Concentrated Aqueous Solutions Of Urea Research Articles

The conformational stability of ovalbumin and lysozyme in the aqueous solutions of various cosolvents

A thermodynamic method is reported to monitor the conformational stability of lysozyme and ovalbumin in the presence of various cosolvents. Heats of dilution of the proteins in concentrated aqueous solutions of urea, ethanol or glucose have been determined at 298.15 K by flow microcalorimetry. The pairwise enthalpic interaction coefficients of the proteins in the different solvent media are derived: They allow to gain information about the influence of the cosolvents on the interactions between two interacting hydrated molecules of a protein, hence on its conformational stability. The two proteins behave very differently in the various cosolvents. In glucose, the coefficients for ovalbumin are positive up to 3 mol kg−1 of cosolvent and then negative, while those for lysozyme are negative up to 6 mol kg−1. In urea, coefficients for ovalbumin are positive, while negative for lysozyme up to 7 mol kg1. In ethanol, coefficients for ovalbumin are almost invariant, even at the highest concentrations of cosolvent, underlining that the hydration shell of the protein is such to maintain essentially unaltered the native conformation. For lysozyme, coefficients are negative and almost invariant up to 20 mol kg−1 ethanol: Then, a jump occurs toward much more large and negative values. The observed behaviors are rationalized also on the basis of the results previously obtained for small model molecules in concentrated solutions of urea, ethanol or glucose. The differences between the two proteins are explained in terms of the effects of the cosolvents on hydrophilic and hydrophobic interactions and account for the structural characteristics of each protein. In fact, notwithstanding both are globular proteins, they are differently packed and that could make them to react differently toward the action of a given cosolvent.

Parsing the Contributions of Polypeptide Backbones and Sidechains to Denaturation in Concentrated Aqueous Solutions of Urea and Guanidinium Chloride

The mechanistic details of protein denaturation are relevant for understanding the nature of the collapse transition that is induced by dilution from denaturing to predominantly aqueous solutions. Concentrated solutions of urea and GdnCl are thought to be good solvents for generic proteins. Here we report results from a series of systematic molecular dynamics simulations. We show that the solvent quality of a denaturing solution depends on the chemical details of the polypeptide system. Polyglycine prefers collapsed states in highly concentrated aqueous solutions of GdnCl, implying that these solvents are poor solvents for polypeptide backbones. The induction of chain expansion in 6 M GdnCl requires the addition of specific categories of sidechains including those with aromatic groups, primary amides, and charged groups. Polyglycine expands in highly concentrated aqueous solutions of urea. However, we show that intra-chain and chain-solvent interactions are almost perfectly counterbalanced in 8 M urea, implying that these conditions are theta solvents for generic polypeptide backbones. The degree of chain expansion can be enhanced in urea by the addition of sidechains that interact favorably with urea molecules. Polypeptide backbones and sidechains contribute to chain expansion through preferential interactions with denaturant molecules in 6 M GdnCl and 8 M urea, although the contributions from sidechain and backbone specific interactions are different in the two milieus. Importantly, we observe that the degree of expansion falls short of the upper limit that is achievable for self-avoiding random walks. This implies that the degree of chain expansion can be sequence-specific and be impacted by residual intra-chain attractions. This should influence the details of the collapse transition upon dilution from denaturants.

Second-harmonic scattering in aqueous urea solutions: evidence for solute clusters?

Measurements of second-harmonic scattering (SHS) from concentrated aqueous solutions of urea are reported for the first time using scanning microscopy. SHS signal was measured as a function of solution concentration (C) over a range of saturation conditions from undersaturated (S = 0.15) to supersaturated (5 = 1.86), where S = C/C(sat) and C(sat) is the saturation concentration. The results show a non-linear increase in SHS signal against concentration, with local maxima near S = 0.95 and 1.75 suggesting a change in solution structure near these points. Rayleigh scattering images indicate the presence of particles in nearly saturated (S = 0.95) urea solutions. Time-dependent SHS measurements indicate that signals originate from individual events encountered during scanning of the focal volume through the solution, consistent with second harmonic generation (SHG) from particles. SHG from aqueous dispersions of barium titanate (BaTiO3) nanoparticles with diameters <200 nm, showed signals approximately 20 times larger than urea solutions. The results suggest the existence of a population of semi-ordered clusters of urea that changes with solution concentration.

