Partial molal volumes of transfer of some amino acids from water to aqueous 1,4-dioxane solutions at K

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1 Indian Journal of Chemistry Vol. 39A, October 2000, pp Partial molal volumes of transfer of some amino acids from water to aqueous 1,4-dioxane solutions at K T S Banipal & Gagandeep Singh Department of Pharmaceutical Sciences, GuruNanak Dev University, Amritsar , India B S Lark Departmen\ of Chemistry, Guru Nanak Dev University, Amritsar , India Received 25 November 1999; revised 23 February 2000 Apparent molal volumes of glycine, DL-n-alanine, L-valine, L-Ieucine and L- phenylalanine in water and in 2.5, 5, 10, 20,25% aqueous solutions of l,4-dioxane have been obtained from densities of their solutions at K measured by using precise vibrating tube digital ~ensitymeter. The estimated partial molal volumes (V 2 0 ) at infinite dilution have been used to obtain corresponding transfer volumes (V 2.. tr ) from water to different l,4-dioxane-water mixtures which have been found to be positive for glycine, and both positive and negative for the remaining amino acids over the l,4-dioxane concentration range studied. The data have been interpreted in terms of solute-cosolute interactions. Concentration dependent behaviour of l,4-dioxane in aqueous solutions have also been taken into account while interpreting the V 2,tr values of amino acids. Denaturation of globular proteins in aqueous solutions is a fundamental biological process which till date is not completely understood and continues to be a subject of extensive investigations 1-6. During the denaturation process, various structural changes occur in protein solutions. The knowledge of solute-solvent and solute-solute interactions in various solvents is thus, prerequisite to understanding the process of denaturation. As the proteins are large complex molecules, direct study of their interactions is a difficult task. One useful approach to understand these interactions is to study their model compounds. Consequently, in recent years, there has been considerable interest in the determination of various thermodynamic properties of amino acids, small peptides and their derivatives in pure aqueous and. d I' 4-11 IDlxe aqueous so uttons. Mixed aqueous solvents are used extensively in chemistry and other fields to control factors like stability, reactivity and solubility of systems 9 1O 1,4- Dioxane isa widely used solvent because it is a nonhydrogen bonded cyclic ether, miscible with water in all proportions with a boiling point close to that of water. Moreover, its dipole moment is close to zero (0.45 D) and the dielectric constant can be varied over a wide range (2.2-78) in its mixtures with water 12 In the present paper, the apparent molal volumes of glycine, DL-a.-alanine, L-valine, L -leucine and L - phenylalanine in water and in 2.5, 5, 10, 20 and 25% aqueous solutions of 1,4-dioxane have been determined from density measurements at K. The data have been used to estimate partial molal volumes of transfer for amino acids from water to aqueous 1,4-dioxane solutions- and assess the relative domination of various contributing factors. Materials and Methods Five amino acids, namely, glycine (0-7126), DL - a.-alanine (A-7502), L -valine (V-0500), L -leucine (L -8000) and L -phenylalanine (P-2126) of highest purity were obtained from Sigma Chemical Co. and used as such without further purification. l,4-dioxane (CDH,AR) was refluxed and then distilled over sodium metal using aim long fractionating glass column. The middle fraction distilling at 373 K was collected for use. Solutions of amino acids were prepared in deionized, doubly distilled water and : degassed before use. All the solutions were made on weight basis and all the weighing were done on the Mettler Balance with an accuracy of ± 1 x 10'"' g. Densities of the solutions were measured with a

2 1012 INDIAN J CHBM. SBC. A. OCTOBBR Table l--densities (d) and apparent molal volumes (tv) of some amino acids in water andin aqueous 1.4-dioxane mixtures at K m, mol kg-l. d,kg.m- 3 +,xlo'.m 3 mor 1 m,molkg-l d,kg.m- 3 +,xlo'.m 3 mor 1 Glycine in waler Glycine in 25% aqueous 1.440JUllle _ Glycine in 2.5% aqueous 1.4-dioJUllle DL-a-Alanine in waler _ ~8 DL-a-Alanine in 2.5% aqueous 1.4-dioXQlle Glycine in 5% aqueous 1.4-dioXQlle : Glycine in 10% aqueous l,4-dioxqlle _ DL-a-Alanine in 5% aqueous l,4-dioxqlle Glycine in 20% aqueous l,4-dioxqlle _ DL-a-Alanine in 10% aqueous 1.4-dioXQlle Contd

