Journal of the Chinese Chemical Society, 2007, 54, 667-672

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Thermodynamic Studies on Amino Acid Solvation in Aqueous Urea Someswar Chatterjee and I. Basumallick* Department of Chemistry, Visva-Bharati, Santiniketan-731235, India

The present paper discusses the behaviour of transfer free energy of some amino acids from water to 4M, 6M and 8M aqueous urea. Dissection of transfer free energy into cavity term, interaction term and electrical term reveals that cavity forming free energy of transfer DG0t (cav) plays an important role in dictating actual interaction of amino acids in aqueous urea. Cavity forming free energy of transfer has been estimated by using Scaled Particle Theory (SPT). Keywords: Cavity forming free energy; Zwitter ion; Scaled Particle Theory; Hydrophobic-interaction.

INTRODUCTION Much attention1-3 is now being paid to the thermodynamic studies of solute-solvent interaction in mixed solvents. The present paper on amino acid solvation in aqueous urea is a continuation of our earlier work4 on amino acid solvation in aqueous alcohols. This is because denaturation of protein by aqueous urea has been exhaustively studied;5-12 however, the understanding of the mechanism of the unfolding of protein can hardly be considered as settled. Among various proposed mechanisms (i) weakening the inter peptide hydrogen bond5 (ii) alternation of water structure by urea17 and (iii) reduction of hydrophobic interaction5,14,15 are attractive. However, in order to understand in depth the dynamics of solvent perturbation of protein structure, i.e. estimation of the driving forces for urea induced unfolding, it would be of interest of know the thermodynamics of solution of an individual amino acid, basic building blocks of protein, in aqueous urea. From time to time attention has been given to determine the various thermodynamic properties such as partial molar volume,16 enthalpy of solution,17 partial molar heat capacity,18 and subilities5 of various amino acids in aqueous urea. The purpose of such studies is to gain the basic aspects of amino acid hydration. Tanford and Nozaki5 reported free energies of some amino acids from water to aqueous urea from solubility measurements. But correlation of experimental transfer free energies at different concentrations of urea for different amino acids is a difficult task in terms of hydrophilicity or hydrophobicity of the amino acids. This may be due to

the fact that experimental transfer free energies are composite in nature and consist of electrical free energies, cavity forming free energies and free energies of interaction. It is expected that the free energies of interaction of these amino acids will bear a good correlation with the hydrophilic-hydrophobic ratio of the amino acids. Therefore, in this paper an attempt has been made to dissect experimental transfer free energies into free energies of interaction, cavity free energies and electrical free energies of transfer. It may be mentioned that similar dissection of transfer free energies of different solutes19-21 including amino acids4 gives better understanding of solutesolvent interactions. The amino acids studied here are Glycine, Alanine, Threonine, Valine, Proline, Glutamine, Histidine, Leucine, Methionine, Cysteine, Asparagine, Phenylalanine, Tyrocine and Tryptophan in 4M, 6M and 8M aqueous urea.

CALCULATIONS The experimental transfer free energies have been dissected into the cavity part, electrical part and interaction part as equation (1) DG0t (expt) = DG0t (cav) + DG0t (ele) + DG0t (int) (1) Scaled Particle Theory (SPT) has been applied in computation of transfer cavity forming free energy from water to aqueous urea of zwitter ionic solute-like amino acid using the following equation (2).4,19-21

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DG0t (cav) = Gc + RT ln (RT/V)

(2)

where Gc = RT [-ln (1-Z) + {3X/(1-Z)} D + {3Y/(1-Z)} D2 + {9X2/4(1-Z)2}D2] Z = pNA (z1a3 + z2b3) / 6V X = pNA (z1a2 + z2b2) / 6V Y = pNA (z1a + z2b) / 6V V = M/d In these expressions NA is Avogadro’s number, M and d are molarmass and density of the solvent, respectively, z1 and z2 are the molefraction of water and urea, respectively. ‘D’, ‘a’ and ‘b’ are the hard sphere diameter of amino acid, water and urea, respectively. Hard sphere diameters of amino acids have been calculated from reported partial volume data 16,22 and using Farrel’s treatment.23

