyte-dependent protein stability. When combined with Kirkwood-Buff theory, the Transfer Model provides a space-resolved solvation pattern of the peptide unit, amino acids, and the folding/unfolding equilibrium of proteins in the presence of osmolytes. We find that the major solvation effects on protein side-chains originate from the osmolytes, and that the hydration mostly depends on the size of the side-chain. The peptide backbone unit displays a much more variable hydration in the different osmolyte solutions. Interestingly, the presence of sucrose leads to simultaneous accumulation of both the sugar and water in the vicinity of peptide groups, resulting from a saccharide accumulation that is less than the accumulation of water, a net preferential exclusion. Only the denaturing osmolyte, urea, obeys the classical solvent exchange mechanism in which the preferential interaction with the peptide unit excludes water.     

Volume changes accompanying the thermal denaturation of deoxyribonucleic acid. I. Denaturation at neutral pH

Dilatometric measurements were made to determine the change in apparent specific volume φ of DNA resulting from thermal denaturation in neutral solution, φ increased continuously with temperature in the range 10–85°C. No deviations from a monotonically rising curve were observed in the φ versus temperature profile in the region of the melting temperature. The results are interpreted in terms of a partial loss of the preferentially bound DNA hydration shell. The nature of the well known buoyant density difference between native and denatured DNA was investigated by evaluating the densities in a series of cesium salt gradients at constant temperature. Extrapolation of the results to zero water activity indicates that the partial specific volumes of anhydrous native and denatured DNA are equal. The density difference at nonzero water activities is attributed to decreased hydration in the denatured state. The absence of a related change in φ accompanying the denaturation in the dilatometric experiments suggests that the probable volume change associated with loss of bound water during denaturation is accompanied by other compensatory volume effects. The possible nature of these volume effects is discussed.     

Preferential interactions in aqueous solutions of urea and KCl

A quantitative characterization of the thermodynamic effects due to interactions of salt ions and urea in aqueous solution is needed for rigorous analyses of the effects of changing urea concentration on biopolymer processes in solutions that also contain salt. Therefore, we investigate preferential interactions in aqueous solutions containing KCl and urea by using vapor pressure osmometry (VPO) to measure osmolality as a function of the molality of urea (component 3) over the range 0.09相似文献   

Interaction of 2,2,2-trifluoroethanol with proteins: calorimetric, densimetric and surface tension approach

The thermal denaturation of hen egg-white lysozyme was studied in the presence of 2,2,2-trifluoroethanol (TFE) at various pH values using micro differential scanning calorimetry. Quantitative thermodynamic parameters accompanying the thermal transitions were evaluated. It is observed that thermal unfolding of lysozyme in the presence of TFE upto a concentration of 4.0 mol dm(-3) follows a two-state denaturation mechanism as indicated by the equality of van't Hoff and calorimetric enthalpies. The finer details of interaction were studied by measuring the partial molar volume of some constituent amino acids and glycine peptides from water to aqueous TFE at 298.15 K. The physico-chemical properties of aqueous TFE: apparent molar heat capacities, apparent molar volumes and surface tension were measured to understand the intrinsic properties of the cosolvent as well. From the correlation among the thermal unfolding data on lysozyme in aqueous TFE, calculated preferential interaction parameters, physico chemical properties of aqueous TFE and partial molar volumes of transfer, it is concluded that both solvent mediated effect and direct interaction constitute the mechanism of TFE-protein interactions.     

Volume changes accompanying collagen denaturation

A dilatometric technique is employed to measure the volume changes occurring on denaturation of collagen solutions and tendons. Partial specific volumes of tropocollagen are calculated to be 0.686 ml./g. in water and 0.689 ml/g in 0.1M citrate buffer. From data on non-aqueous solutions, it is estimated that most of the volume change arises from changes in polymer configuration rather than from changes in solvation.     

Effect of calcium chloride on the conformation of proteins. Thermodynamic studies of some model compounds

Partial molar heat capacities (CP2 degrees) and volumes (V2 degrees) for some amino acids and peptides were measured in 1 M aqueous calcium chloride solutions at 298.15 degrees K using a Picker flow microcalorimeter and an oscillating-tube digital density meter. Using the data for these amino acids and peptides in water, the corresponding partial molar heat capacities of transfer (CP2,tr degree) and volumes (V2,tr degree) from water to 1 M aqueous calcium chloride were deduced. These thermodynamic parameters are significantly positive, indicating that strong interactions occur between the ions of calcium chloride and the charged centres of these amino acids and peptides. A comparison has been made with a similar transfer of these compounds to sodium chloride solutions. The thermodynamic parameters of the transfer of peptide group (-CONH) are much more positive in calcium chloride than in sodium chloride solutions. The implication of this result for the ability of calcium chloride to act as a stronger destabilizer of protein conformation is discussed.     

Differences in the processes of beta-lactoglobulin cold and heat denaturations.