3 BANIPAL et al.: PARTh\L MOLAL VOLUMES OF AMINO ACIDS 1013 Table l-oensities (d) and apparent molal volumes (+.. ) of some amino acids in water and in aqueous l,4-dioxane mixtures at K-Contd '. m, mol kg-l d,kg.m 3... xl0 6, m 3 morl m, mol kg-i d,kg.m xlff, m 3 morl DL-a-Alanine in 20% aqueous l,4-dioxane L-Valine in JO%aqueous 1.4-dioxane DL-a-Alanine in 25% aqueous l,4-dioxane L-Valine in 20% aqueous 1.4-dioxane L-Valine in water L-Valine in 25% aqueous 1.4-dioxane L-Valiru: in i5%aqueous 1.4-dioxane : L-Leucine in water L-Valine in 5% aqueous 1.4-dioxane L-Leucine in 2.5% aqueous J.4-dioxane Contd

4 1014 INDIAN J CHEM, SEC. A, ocrober 2000 Table l-densities (d) and apparent molal volumes (~) of some amino acids in water and in aqueous l,4-dioxane mixtures at K-Contd m, mol kg 1 d, kg.m- 3 cl>v x 10 6, m 3 mor l m, mol kg-i d, kg.m- 3 ~ xl0 6, m 3 mor l L-Phenylalanine in 2.5% aqueous l,4-dioxane L-Leucine iii 5% aqueous l,4-dioxane _ L-Phenylalanine in 5% aqueous l,4-dioxane L-Leucine in 10% aqueous 1,4-dioxane L-Phenylalanine in 10% aqueous 1,4-dioxane _ _ L-Leucine in 20% aqueous 1,4-diomne lot L-Phenylalanine in 20% aqueous l,4-dioxane L-Leucine in 25% aqueous 1,4-dioxane L-Phenylalanine in 25% aqueous l,4-dioxane L-Phenylalanine in water

5 BANIPAL et al.: PARTIAL MOLAL VOLUMES OF AMINO ACIDS 1015 vibrating-tube digital densitymeter (Model DMA 60/602 Anton Paar, Austria). The details of its principle and working have been described elsewhere!3. Temperature of water flowing through the densitymeter cell was controlled within ± 0.01 K using an efficient temperature bath (Heto BirkerodlDenmark). The densitymeter was calibrated with dry air and water and all the measurements were made relative to pure water. The working of densitymeter was checked by measuring the densities of aqueous sodium chloride solutions and an excellent agreement was found with literature values 13,l4. The precision and accuracy of density measurements were better than lxlo-6, 3x 10-6 g cm 3 respectively. Results and Discussion Densities of solutions of amino acids in water and in 2.5, 5, 10, 20 and 25% (v/v) aqueous 1,4-dioxane solutions at K as a function of molality are given in Table 1. The apparent molal volume of a solute in a given solvent with density, tk, (Table 1) was calculated from the equation q,y = MId -[(d-tk)i000/mddoj... (1) where M is the molecular mass of the solute, m is molality (mol kg l of solvent) of the solution having density d. Apparent molal volume of a solute at infinite dilution (q,. ') is its corresponding partial molal volume, i.e, V 2 0, and, in the cases, where q,y values showed negligible concentr~tion dependence, it was estimated to be the average of various q,y values. For most of the cases q,y data showed concentration dependence and corresponding V 2 0 values were determined by the least squares fitting of the data to Eq.2, - 0 q,y= V 2 + Sym... (2) where Sv is the slopels. The V 2 values in water and in aqueous 1,4-dioxane solutions have been summarised in Table 2 along with the literature values of V 2 in water. Good agreement between the experimental. and literature values has been observed7, Partial molal volume of transfer of a solute from water to aqueous 1,4-dioxane solutions at infinite dilution (V 2,tr ) has been estimated as follows, V -o 2,tr = V02 (in aqueous 1,4-dioxane solution) - V02 (in water)... (3) Values of V \tr are illustrated in Fig. 1. V 2,tr values are both positive and negative over the concentration range of 1,4-dioxane studied except for glycme. V \tr values are positive for all the amino acids in 2.5 and 5% aqueous l,4-dioxane solutions Table 2-Partial molal volume at infinite dilution ( V. 2 ) of some amino acids in water and aqueous 1,4-dioxane solutions at K. Compo V02X 106,m 3.mor l water 2.5% 5% 10% 20% 25% Glycine (2.03", 0.03,,) (0.823", 0.02,,) (0.81",0.02,,) (0.93, 0.02,,) (1.28", 0.02") (0.65",0.02,,) 43.20",43.19 d, 43.14", f DL-a-Alanine (1.33", 0.02,,) (0.738", 0.02,,) (0.66",0.04,,) (0.49",0.03,,) (0.94",0.04") 60.50<1, 60.62", 60.39' L-Valine (-7.18", 0.03") (-7.16", 0.03,,) (5.94",0.02,,) (8.54", 0.02") d, 90.98" 90.8aI' L-Leucine (4.20",0.01,,) ( 8.31",0.03,,) (8.08", 0.02,,) (8.05", 0.03,,) (6.32', 0.04,,) d, " L Phenylalanine (40.70",0.04,,) (21.07", 0.03,,) ( , 0.03,,) (20.60", d 0.02,,) Slope, b standard deviation, ~ef. 7; dref. 16; "Ref. 17; fref. 18; iref. 19; ~ef. 20

