Solvation of biomolecules in aqueous kosmotrope solutions - The energetics of the glycerol interaction with amino acids in water

Accepted Manuscript Solvation of biomolecules in aqueous kosmotrope solutions - The energetics of the glycerol interaction with amino acids in water

Andrey V. Kustov, Olga A. Antonova, Nataliya L. Smirnova PII: DOI: Reference:

S0167-7322(16)34068-5 doi: 10.1016/j.molliq.2017.02.065 MOLLIQ 6978

To appear in:

Journal of Molecular Liquids

Received date: Accepted date:

15 December 2016 17 February 2017

Please cite this article as: Andrey V. Kustov, Olga A. Antonova, Nataliya L. Smirnova , Solvation of biomolecules in aqueous kosmotrope solutions - The energetics of the glycerol interaction with amino acids in water. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Molliq(2017), doi: 10.1016/j.molliq.2017.02.065

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ACCEPTED MANUSCRIPT Solvation of biomolecules in aqueous kosmotrope solutions - the energetics of the glycerol interaction with amino acids in water Andrey V. Kustov*, Olga A. Antonova, Nataliya L. Smirnova

United Physico-Chemical Centre of Solutions of G.A. Krestov Institute of Solution Chemistry of

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Russian Academy of Sciences, Akademicheskaya str. 1, 153045, Ivanovo, Russian Federation

Ivanovo State University of Chemistry and Technology, Sheremetev av. 7,153012, Ivanovo,

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Received

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Russian Federation

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*Corresponding author: DSc, The Leading Scientific Researcher, Andrey V. Kustov.

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Tel, Fax: +7(4932)327256. E-mail address: [email protected]

ACCEPTED MANUSCRIPT Abstract This paper focuses on the energetics of glycine, L-alanine, L-phenylalanine and benzene interaction with glycerol (Gl) which is one of the most important stabilizing agents of globular proteins in water. The solute-glycerol parameters of pair and triplet interactions have been determined from calorimetric data at 298 and 313 K using standard thermodynamic procedures and compared with previously reported values for ethylene glycol (Eg). The biomolecule

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interaction with both kosmotropes is found to be enthalpically unfavorable (hGl-S>>0). The larger hydrophobicity of the solute the larger is the enthalpy of pair interaction. The total interaction is

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also slightly repulsive in cold water (gGl-S>0) but reveal a tendency to become attractive at elevated temperatures due to a favorable entropic term (-TsGl-S<0). The existence of a rather good

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linear relationship between enthalpic parameters for Eg and Gl solutions has been found. This allows to predict the behavior of different amino acids in aqueous kosmotrope solutions at

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different temperatures.

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Keywords: Amino acids; Kosmotropes; Aqueous solutions; Pair and triplet interactions

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ACCEPTED MANUSCRIPT 1. Introduction Stability of biopolymers in living systems depends on many factors, the most important of which is believed to be the polymer interaction with surrounding solvent molecules. The central importance of these interactions for stabilizing specific structure of proteins and other macromolecules has long been recognized [1-3]. Both the hydrophobic interaction between apolar amino acid residues and solvent-protein attractive forces including hydrogen bonding, ionic and van der Waals interactions contribute to the stabilization of native conformations [1].

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The appearance of hydrated ions or small organic molecules in the protein nearest vicinity may change the microstructure and local composition of this region [3]. One type of additives (from

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here on they will be called co-solvents) accumulates at the protein surface in significant excess

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over its concentration in bulk water. Upon reaching a certain concentration this process can induce protein denaturation [1-3]. Guanidine chloride and urea are typical denaturants binding with peptide groups at the protein surface [2, 3]. Another type of organic co-solvents including

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sugars, polyols and related compounds is well incorporated into the three-dimensional H-bond network of water. These species influence slightly the protein-water interaction. They induce

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preferential hydration of the protein surface at higher concentrations and stabilize native structures due to the minimization of the surface contact between protein and non-electrolyte molecules [2]. It is not surprising that glycerol is widely used by biochemists to stabilize the

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activity of enzymes and enhance the thermal stability of proteins for many years [2, 3]. Thus,

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hydrophilic chaotropes such as urea stabilize the unfolded conformation of biomolecules, whereas hydrophilic kosmotropes indirectly stabilize more folded conformations. During the last decade we have been involved in the extensive study on thermodynamics of

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interactions between single blocks of macromolecules – amino acids and non-electrolytes in an aqueous medium (see [4-7] and references therein). The main goal of those investigations was to