A calorimetric study of the interactions in the aqueous solutions of lysozyme in the presence of denaturing cosolvents

A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. Heats of dilution of the protein in concentrated aqueous solutions of urea or ethanol have been determined at 298.15 K by flow microcalorimetry. The pairwise enthalpic interaction coefficients of the protein in the different solvent media are derived. These parameters allow to gain information about the influence of the cosolvents on the interactions acting between two interacting hydrated molecules of lysozyme, hence on the denaturation process. At increasing urea concentration, up to about 6 mol kg−1, the values of the interaction coefficients are large and negative and remain almost unaltered. The invariance of the coefficients underlines that, even in highly concentrated urea, the hydration shell of the protein is such to maintain essentially unaltered the native conformation. At higher urea concentrations, a sudden change in the sign of the coefficients monitors the variation in the interactions between two hydrated denatured protein molecules.The same trend is found when ethanol is the cosolvent. At increasing concentration of the cosolvent, coefficients are, at first, almost invariant. After that, denaturation occurs, detected as a jump toward much more negative values. The results obtained are rationalized on the basis of those previously found for small model molecules in concentrated solutions of urea or ethanol. The thermodynamic framework allows useful comments to be made on the possible mode of action of the two cosolvents on the stability of proteins in solution.

Solvent effects on the complexation of 1-alkanols by parent and modified cyclodextrins. Calorimetric studies at 298 K

The formation of complexes of parent and alkylated cyclodextrins (CDs) with 1-heptanol and 1-octanol has been studied calorimetrically at 298 K in water and in concentrated aqueous solutions of urea. The forces involved in the association process are discussed in the light of the signs and values of the thermodynamic parameters obtained: association enthalpy, binding constant, Gibbs free energy, and entropy. It was inferred that: (i) in water, the formation of complexes for parent and substituted α-cyclodextrins (αCDs) is determined by enthalpy. For parent and substituted β-cyclodextrins (βCDs), instead, hydrophobic interactions are the prevailing forces determining complexation, as indicated by the small and negative or positive enthalpies and by the high and positive entropies. (ii) In urea, hydrophilic interactions are attenuated. The formation of complexes with alkylated CDs does not occur. (iii) The analysis of the thermodynamic properties confirms that inclusion is a process dominated by hydration phenomena. Modifications experienced by the solvent water in the hydration shells of the interacting substances upon association determine the formation of the complexes.

Size and temperature dependence of hydrocarbon solubility in concentrated aqueous solutions of urea and guanidine hydrochloride

At 25°C, methane and ethane are more soluble in water than in 7 M aqueous urea or 4.9 M aqueous guanidine hydrochloride (GuHCl); the reverse is true for larger hydrocarbons. In addition, the hydrocarbon solubility in 7 M aqueous urea or 4.9 M aqueous GuHCl increases compared with that in water on raising the temperature in the range of 5–45°C. These experimental data have not yet been rationalized. Using a well-founded theory of hydrophobic hydration, the present analysis indicates that the transfer of hydrocarbons from water to 7 M aqueous urea or to 4.9 M aqueous GuHCl is favored by the difference in the solute–solvent van der Waals interaction energy, and contrasted by the difference in the work of cavity creation. At room temperature, on increasing the hydrocarbon size, the first contribution rises in magnitude more rapidly than the second contribution, accounting for the threshold size occurrence. Moreover, the second contribution decreases in magnitude with an increase in temperature, becoming less unfavorable, while the first contribution is practically constant in the range of 5–45°C. The different temperature dependence of the work of cavity creation in such solvent systems is due to the fact that the density of 7 M aqueous urea and 4.9 M aqueous GuHCl decreases more rapidly than that of water when raising the temperature. The relationship between the density of a liquid and the work to create a cavity in it is discussed in detail.Key words: work of cavity creation, solute-solvent van der Waals interaction energy, H-bond reorganization.