6 1016 INDIAN J CHEM, SEC. A, OCTOBER r ~ Glycine ' b--- DL-oG -Alanine '-0-.- L - Vali ne 1.5. ~ L-Leucine \ L -Phenylalanine 1.0 /0 0.5.!:;.. 0::.-. ON I> L L10---'15---2'-O--2SJ.--.J -4 of 1,4'- Dioxane Fig. 1-V 2.tI versus % of l,4-dioxane with the exception of very small negative values for DL-a-alanine in 2.5% aqueous l,4-dioxane solution. The magnitude of ifo 2 tr decreases from glycine to DLa-alanine in 2.5 and 5% and then increases regularly from DL-a-alanine to L -valine to L -leucine to L - phenylalanine. In 10, 20 and 25% aqueous 1,4- dioxane solutions, if \tr values are positive for glycine and DL-a-alanine (the magnitude is higher for glycine) and negative for L -valine, L -leucine andl - phenylalanine. To rationalise the behaviour of amino acids in aqueous l,4-dioxane solution, it will be worthwhile to first discuss the behaviour of 1,4-dioxane in aqueous solution. From heat capacity and </lv data Kiyohara et a1. 2! have reported that 1,4-dioxane behaves like a hydrophobic solute in highly concentrated region (5m) whereas in lower concentration range (lm) its interaction with water is not so strong and does not involve any structural changes. Kay and Broadwater 22 have reported that dipolar nature of l,4-dioxane interferes with the process by which hydrocarbon segments of apolar molecule bring about an enhancement of long range order in aqueous mixtures. Gancl 3 considered it only as inert while Harnann 24 showed that it is not essentially inert. From it's positive compressibility of electrostriction, Lo Surdo et al.25 have concluded that 1,4 dioxane is only slightly hydrated in aqueous solution. From viscosity data Lark et al.!9 have indicated that l,4-dioxane is a very weak structure-breaking solute. Tada et al.26 from conductance measurements have suggested that l,4-dioxane is a structure breaker. Consequently it appears that, at small percentages, it might be behaving as a weak structure breaker and at higher percentages as a hydrophobic solute.. Now to explain partial molal volume data, different models have been used. Franks et al. 27 have shown that the partial molal volume of a non-electrolyte is a combination of the intrinsic volume of non-electrolyte. and volume due to it's interaction with the solvent. The intrinsic volume has been considered to be made up of two types of contributions. V int = Vvw + V void Shahidi et al.28 modified this equation to include the contribution of interactions of a non-electrolyte solute with the solvent. where O's is the shrinkage in volume produced by the interactions of hydrogen bonding groups present in the solute with water molecules and n is the potential number of hydrogen bonding sites in the molecule. For electrolytes and zwitterionic solutes the shrinkage is caused by electrostriction and finally if 2 can be evaluated as It has been assumed 29 that Vvw and V void have the same magnitude in water and in mixed solvents for the same class of compounds. Therefore the observed positive if 2.tr values can be attributed to the decrease in volume of shrinkage and negative if 2.tr values to the increase in volume of shrinkage in the presence of 1,4-dioxane. This may be attributed to various types of interactions occurring between amino acids and the 1,4-dioxane molecules having different contributions to if\tr. if 2.tr values can further be rationalised by cosphere overlap model developed by Gurney30 and. Frank and Evans 3!. According to this; properties of water molecules in the hydration cosphere depend on the nature of solute molecules. When two solute. particles come close enough such that their cospheres overlap, some of the cosphere material is displaced and this is accompained by changes in thermodynamic parameters. The following types of