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obtain the experimental information on the temperature dependence of the solute - co-solvent interactions in diluted aqueous solutions in the hope that such studies may contribute to better understanding of the protein-solvent interactions. In particular, we have shown that the pair interaction of aromatic amino acids such as L-phenylalanine and L-histidine with urea is attractive in a wide temperature range, preferential orientations between polar groups of solute molecules being observed [4, 5]. Enthalpic and entropic parameters have revealed the extreme temperature dependence, so that the heat capacity of amino acid –urea interaction changed its sign. In contrast, the L-histidine - glycerol interaction in water was found to be repulsive and accompanied by zero heat capacity change in the temperature range of 288-328 K [6]. This 3

ACCEPTED MANUSCRIPT phenomenon has been attributed to the existence of a delicate balance between hydrophobic and hydrophilic interactions in kosmotrope solutions. The similar behavior was found for amino acids in aqueous solutions of ethylene glycol [7]. Here our efforts are mainly directed towards obtaining the information on glycerol interactions with glycine, L-alanine, L-phenylalanine and benzene at 298 and 313 K using a virial expansion technique to elucidate some of the features which contribute to stability of

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proteins in an aqueous solution of kosmotropes. 2. Experimental

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Water was distilled twice to reach the electric conductivity of 110-5 S·m-1. Glycerol (Sigma–

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Aldrich, > 99.5%) was stored with freshly dried 4 Å molecular sieves and then used without further purification. Glycine (Sigma–Aldrich, >99%), L-alanine (Fluka, >99%) and L-

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phenylalanine (Carl Roth GmbH, >99.5%) were dried in vacuum at 343 K for several days and used without further purification. Benzene (Reachem, for chromatography, >99.5%) was used as

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supplied. Aqueous glycerol solutions were prepared by weight from freshly bidistilled water and the pure alcohol with accuracy of XGl=0.0001 mol fraction. Enthalpies of solution (Hsolm, kJ mol-1) were measured with the self-built isoperibol

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automated ampoule calorimeter equipped by titanium vessels of various capacities. The detection

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limit of the apparatuses was 10 µK. The temperature instability in the thermostat was less than 1 mK. Enthalpies of solution were measured by a comparative method with the digital Standard Temperature Measuring Instrument [4-7]. The calorimeter was tested by measuring enthalpies of

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solution of 1-propanol in water at 298.15 K. Our result Hsol0= -10.200.06 kJ mol-1 was found to be in a very good agreement with the recommended value of -10.160.02 kJ mol-1 [8].

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Duration of dissolution of solutes in our cell equipped effective stirring was found to be equal to twenty minutes for benzene and about three-ten minutes for amino acids. The measurements for benzene required special accuracy due to low solute solubility and a correct estimation of side effects associated with an ampoule crushing on the vessel bottom and partial solute vaporization into a free space of the ampoule.

3. Results Standard enthalpies of solution (Hsol0, kJ mol-1) for benzene and amino acids in water given in Tables 1, 2 represent the result of five or more independent measurements in the range of solute 4

ACCEPTED MANUSCRIPT molalities of 0.002-0.03 mol kg-1, where the Hsolm values do not depend on a solute concentration. The Hsolm  Hsol0 values in water-Gl mixtures reflect the result of one experiment in the same molality range. Tables 1, 2 show that our results in pure water are in a very good agreement with the most reliable literature values [9-15].

4. Discussion

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4.1. Enthalpies of transfer at 298 and 313 K Fig. 1 illustrates the curves Htr0 vs. XGl for amino acids and benzene. The solute transfer from

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water to aqueous Gl solutions is seen to be accompanied by a positive enthalpy change. It is almost independent of the temperature at low glycerol concentrations but depends on the amino

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acid side-chain nature. For hydrophobic L-alanine and, especially, L-phenylalanine the enthalpic cost is significantly larger than that for more hydrophilic L-histidine or glycine. The transfer of

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benzene is also energetically unfavorable, the curves Htr0 vs. XGl passing through noticeable maxima at both temperatures. The experimental curves for L-phenylalanine indicate that the

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amino acid should reveal the similar behavior, however, the extremum position here is slightly shifted to a larger Gl content.