Solubility of 1-Amino-4-Hydroxyanthraquinone in Water-Amide Mixtures

The solubility of a C.I. Disperse Red 15 dye in water and concentrated aqueous solutions of urea and acetamide was studied by isothermal saturation within the 313-353 K temperature range The thermodynamic characteristics of the dye solution were calculated, and the results were analyzed.

Interactions of aminoacids in concentrated aqueous solutions of urea or ethanol. Implications for the mechanism of protein denaturation

Calorimetric measurements were carried out at 298.15 K on binary aqueous solutions of glycine, and on binary and ternary solutions containing the L and D forms of the α-aminoacid leucine at different concentrations of urea or ethanol as cosolvents. The derived pairwise interaction coefficients of the excess enthalpies were rationalized according to the preferential configuration model. The behaviour shown by glycine, when the concentration and the nature of the cosolvent changes, allows important observations on the influence of the medium on hydrophilic interactions. For leucine, differences were found between the values of the homochiral and heterochiral pairwise enthalpic interaction coefficients. This chiral recognition depends on the nature and concentration of the cosolvent, which influences differently hydrophilic and hydrophobic interactions. The results obtained are compared with those found for other model compounds in concentrated aqueous solutions of urea or ethanol: some comments are made on the possible mode of action of ethanol and urea as protein denaturants.

Chiral recognition in solution. Interactions of α-amino acids in concentrated aqueous solutions of urea or ethanol

Enthalpies of dilution of the L and D forms of glutamine, citrulline, and phenylalanine in concentrated aqueous solutions of urea or ethanol were measured calorimetrically at 298 K. Glycine, urea, formamide, and phenol were also studied under the same experimental conditions, to get information about the behaviour of the zwitterion and of the functional group in the side chain of the cited amino acids when the concentration of the cosolvent changes. The derived pairwise enthalpic interaction coefficients for the three amino acids were rationalized according to the preferential configuration model. Indications are that, in concentrated urea, the coefficients for citrulline and glutamine are determined mainly by the interactions between the cosolvent and the hydrophilic groups in the molecule of the amino acids. For phenylalanine, coefficients are less positive than in water, because the presence of urea, which solvates preferentially the zwitterions, attenuates hydrophobic interactions between the benzene rings. In ethanol, coefficients for the three amino acid become negative or more negative than in water, because in this medium hydrophilic interactions are enhanced. Chiral recognition, namely the difference in the values of homo- and heterochiral interaction coefficients, was detected only for phenylalanine in urea. Hence, the nature of the cosolvent, influencing differently hydrophilic and hydrophobic interactions, can lead to the detection of chiral recognition also for those systems that, as phenylalanine, do not present this effect in pure water.Key words: α-amino acids, excess functions, molecular interactions, preferential configuration.

Chiral recognition in solution. Interactions of α-amino acids in concentrated aqueous solutions of urea or ethanol

Enthalpies of dilution of the L and D forms of glutamine, citrulline, and phenylalanine in concentrated aqueous solutions of urea or ethanol were measured calorimetrically at 298 K. Glycine, urea, formamide, and phenol were also studied under the same experimental conditions, to get information about the behaviour of the zwitterion and of the functional group in the side chain of the cited amino acids when the concentration of the cosolvent changes. The derived pairwise enthalpic interaction coefficients for the three amino acids were rationalized according to the preferential configuration model. Indications are that, in concentrated urea, the coefficients for citrulline and glutamine are determined mainly by the interactions between the cosolvent and the hydrophilic groups in the molecule of the amino acids. For phenylalanine, coefficients are less positive than in water, because the presence of urea, which solvates preferentially the zwitterions, attenuates hydrophobic interactions between the benzene rings. In ethanol, coefficients for the three amino acid become negative or more negative than in water, because in this medium hydrophilic interactions are enhanced. Chiral recognition, namely the difference in the values of homo- and heterochiral interaction coefficients, was detected only for phenylalanine in urea. Hence, the nature of the cosolvent, influencing differently hydrophilic and hydrophobic interactions, can lead to the detection of chiral recognition also for those systems that, as phenylalanine, do not present this effect in pure water.Key words: α-amino acids, excess functions, molecular interactions, preferential configuration.