7 BANIPAL et al.: PARTIAL MOLAL VOLUMES OF AMINO ACIDS 1017 interactions are possible: (i) ion-dipolar interactions occurring between zwitterionic centres of amino acids and diplolar parts of 1,4-dioxane (ii) hydrophobicdipolar interaction between non-polar parts of amino acids and dipolar part of 1,4-dioxane, and, (iii) hydrophobic - hydrophobic interactions occurring between non-polar parts of amino acids and hydrophobic parts of 1,4-dioxane. According to the cosphere overlap model, the first. type of interactions results in positive volumes of transfer whereas the other two result in negative volumes of transfer. Therefore, the presently obtained positive values for glycine and DL-a-alanine,(except in 2.5% aqueous 1,4-dioxane solution), L-valine, L leucine and L-phenylalanine upto == 5% aqueous 1,4- dioxane solutions show that the first type of interactions (due to ion-dipolar) dominate over the other two types. The negative values for L-valine, L leucine and L-phenylalanine (due to increasing hydrophobicity) at higher, (10, 20 and 25%) concentration range suggest that the second and third types of interactions overweigh the eff~t of the first. This is in line with the earlier conclusion drawn on the basis of volume of shrinkage that various solutecosolute interactions occur in these systems which contribute to different extents, depending upon the particular amino acid and 1,4-dioxaneconcentration. Keeping in view the concentration dependent behaviour of 1,4-dioxane in water the volumes of transfer of amino acids from water to aqueous 1,4- dioxane solutions can further be rationalised. The Y \tr values observed from increasing positive glycine to L-phenylalanine (except very small negative value in the case of DL-a-alanine at 2.5% ) in the lower percentage region, i.e., up to == 5% of aqueous 1,4-dioxane may be attributed to contributions from ion-dipolar interaction whereas the increasing hydrophobic hydration may be attributed to the increase of non-polar side chain in the amino acids. The negative y0 2,tr volume of the order of - O.l1x 10-6 m 3 mor l, in the case of DL-a.-alanine at 2.5% of 1,4-dioxane, seems to be a little out of step. This difference is of the order of uncertainty in the measurement of cj)... However it may arise due to weak structure breaking effect of 1,4-dioxane in this range coupled with weak structure making propensity of DLa-alanine as compared to glycine which is a structure breaker. The negative Y \tr values ill the higher concentration range (l0, 20, 25%) of aqueous 1,4-. dioxane solutions may be resulting from the dominating effect of the hydrophobic-hydrophobic interactions which contribute negatively to Y \tr in the case of L-valine, L-Ieucine and L-phenylalanine due to the hydrophobic nature of 1,4-dioxane. The positive Y \tr value in the case of glycine and DL-a-. alanine may certainly be due to the dominating effect of charged end groups of amino acids. Sv values for L-valine in 5 and 10% aqueous 1,4- dioxane solution and for L-Ieucine in 5% aqueous 1,4- dioxane solution are negative which become positive at higher concentrations. These values indicate the 1,4-dioxane induced effect on the solute-solute interactions which have the bearing of both the increasing non-polar part of the amino acids and the dependence of the behaviour of 1,4-dioxane on the concentration in aqueous medium. This fwther strengthens the view that behaviour of 1,4-dioxane is concentration dependent in aqueous medium as change in slopes again occurs in the region of approximately 10% 1,4-dioxane (== 1m) solutions. Kozak et al. 3l have proposed a formalism based on the McMillan-Mayer theory of solutions that permit the formal separation of effect due to interactions between pairs of solute molecules and those due to interactions involving three or more solute molecules. This approach has further been discussed by Friedman & Krishnan 33 and Frank et al. 31 in order to include solute-cosolute interactions in the solvation sphere 2934 d th and used by vanous workers' to stu y e interactions of the amino acids and cosolutes in aqueous medium. According to this treatment, a thermodynamic transfer function at infinite dilution -0 e.g. V 2,tr can be expressed as V \tr = 2 V AB mb + 3 V ABB m B + 4 V ABBB m B +... where A stands for solute and B for cosolute. V AR, V ABB, V ABBB are respectively the pair, triplet, quartet interaction coefficients corresponding to a particular thermodynamic property and fib is the molality of cosolute (l,4-dioxane in present case). Y\tr data have been fitted into the above equation to obtain V AB, V ABB and V ABBB interaction coefficients which are given in Table 3. V AB is positive for glycine which. becomes negative for DL-a-alanine and decreases up. to L-Ieucine and becomes positive again for L phenylalanine. Unlike V AB, the triplet interaction coefficient V ABB is negative for glycine, and is positive for DL-a-alanine and again becomes negative and decreases continuously up to L-phenylalanine.