Fig. 1 shows one interesting feature of amino acid solvation in an aqueous Gl solution

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which is worth noting. While the enthalpy of L-phenylalanine or L-proline transfer increases

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monotonously with the glycerol content, the corresponding values for L-histidine and glycine become nearly constant at XGl>0.05 mol fractions. For the first sight it should signify the formation of labile complexes between amino acid and glycerol molecules. Since the enthalpy of

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transfer shown in Fig. 1 is small but positive, the formation of such species should be driven by a favourable entropy change. However, this is unlikely. Both amino acid and glycol molecules are

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well-hydrated to form such an aggregate in water. Moreover, molecular dynamics simulations show [18] that Gl molecules can bind to protein O and N atoms in 30% glycerol aqueous solutions favouring formation of multiple hydrogen bonds. This favourable energetic effect occurring at high co-solvent concentrations is accompanied by negative enthalpy changes. However, in our case the enthalpy of transfer is positive and does not depend on the temperature (see Fig. 1). This indicates that our results cannot suggest direct amino acid-glycerol binding. Glycerol molecules appear to perturb only slightly hydration shells of such amino acids as long as sufficient bulk water is present in the mixed solvent.

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ACCEPTED MANUSCRIPT 4.2. Solute-glycerol interaction The formally exact theory of solutions originally developed by McMillan and Mayer and then adapted by Kauzmann, Friedman, Wood and Lilley (see [19-22] and references therein) relates thermodynamic properties of a multicomponent system to certain integrals of the potential of mean force associated with the interaction between pairs, triplets and of a high number of solute molecules in a solvent medium. The thermodynamic formalism used here is the same as in our previous efforts [4-7]. The Hsol0–f(XGl) curves have been fitted to second or third order

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polynomials for the whole glycol concentration range studied: Hsol0 = A0 + A1 XGl + A2 XGl2 + A3 XGl3

(1)

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The A1 and A2 coefficients obtained in a least-square fitting routine are related to the enthalpic

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parameters of the solute-glycerol pair (hGl-S) and triplet (hGl-Gl-S) interactions in the following manner [4-7]:

hGl-Gl-S = (A2-A1) M H O 2 / 3 2

(2)

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hGl-S = A1 M H O /2; 2

where M H O is molar mass of water (kg mol-1). These quantities shown in Tables 3, 4 are seen to 2

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be positive. It indicates that the interaction between one solute and one co-solvent molecules in water is enthalpically unfavorable due to a partial overlapping of their hydration spheres [19]. We see that the larger hydrophobicity of the solute, the larger are hGl-S values. This effect is

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much weaker pronounced in our systems than in the case of the interaction between typical

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hydrophobic species – tetraalkylammonium ions and hexamethyl phosphoric triamide in water [23], where the enthalpy of interaction exceeds few thousands J kg mol-1. The triplet interaction between two kosmotrope and one amino acid molecules is enthalpically favourable. The hGl-Gl-S

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values are seen to be several times smaller than corresponding pair interaction parameters suggesting about a rather rapid convergence of a virial expansion.

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Table 3 compares interaction parameters obtained by different scientific groups [6, 9, 12, 24, 25]. We see that our hGl-S value for glycine is in a satisfactory agreement with the quantity reported by Wang et al. [24], but smaller than the parameter given elsewhere [9]. For L-alanine our hGl-S value is also in a satisfactory agreement with literature quantities [12, 24]. The comparison of the enthalpic pair and triplet interaction parameters listed in Tables 3, 4 shows that they are nearly identical at 298 and 313 K excepting the hGl-Gl-S values for most hydrophobic species such as benzene and L-phenylalanine. Taking into account that both the hGlS

and hGl-Gl-S parameters for L-histidine are constant in a wide temperature range [6], we can

assume that for slightly hydrophobic amino acids such as glycine or L-alanine it should be also the case, i.e. these quantities are to be temperature independent. Hence, it should lead to the 6

ACCEPTED MANUSCRIPT temperature independent enthalpies of the solute transfer from water to rather concentrated glycerol solutions until pair and three-particle interactions dominate. It leads to the nearly zero heat capacity change. Fig. 1 illustrates that, however, it is not the case for hydrophobic Lphenylalanine and benzene, where triplet interactions are found to be temperature dependent. It is the main reason of deviating Hsol0–f(XGl) curves for these species at higher Gl concentrations (see Fig.1). Recently [7] we have shown that in the physiological temperature range the interaction

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between Eg and amino acid molecules in water is accompanied by the following changes of thermodynamic parameters gEg-S >0 and hEg-S>TsEg-S>0. The identical changes take place for the

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L-histidine-Gl interaction [6]. It is known that such a combination of parameters is observed for non-electrolytes stabilizing native structure of globular proteins [26]. The study of toluene

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solvation in aqueous kosmotrope solutions has indicated [27] that both Eg and Gl addition to water enhances solubility of hydrocarbon. It leads to negative gEg-S and gGl-S values. Hence, for

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Gl interactions with any apolar solute we have g<0 and Ts>h>0. This combination is usually observed for the systems, where the hydrophobic interaction dominates. It is obvious that apolar

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residues of amino acids give a positive contribution to the energetics of glycerol - amino acid interaction, whereas polar and zwitter-ionic groups contribute in an opposite manner. The comparison of the hGl-S parameters for glycine and L-alanine supports this finding.