Chiral recognition in aqueous solutions: On the role of urea in hydrophilic and hydrophobic interactions of unsubstituted ?-amino acids

Calorimetric and nuclear relaxation time measurements were carried out at 25°C on concentrated aqueous solutions of urea containing the L and D forms of the following α-aminoacids: alanine, α-aminobutyric acid, norvaline, and norleucine. Glycine was also studied under the same experimental conditions. The enthalpic interaction coefficients were rationalized according to the preferential configuration model. The trends of the homochiral coefficients at increasing concentration of urea underline an interaction mechanism not different from that occurring in water. Chiral recognition is the quantity more affected by the nature of the solvent. Among the amino acids studied, only norleucine presents this effect, which vanishes at the highest concentrations of urea because the attenuated electrostatic interactions cannot impose preferential configurations.

Role of cosolvent in hydrophobic interactions. Calorimetric studies of alkanols in concentrated aqueous solutions of urea at 298 K

Calorimetric measurements have been carried out at 298.15 K on aqueous solutions of 7 mol kg–1 urea containing alkan-m-ols and alkan-m,n-diols in order to obtain more information on the influence of urea on the hydrophobic solvation and interactions. The pairwise interaction coefficients of the virial expansion of the excess enthalpies were found to be positive and generally lower than the corresponding values in water. The only exception concerns pentan- and hexan-2- and -3-ols, whose coefficients in the mixed solvent are higher. These coefficients were rationalized according to the preferential configuration model. Urea mainly solvates the hydroxy groups. Hydrophobic interactions are attenuated by the presence of such a large amount of urea, thus indicating that the hydrophobic cosphere of the alkanols must be reduced with respect to that in pure water. The interaction mechanism in water and in concentrated urea solutions does not seem to be alterated. The data show that in 7 mol kg–1 urea the nature of the hydration cosphere of the alkyl chain does not change, so that water still plays the major role in this interaction process between two hydrated molecules.

Excess enthalpies of N-acetylglycineamide and N-acetyl- l-leucineamide in concentrated aqueous solutions of tetramethylurea

A comparison has been made among the values of the enthalpic pairwise interaction coefficients of two N-acetylamides of the amino acids glycine (NAGA) and leucine (NALA) in concentrated aqueous solutions of tetramethylurea (TMU) and urea, in pure water, and in pure liquid amides. The second virial coefficients of the excess enthalpies are found to be negative for both the model peptidic molecules in 4M TMU, as in liquid amides, dimethylformamide (DMF) and fused N-methylacetamide (NMA), substances assumed to mimic the core of globular proteins. This is the reverse of what was found for concentrated aqueous solutions of urea (U), where all the enthalpic second virial coefficients were positive. This suggests a completely different mechanism for the denaturation of protein in the presence of urea and TMU, due to different protein-denaturant interactions.