8 1018 INDIAN J CHEM, SEC. A, OCTOBER 2000 Table 3--Interaction coefficients for various amino acids at K in aqueous I A.. dioxane solutions obtained from Eq. (4) Compo V AD V ABB V ADBB Glycine DL-a-AJanine L-Valine L.Leucine L-Phenylalanine These trends indicate that various types of interactions are contributing to different extents. It may further be seen that the positive contribution of V AB to V \tr in case of L-phenylalanine in the lower concentration region of l,4-dioxane is very prominent which may be attributed to the hydrophobic hydration of the large non-polar group, i.e., phenyl group. In case of L leucine and L.. valine positive contribution is smaller in the lower concentration region which indicates that the disruption of hydration spheres around the charged centres is more pronounced than the hydrophobic hydration. This again supports the previous view that the effect of charged end groups is dominant in the case of glycine and DL.. a.. alanine. Negative values of higher order coefficients suggest that the negative contribution increases with increase in the l,4-dioxane concentration. References I Creighton T E, Protein folding (W H Freeman & Co, New York) Lapanze S, Physicochemical aspects of protein denaturation, (Wiley, New York) Lilley T H, Biochemical thermodynamics, edited by M N Jones, (Elsevier, Amsterdam) 1998, Ch. I. 4 Hedwig G R & Hoiland H, J chem Thermodyn, 23 (1991) 1029; 25 (1993) 349., 5 Hedwig G,R, Pure appj Chem, 66, 3 (1994) Hedwig G.R & Hoiland H, Biophys Chem, 49 (1994) Chalikian T V, Sarvazyan A P & Breslauer K J, J phys Chem, 97 (1993) Bhat R & Ahluwalia J C, J phys Chem, 99 (1985) Wadi R K & Ramasami P, J chem Soc Faraday Trans, 93 (1997) Banipal T S & Sehgal G, Thermochim Acta, 262 (1995) Banipal T S & Kapoor P, J Indian chem Soc, 76 (1999) Havenga E & Leaist D G, J chem Soc Faraday Trans, 94 (1998) Picker P, Trembley E & Jalicoeur C, J sol Chem, 3 (1974) Leduc P A, Fortier J C & Desnoyers J E, J phys Chem, 7 (1974) Millero F J, Chem Rev, 71(1971 ) Millero F J, Lo Surdo A & Shin C, J phys Chem, 82 (1978) Dipola G & Belleau B, Can J Chem, 56 (1978) Lark B S & Bala K, Indian J Chem, 22A (1983) Lark B S, Banipal T S & Bala K, Unpublished results. 20 Duke M M, Hakin A W, Mickay R M & Preuss K E, Can J Chem, 72 (1994) Kiyohara 0, Perron G & Desnoyers J E, Can J Chem, 53 (1975) Kay R L & Broadwater T L, Electrochem Acta, 16 (1971) Gancy A B, Water and aqueous solutions, edited by R A Home, (Wiley Interscience, New York) Hamann S D, J phys Chem, 67 (1963) Lo Surdo A, Shin C & Millero F J, J chem engg Data, 23 (1978) Tada Y, Ibuki K, Tsuchihashi N & Ueno M, J sol Chem, 26 (1997) Franks T, Quickenden M A J, Reid D S & Watson B, Trans Faraday Soc, 66 (1970) Shahidi F, Farrell P G & Edwards J T, J sol Chem, 5 (1976) Mishra A K & Ahluwalia J C, J chem Soc Faraday Trans I, 77 (1981) Gurney R W, Ionic process in solutions, (McGraw Hill, New York) Frank H S & Evan M W, J chem Phys, 13 (1945) Kozak J J, Knight W & Kauzmann W, J chem Phys 68 (1968) Friedman H L & Krishnan C V, Water - A comprehensive treatise,edited by F Franks, (Plenum, New York) Vol 3, Ch. I. 34 Lark B S & Bala K, Nat Acad Sci Letters, 12 (1989) 155.