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The comparison of these and previously reported results indicates that both in Eg and Gl

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solutions we have the following combination of the interaction parameters g>0, h>Ts>0. Hence, one can assume that similar mechanisms should be involved into the interaction between amino acid and glycol molecules. Fig. 2 supports this idea highlighting the existence of linear changes

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of the hEg-S and hGl-S values:

R2 = 0.963, sf = 72 J kg mol-2

(3)

hEg-S(313 K) = 36(40)+1.095(0.1) hGl-S,

R2=0.979, sf = 50 J kg mol-2

(4)

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hEg-S(298 K) = 65(57)+1.151(0.2) hGl-S,

We see from eqs. (3, 4) that corresponding enthalpic parameters in Eg and Gl solutions are nearly identical. It indicates that OH- and CH-groups contribute in an opposite manner to the enthalpies of interaction. The hEg-S quantities are, however, slightly larger, which signifies the slight prevalence of a polar group contribution. The relationships depicted in Fig. 2 are seen to cover solutes with small (glycine) and high (L-phenylalanine, benzene) hydrophobicity, which has some predictive value. If the hEg-S or hGl-S parameter is available, the corresponding value for other kosmotrope can be easily estimated. It is important that the coefficients of eqs. (3, 4) are nearly identical. This fact gives the additional opportunity to estimate corresponding enthalpic 7

ACCEPTED MANUSCRIPT parameters, for example, at unexplored temperatures at least within the physiological temperature range. 5. Conclusion In conclusion, we would like to mention the following, as a result of this and previous calorimetric studies using biomolecules which, in our opinion, have some importance for understanding the mechanisms of the protein-kosmotrope interaction. (a) The amino acid pair

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interaction both with Eg and Gl is accompanied by positive changes of the enthalpy, entropy and free energy. Enthalpic and entropic parameters appear to be temperature independent over the

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physiological temperature range. For hydrophobic solutes such as L-phenylalanine or benzene the total interaction is more attractive (the free energy of pair interaction is smaller) than for

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hydrophilic species. Due to a favourable entropy change this effect should be much stronger pronounced in hot water. Hence, proteins with a larger content of aromatic residues on their

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outer surface should reveal larger affinity to glycols and lower thermal stability. (b) There is a quite clear, if not precise, the existence of the linear relationship between enthalpies of the solute

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pair interaction with ethylene glycol and glycerol in water. It suggests that similar mechanisms are involved into pair correlations for both cases. This result has a predictive value providing a rapid estimation of corresponding interaction parameters for unexplored biomolecules in

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aqueous kosmotrope solutions.

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[6] A.V. Kustov, N.L. Smirnova, R. Neueder, W. Kunz, Amino acid solvation in aqueous kosmotrope solutions: Temperature dependence of the L-histidine - glycerol interaction, J.

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[10] B. Palecz, Enthalpies of solution and dilution of some L-α-amino acids in water at 298.15 K, J. Thermal Anal. Calorim. 54 (1998) 257-263.

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ACCEPTED MANUSCRIPT [14] A.V. Kustov, A.V. Bekeneva, O.A. Antonova, V.P. Korolev, Enthalpic pair-interaction coefficients of benzene, aniline and nitrobenzene with N,N-dimethylformamide and acetonitrile in water at 298.15 K, Thermochim. Acta 398 (2003) 9-14. [15] D. Hallén, S.-O. Nilsson, I. Wadsö, A new flow-microcalorimetric vessel for dissolution of small quantities of easily or slightly soluble liquids, J. Chem. Thermodyn. 21 (1989) 529537. [16] V.P. Korolev, O.A. Antonova, N.L. Smirnova, Dissolution enthalpies of L-proline and its

298.15 K, Russ. J. Chem. A 84 (12) (2010) 2056-2060.

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interactions with methanol, 2-propanol, ethyleneglycol, and glycerol in aqueous solution at

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[17] V.P. Korolev, O.A. Antonova, N.L. Smirnova, Thermodynamic characteristics, structure and interactions of L-proline in aqueous alcohols and urea solutions, J. Struct. Chem. 55

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(2014) 344-349.