Solvent parameters and diameters used in this calculation are taken from well-reported literature data4,23-25 and are displayed in Table 1. Cavity forming free energies of transfer of these amino acids are shown in Table 2. Admittedly, authenticity of these cavity forming free energies data will be guided by the uncertainties associated with cavity free energies terms. Major uncertainties for cavity free energies are incorporated via solute and solvent diameters. However in the present calculation these molecular parameters have been taken from standard literature data or have been derived from literature data using wellestablished relations. For the sake of satisfaction we have calculated cavity terms of these amino acids varying cavity diameter to ±0.2 Å and solvent diameter to ±0.05 Å unit and for such variation cavity forming free energies change only ±0.05 and ±0.12 KJ/mole, respectively. It is also noted that even after such variation an unfavorable cavity effect is observed for all the amino acids in these media. Very recently, Graziano26 has shown that variation of hard sphere

Table 1. Solvent parameters used in calculation of DG°t (cav.) and DG°t (ele.) at 298.15 K Solvent

Hard sphere diameter4,23 in Å

Water Urea

Mol% of Molarmass Molarity co-solvent

2.76 07.77 12.80 18.95

4.24

18.04 21.30 23.41 25.99

4 6 8

Density25 in gm.cm-3

Dielectric constant24

00.9973 1.058 1.088 1.118

78.4 87.9 93.2 96.8

Table 2. Transfer cavity forming free energies DG°t (cav) and transfer free energies of interaction DG°t (int) in KJ/mol from water to aqueous urea at 298.15 K DG°t (int.)

DG°t (cav.) Amino acids

Glycine Alanine Threonine Valine Proline Glutamine Histidine Luecine Methionine Cysteine Asparagine Phenylalanine Tyrocine Tryptophan

Diameter (Å)

3.85 4.20 4.86 5.00 4.97 5.19 5.28 5.40 5.39 4.79 5.57 5.60 5.69 5.98

Urea

Urea

4M

6M

8M

4M

6M

8M

0.26 0.36 0.57 0.62 0.61 0.69 0.72 0.77 0.76 0.55 0.83 0.84 0.88 0.98

0.39 0.55 0.87 0.95 0.93 1.05 1.10 1.16 1.16 0.84 1.26 1.28 1.33 1.51

0.54 0.75 1.20 1.30 1.27 1.44 1.50 1.60 1.59 1.15 1.73 1.75 1.83 2.07

-0.67 -0.62 -0.09 -0.05 -0.23 -0.32 -0.47 -0.50 -0.78 -0.57 -0.59 -1.29 -1.61 -2.18

-1.09 -0.97 -0.23 -0.09 -0.10 -0.37 -0.49 -0.62 -1.05 -0.73 -0.78 -1.78 -2.28 -3.09

-1.31 -1.15 -0.22 -0.12 -0.26 -0.55 -0.72 -0.98 -1.45 -1.01 -1.15 -2.42 -3.07 -4.08

Amino Acids Solvation in Aqueous Urea

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diameter of solute to the extent of 0.3 Å does not affect the physical reliability of the results of SPT calculation. Thus, the question of misuse of SPT may not be significant here. When amino acids are transferred from water to aqueous urea the dielectric constant of media change appreciably, so that free energy of transfer due to electrostatic effect DG0t (ele) has been calculated using Scatchard and Kirkwood expression4,27-28 of zwitter ion as shown in equation (3) DG0t (ele) = k2 (1/D2 – 1/D1)

(3)

where D1 and D2 are the dielectric constant of water and urea-water, respectively, and k2 is constant. The deduction of the value of k2 has been discussed elsewhere.4 The values of DG0t (ele) are -0.90 KJ/mol, -1.34 KJ/mol and -1.61 KJ/mol for 4M, 6M and 8M, respectively. Transfer free energies of interaction DG0t (int) values have been calculated from experimental free energies of transfer after subtracting the cavity forming free energy and electrical effect DG0t (ele), i.e. DG0t (int) = DG0t (expt) - DG0t (cav) - DG0t (ele) (4) The values of DG0t (int) are displayed in Table 2.