Dilution enthalpies of alkanols in concentrated aqueous solutions of urea at 25�C

Enthalpies of dilution of some aliphatic alcohols were determined at 25°C in aqueous 7M urea solutions by flow microcalorimetry. The excess enthalpies were expressed as power expansion series in molalities referred to 1 kg of constant composition urea-water mixture. This urea-water mixture was utilized throughout as a mixed solvent. The values of the second enthalpic virial coefficients were all found to be positive and generally lower than the corresponding values in water. Large differences were encountered, as in water, by comparing normal and branched isomeric propanols and butanols. For one system it was possible to measure the third coefficients, which were also positive. The second enthalpic coefficients were found to increase with the molecular weight of the alkanols. These facts suggest that in the presence of a large concentration of urea, the excess enthalpies are mainly determined by apolar interactions. This is surprising and potentially rich in consequences for a better understanding of the interactions among amino acid residues distantly situated in the primary sequences but topologically near in the loops of globular proteins. An analysis, carried out using the Savage-Wood additivity group method, shows that the enthalpic contributions (that appear to play a crucial role in water in making the polar interaction to be favorable) become essentially unfavorable in urea-water solvent. The hypothesis that the peptide-peptide interactions are prevented by the preferential solvation of urea is also discussed.

Thermodynamic behaviour of some uncharged organic molecules in concentrated aqueous urea solutions and other polar solvents

A comparison has been made between the values of the enthalpic pairwise interaction coefficients of several organic molecules (peptides, amides and alcohols) in water, in concentrated aqueous solutions of urea, in N, N-dimethylformamide and in liquid N-methyl-acetamide. The second virial coefficients of the excess enthalpies are found to be positive for all the systems studied in water-urea mixtures. A preliminary analysis, carried out using the Savage and Wood group additivity approach, suggests that, in concentrated aqueous solutions of urea, this arises from the peptide-peptide or hydroxyl-hydroxyl, and the apolar-apolar group contributions, all being positive, and these overwhelm the negative contributions from the polar-apolar group interactions. A remarkable feature is the inversion of the signs of the peptide-peptide, hydroxyl-hydroxyl, peptide-methylene and hydroxyl-methylene, group interactions when compared with those which prevail in pure water. This suggests a completely different solvation and interaction mechanism in concentrated urea solutions. The enthalpic contributions from apolar-apolar group interactions in the mixed solvent, in turn, are higher than the values found in water. Some comments are made on the behaviour of some of the above solutes, in the liquid amide solvents N, N-dimethylformamide and N-methylacetamide.

Enthalpies of dilution of bifunctional alcohols in concentrated aqueous solutions of urea at 298.15 K

The enthalpies of dilution of aliphatic bifunctional alcohols were measured in 7 m urea aqueous solutions at 298.15 K by flow microcalorimetry. The excess enthalpies were expressed as a power expansion series in the molality of the alcohol, referred to 1 kg of the mixed urea-water solvent. The values of the second and the third coefficients were all found to be positive and lower than the corresponding values in water. They show an approximately linear dependence on the square of the number of methylene groups. This fact suggests that in the presence of a large amount of urea excess enthalpies are determined primarily by methylene-methylene interactions. This is quite surprising and of remarkable interest on account of its obvious biological implications, since a kind of “lipophobic” or some modified hydrophobic effect seems still to be effective in concentrated urea solutions.

Formation and stabilization of the collagen-fold

The mutarotation of ichthyocol gelatin at low temperatures has been examined over a wide range of protein concentrations in the region below the gelation level. The rates of mutarotation as well as the final values of specific rotation are shown to be independent of concentration suggesting that the configurational changes responsible for rotation depend on intramolecular events. Some type of hydrogen bonding appears to be involved in the formation and stabilization of the collagen-fold since no mutarotation is observed in concentrated aqueous solutions of urea or guanidine-HCl. Moreover, the melting profiles of chilled gelatin solutions in H 2O and D 2O reveal a significant increase in melting temperature (3.7 °C.) in D 2O over that in H 2O. Rate measurements of mutarotation reveal that (1) the process obeys second-order kinetics with respect to the amount of gelatin in the unfolded configuration; and (2) the rate of mutarotation at a fixed low temperature is much greater in D 2O than in H 2O. A mechanism for the formation of the collagen-fold is proposed in which water molecules act to stabilize the collagen-fold configuration through formation of doubly hydrogen-bonded bridges between adjacent carbonyl groups. The kinetics are interpreted in terms of an intramolecular crystallization process originating from preformed nuclei.