[18] V. Vagenende, M.G.S. Yap, B.L. Trout. Molecular anatomy of preferential interaction

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coefficients by elucidating protein solvation in mixed solvents: Methodology and application for lysozyme in aqueous glycerol, J. Phys. Chem. B 113 (2009) 11743–11753.

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[19] H.L. Friedman, C.V. Krishnan, Studies of hydrophobic bonding in aqueous solutions. Enthalpy measurements and model calculations, J. Solut. Chem. 2 (1973) 119-140. [20] J.J. Savage, R.H. Wood, Enthalpy of dilution of aqueous mixtures of amides, sugars, urea,

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ethylene glycol and pentaerythritol at 25 0C. Enthalpy of interaction of the hydrocarbon,

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amide and hydroxyl functional groups in dilute aqueous solutions, J. Solut. Chem. 5 (1976) 733-750.

[21] Ph.J. Cheek, T.H. Lilley, The enthalpies of interaction of some amides with urea in water at

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250C, J. Chem. Soc., Faraday Trans. I 84 (1988) 1927–1940. [22] Yu.M. Kessler, A.L. Zaitsev, Solvophobic Effects, Ellis Horwood, Chichester, 1994.

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[23] A.V. Kustov, V.P. Korolev, Temperature and length scale dependence of the tetralkylammonium ion-amide interaction, J. Phys. Chem. B 112 (2008) 2040–2044. [24] Rui Wang, Ni Cheng, Jingjing Jiao, Min Liu, Li Yu. Studies of interparticle interactions among some L--amino acids and 1,2(1,3)-propandiols in aqueous solution at 298.15 K, Thermochim. Acta. 565 (2013) 34-38. [25] Y. Li, Z. Yingyuan, L. Yonghui, J. Jing, W. Xiaoqing, Heterogeneous interaction between zwitterions of amino acids and glycerol in aqueous solutions at 298.15 K, J. Thermal. Anal. Calorim. 104 (2011) 797-803.

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ACCEPTED MANUSCRIPT [26] G. Castronuovo, V. Elia, M. Niccoli, F. Velleca, Calorimetric studies of hydrophobic interactions of alkanols in concentrated aqueous solutions of glucose. Implications for the mechanism of protein stabilization by sugars, Thermochim. Acta. 389 (2002) 1-9. [27] E. Carrillo-Nava, V. Dohnal, M. Costas, Infinite dilution activity coefficients for toluene in aqueous solutions of protein stabilizers glycerol, ethylene glycol, glucose, sucrose and

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trehalose, J. Chem. Thermodyn. 34 (2002) 443-456.

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ACCEPTED MANUSCRIPT Figure captions Fig. 1. Enthalpies of transfer of glycine (,), histidine (,  - 308.15 K [6]), alanine (,), proline ( - [16],  - [17]), phenylalanine (▲, - [6]) and benzene (, ), from water to aqueous glycerol solutions at 298.15 K (dark symbols) and 313.15 K (empty symbols).

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Fig. 2.The relationship of hEg-S and hGl-S parameters in water for glycine (,), histidine (,  - 308.15 K [6]), alanine (,), proline ( - [16],  - [17]), phenylalanine (▲, - [6]) and benzene (, ) at 298.15 K (dark symbols, solid line – eq. (3)) and 313.15 K (empty symbols, dashed line – eq. (4)).

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Fig. 1

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Fig. 2

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ACCEPTED MANUSCRIPT Table 1 Enthalpies of solution (Hsol0, kJ mol-1) of glycine, L-alanine, L-phenylalanine and benzene in aqueous glycerol solutions at 298.15 K Alanine

14.190.02b 14.200.06 [9,10]

0

Benzene

XGl

Hsol0

XGl

Hsol0

0

8.300.03 [6] 8.23 [4] 8.280.08 [13]

0

2.140.07 [7] 2.04 [14] 2.220.01 [15]