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RESULTS AND DISCUSSION Fig. 1 shows variation of experimental5 free energies of transfer of amino acids with composition of aqueous urea. The positive values of DG0t (expt) for glycine and alanine; and negative values of DG0t (expt) for other amino acids indicate stabilization of amino acids except glycine and alanine in aqueous urea. While alanine is most destabilized on transfer, tryptophan is least. The observed stabilization and destabilization increase with increasing the concentration of urea. It may be noted that stabilization of amino acids in aqueous urea is mainly guided by hydrophobicity of different side chains. This is indicated from the fact that the favourable free energy of transfer of amino acids increases with the increase of the size of the attached hydrophobic part as side chain. However transfer free energies for methionine and cystine are almost equal though their attached side chains are different; similarly at low concentrations of urea, transfer free energies of asparagines are equal to the former two amino acids. Again, alanine with a larger hydrophobic side chain gets more destabilized than glycine upon transfer to aqueous urea. To explain these anomalous behaviors of transfer free energies we have taken into account the effect of change of dielectric constant of the media through DG0t (ele) electri-

Fig. 1. Transfer free energies of amino acids DG0t (expt)5 from water to aqueous urea at 298.15 K.

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cal term and cavity creation energy term DG0t (cav). Table 2 shows that transfer cavity forming free energies of these amino acids DG0t (cav) are unfavourable. This is pictorially shown in Fig. 2. It may be noted that a similar result was reported by Graziano29 from study of the transfer cavity forming free energy of some hydrocarbons from water to 7M aqueous urea. Analysis of the cavity equation indicates that cavity forming free energies values are guided by hard sphere diameter of solute, solvent and density of the medium. On addition of urea to water, both hard sphere diameter and density of the solution increase. These two things affect DG0t (cav) in an opposite way, but the latter one predominates and results in larger cavity energy in urea-water than of that in water. This has been explained by Trzesniak et al.30 Because urea is strongly hydrated in aqueous solution it is difficult to create a cavity in aqueous urea compared to bulk water. Again increased surface tension,31 direct proportionality between work of cavity creation and surface tension32 indicates unfavorable cavity energy in aqueous urea. While cavity forming free energies are unfavourable,

Chatterjee and Basumallick

the electrical free energies of transfer DG0t (ele) values are favourable during transfer of amino acids from water to aqueous urea due to higher dielectric constant. Fig. 3 shows the variation of transfer free energies of interaction of amino acids with mol% of urea. The transfer free energies of interaction for glycine and alanine now seem to be guided by the hydrophobicity of the side chain. It may be noted that, although favourable interaction energies are primarily guided by the hydrophobicity of the side chains, other interactions like overlap of different hydration spheres are to be considered for further insight. The observed stabilization of hydrophobic side chains in aqueous urea may be explained in terms of better hydrophobic hydration of apolar side chains of amino acids in aqueous urea where more free water molecules are available because of the structure breaking ability of urea. In conclusion, it may be stated that experimental free energy of transfer of amino acids from water to aqueous urea need to be dissected into various components including cavity component to gain an idea about the effective interaction experienced by the amino acids in these protein

Fig. 2. Transfer cavity forming free energies DG0t (cav) of amino acids from water to aqueous urea at 298.15 K.

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Fig. 3. Transfer free energies of interaction DG0t (int) of amino acids from water to aqueous urea at 298.15 K.

denaturing media.

ACKNOWLEDGEMENTS We thank the Dept of Chemistry, Visva-Bharati for providing useful computational facilities.

Received May 3, 2006.

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