The changes in beta-lactoglobulin upon cold and heat denaturation were studied by scanning calorimetry, CD, and NMR spectroscopy. It is shown that, in the presence of urea, these processes of beta-lactoglobulin denaturation below and above 308 K are accompanied by different structural and thermodynamic changes. Analysis of the NOE spectra of beta-lactoglobulin shows that changes in the spin diffusion of beta-lactoglobulin after disruption of the unique tertiary structure upon cold denaturation are much more substantial than those upon heat denaturation. In cold denatured beta-lactoglobulin, the network of residual interactions in hydrophobic and hydrophilic regions of the molecules is more extensive than after heat denaturation. This suggests that upon cold- and heat-induced unfolding, the molecule undergoes different structural rearrangements, passing through different denaturation intermediates. From this point of view, cold denaturation can be considered to be a two stage process with a stable intermediate. A similar equilibrium intermediate can be obtained at 35 degrees C in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of the intermediate. However, these processes differ in cooperativity, showing noncommensurate sigmoidal-like changes in efficiency of spin diffusion, ellipticity at 222 nm, and partial heat capacity. The disruption with cooling is accompanied by cooperative changes in heat capacity, whereas with heating the heat capacity changes only gradually. Considering the sigmoidal shape of the heat capacity change an extended heat absorption peak, we propose that the intermediate state is stabilized by enthalpic interactions.     

Distinctive Solvation Patterns Make Renal Osmolytes Diverse

The kidney uses mixtures of five osmolytes to counter the stress induced by high urea and NaCl concentrations. The individual roles of most of the osmolytes are unclear, and three of the five have not yet been thermodynamically characterized. Here, we report partial molar volumes and activity coefficients of glycerophosphocholine (GPC), taurine, and myo-inositol. We derive their solvation behavior from the experimental data using Kirkwood-Buff theory. We also provide their solubility data, including solubility data for scyllo-inositol. It turns out that renal osmolytes fall into three distinct classes with respect to their solvation. Trimethyl-amines (GPC and glycine-betaine) are characterized by strong hard-sphere-like self-exclusion; urea, taurine, and myo-inositol have a tendency toward self-association; sorbitol and most other nonrenal osmolytes have a relatively constant, intermediate solvation that has components of both exclusion and association. The data presented here show that renal osmolytes are quite diverse with respect to their solvation patterns, and they can be further differentiated based on observations from experiments examining their effect on macromolecules. It is expected, based on the available surface groups, that each renal osmolyte has distinct effects on various classes of biomolecules. This likely allows the kidney to use specific combinations of osmolytes independently to fine-tune the chemical activities of several types of molecules.     

A simple model for solvation in mixed solvents. Applications to the stabilization and destabilization of macromolecular structures

The properties of a simple model for solvation in mixed solvents are explored in this paper. The model is based on the supposition that solvent replacement is a simple one-for-one substitution reaction at macromolecular sites which are independent of one another. This leads to a new form for the binding polynomial in which all terms are associated with ligand interchange rather than ligand addition. The principal solvent acts as one of the ligands. Thermodynamic analysis then shows that thermodynamic binding (i.e., selective interaction) depends on the properties of K'-1, whereas stoichiometric binding (site occupation) depends on K'. K' is a 'practical' interchange equilibrium constant given by (f3/f1)K, where K is the true equilibrium constant for the interchange of components 3 and 1 on the site and f3 and f4 denote their respective activity coefficients on the mole fraction scale. Values of K' less than unity lead to negative selective interaction. It is selective interaction and not occupation number which determines the thermodynamic effects of solvation. When K' greater than 100 on the mole fraction scale or K' greater than 2 on the molality scale (in water), the differences between stoichiometric binding and selective interaction become less than 1%. The theory of this paper is therefore necessary only for very weak binding constants. When K'-1 is small, large concentrations of the added solvent component are required to produce a thermodynamic effect. Under these circumstances the isotherms for the selective interaction and for the excess (or transfer) free energy are strongly dependent on the behavior of the activity coefficients of both solvent components. Two classes of behavior are described depending on whether the components display positive or negative deviations from Raoult's law. Examples which are discussed are aqueous solutions of urea and guanidinium chloride for positive deviations and of sucrose and glucose for negative deviations. Examination of the few studies which have been reported in the literature shows that most of the qualitative features of the stabilization of proteins by sugars and their destabilization by urea and guanidinium chloride are faithfully represented with the model. This includes maxima in the free energy of stabilization and destabilization, decreased and zero selective interaction at high concentrations, etc. These phenomena had no prior explanation. Deficiencies in the model as a representation of solvation in aqueous solution are discussed in the appendix.     

A dilatometric study of the denaturation of bovine serum albumin by guanidine hydrochloride

The denaturation of bovine serum albumin by guanidine hydrochloride was studied using the dilatometric method. From dilatometric measurements the differences between the partial specific volume of the protein in denaturant solutions and water, respectively, were determined. The differences reflect the extent of unfolding as well as the binding of the denaturant. From the differences and the known partial specific volume of the native protein, the partial specific volumes at individual denaturant concentrations were obtained.