0.006400 14.22

7.750.05 7.670.05 [10] 7.780.04 [11] 7.660.05 [12] 0.01010 7.99

0.01806 9.21

0.005010 2.53

0.01247

14.23

0.02996 8.44

0.03306 9.79

0.01002

2.84

0.02091

14.24

0.05020 8.84

0.04996 10.52

0.01503

3.12

0.03170

14.26

0.08020 9.29

0.06123 10.95

0.03001

4.00

0.04109

14.27

0.1004

0.07825 11.60

0.04495

4.57

0.04860

14.27

0.08914 11.90

0.05502

4.81

0.06069

14.28

0.07002

5.12

0.07033

14.28

0.07500

5.30

0.08040

14.27

0.08997

5.50

0.09020

14.26

0.1100

5.63

0.1038

14.24

0.1250

5.53

0.1499

5.32

0.1750

4.75

0.2002

4.45

0.3001

3.39

9.45

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0

Hsol0

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XGl

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Hsol0

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XGla

Phenylalanine [6]

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Glycine

a b

– Gl mol fractions. - Errors from here on represent the twice standard deviation.

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ACCEPTED MANUSCRIPT Table 2 Enthalpies of solution of glycine, L-alanine, L-phenylalanine and benzene in aqueous glycerol solutions at 313.15 K Glycine

Alanine Hsol0

13.530.03 13.500.05 [7] 0.005739 13.55

0

8.190.04 0

0.01010 8.42

11.010.05 11.000.05 [7] 0.01160 11.61

0.01005

13.56

0.03020 8.82

0.02690 12.26

0.01001

6.30

0.02526

13.59

0.05020 9.28

0.04220 12.84

0.01519

6.59

0.03131

13.60

0.08120 9.75

0.05690 13.37

0.02020

6.84

0.04049

13.61

0.1004

0.07380 13.85

0.02999

7.48

0.05085

13.61

0.09190 14.12

0.04004

7.86

0.06051

13.62

0.06040

8.53

0.07501

13.61

0.08517

9.04

0.08082

13.61

0.09010

9.16

0.09053

13.59

0.1101

9.40

0.09935

13.58

0.1195

9.45

0.1300

9.40

0.1497

9.30

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9.95

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0.1189

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0

XGl

Hsol0

Benzene

XGl

XGl

Hsol0

Phenylalanine

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14.55

XGl

Hsol0

0

5.600.06 [7]

0.005010 6.01

ACCEPTED MANUSCRIPT Table 3 Enthalpic parameters of pair (hGl-S/J kg mol-2) and triplet (hGl-Gl-S/J kg2 mol-3) interaction of a solute (S) with glycerol (Gl) in water at 298.15K hGl-S

hGl-Gl-S

Glycine

24(1)

-2.6(0.2)

-42(125) [24]

-2.2 [25]

252 (44) [9]

-

235(4)

-12.6(0.6)

132 (128) [24]

-33.3 [25]

355(7) [12]

-19.0(0.8) [12]

Histidine

200(9) [6]

-29.4(2.3) [6]

L-Proline

-99(275) [24]

-

362(9) [16]

-13.5(1.1) [16]

Phenylalanine

442(15) [6]

-15.7(2.1) [6]

Benzene

586(19)

RI

SC

NU

-41.0(2.4)

AC

CE

PT E

D

MA

L-Alanine

17

PT

Solute

ACCEPTED MANUSCRIPT Table 4 Enthalpic parameters of pair (hGl-S/J kg mol-2) and triplet (hGl-Gl-S/J kg2 mol-3) interaction of a solute (S) with glycerol (Gl) in water at 313.15K hGl-S

hGl-Gl-S

Glycine

25(1)

-2.8(0.1)

L-Alanine

220(8)

-10.0(1.2)

L-Proline

332(5) [17]

-11.9(0.6) [17]

Histidine

203(6) [6]a

-28.8(4.9) [6]a

Phenylalanine

462(12)

-25.4(1.3)

Benzene

579(18)

-37.0(2.0)

RI

CE

PT E

D

MA

NU

SC

– calculated as the mean value from the hGl-S quantities at 308 K and 318 K [6].

AC

a

PT

Solute

18

ACCEPTED MANUSCRIPT

800

Phe Phe

600

B

PT

313 K Pro Pro

Ala His Ala

SC

200 Gly

RI

His

400

NU

hEg-S(W+Eg), J kg mol

-2

298 K B

0

200

MA

0

400

Graphical abstract

AC

CE

PT E

D

hGl-S(W+Gl), J kg mol

19

600 -2

ACCEPTED MANUSCRIPT

Highlights Enthalpies of amino acid solution in water-Gl mixtures were measured at 298 and 313 K.

-

Pair and triplet interaction parameters were computed.

-

The findings were discussed in terms of a virial expansion technique.

AC

CE

PT E

D

MA

NU

SC

RI

PT

-

20