Thermodynamics of Diglycine and Triglycine in Aqueous NaCl Solutions: Apparent Molar Volume, Isentropic Compressibility, and Refractive Index

$Page 1: Thermodynamics of Diglycine and Triglycine in Aqueous NaCl Solutions: Apparent Molar Volume, Isentropic Compressibility, and Refractive Index$
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Journal of Solution Chemistry [josc] PP1201-josl-486007 April 12, 2004 11:19 Style file version June 5th, 2002

Journal of Solution Chemistry, Vol. 33, No. 1, January 2004 (C© 2004)

Thermodynamics of Diglycine and Triglycine inAqueous NaCl Solutions: Apparent Molar Volume,Isentropic Compressibility, and Refractive Index

Ana Soto,1 Alberto Arce,1 and Mohammad K. Khoshkbarchi2

Received April 7, 2003; revised December 7, 2003

New experimental data for the density, speed of sound, and refractive index of aqueoussolutions of diglycine+ NaCl and triglycine+ NaCl have been reported. The apparentmolar volume and apparent molar isentropic compressibility of these peptides at variousNaCl concentrations have been calculated from the measured properties. The resultsshow that both peptides exhibit a positive volume transfer to solutions with higherNaCl concentrations and a negative apparent isentropic compressibility in the presenceof NaCl. These effects indicate that the apparent volumes of the peptide moleculesare larger in solutions with higher NaCl concentrations and that the water moleculesaround the peptide molecules are less compressible than the water molecules in the bulksolvent. These effects are attributed to the doubly charged nature of the peptides and theinteractions between the charged groups and hydrocarbon backbone of peptides withthe ions.

KEY WORDS: NaCl; diglycine; triglycine; molar volume; isentropic compressibility.

1. INTRODUCTION

Development and design of new separation and purification processes forbiomolecules require accurate knowledge of their thermodynamic properties insolution. Peptides are important biomolecules due to their wide range of applicationin drug production that is a result of their ability to act as hormones and their roleas signal transmitters in cell communication. Peptides are also among the buildingunits of complex biomolecules such as proteins. Therefore, a systematic studyof their thermodynamic properties can provide valuable information about their

1Department of Chemical Engineering, University of Santiago de Compostela, Santiago E-15706,Spain.

2Aspen Technology Inc., Suite 900, 125 9th Ave., Calgary, AB, Canada.

11

0095-9782/04/0100-0011/0C© 2004 Plenum Publishing Corporation

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12 Soto, Arce, and Khoshkbarchi

behavior in solution and purification, as well as insight into conformational stabilityand interactions of larger biomolecules in solution. Thermodynamic properties ofpeptides have been the subject of several studies.(1–6) In previous studies, we haveshown that the interactions between simple amino acids, such as glycine andDL-alanine, and ions could strongly influence their physicochemical propertiesin aqueous solutions.(7,8) We have undertaken a series of systematic theoreticaland experimental studies to investigate the effect of ions on the thermodynamicproperties of biomolecules.

In this study the apparent molar volumeVφ and isentropic compressibilityKφ

of diglycine and triglycine in aqueous NaCl solutions have been investigated. Newexperimental data at 25◦C for the density, speed of sound, and refractive index forthese systems are reported and compared.

2. MATERIALS AND METHODS

Sodium chloride with nominal purity of greater than 99.8 mass% was pur-chased from SIGMA (Madrid, Spain) and was oven-dried for 72 h prior to use.Diglycine and triglycine, with a purity of greater than 99.0 mass%, were alsopurchased from SIGMA (Madrid, Spain) and were used without further purifica-tion. Deionized water produced by a Milli-Q water deionizer was used in all ex-periments. The deionized water was ultrasonically degassed prior to use. The pHof the deionized water was 7.0 as measured with a Mettler Toledo MP225 pH meter.The high quality of the water used in the experiments and its neutral pH minimizedthe possibility of formation of charged peptide molecules.

All the solutions were prepared by weight on a Mettler AE 240 balanceprecise to within±0.0001 g. To prepare the solutions under investigation, aqueoussolutions of each salt at different concentrations were first prepared. A knownamount of each peptide was then added to the NaCl solution and stirred until ahomogeneous solution was obtained.

The densities of the mixtures were measured to within±0.00001 g-cm−3 us-ing an Anton Paar DMA-60/602 densimeter. The temperature was controlled usinga Heto Therm ultrathermostat and measured with a precision of±0.001 K using aCKT 100 Anton Paar precision thermometer. The speed of sound was measured towithin±1.0 m-s−1 using an Anton Paar DSA 48 densimeter and a sound analyzer.Both densimeters were calibrated with air and water. Refractive indices were mea-sured with an Atago RX-1000 refractometer to within±0.0001. All measurementswere repeated at least three times for each sample and were found to be repeatableto within the precision quoted for the apparatus.

3. RESULTS AND DISCUSSIONS

Tables I and II present the experimental results of the density, speed of soundand refractive index measurements at 25◦C for diglycine+ NaCl + water and

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Table I. Measured Values of the Density, Speed of Sound, and Refractive Indexand Calculated Values of Apparent Molar Volume and Isentropic Compressibility

in Diglycine+ NaCl+Water Systema

Diglycine Speed of Refractiveconcentration Density sound index 106Vφ 1015Kφ

No NaCl0.0000 997.04 1497 1.3325 — —0.3019 1013.32 1521 1.3399 77.007−368430.5886 1027.78 1542 1.3465 77.582−336130.9092 1043.01 1566 1.3534 78.050−319191.1718 1054.75 1584 1.3588 78.429−300091.5056 1068.85 1607 1.3652 78.852−28234

0.2m NaCl0.0000 1005.20 1509 1.3345 — —0.3005 1020.98 1533 1.3417 78.236−339860.5796 1034.75 1553 1.3480 78.665−305860.8808 1048.75 1575 1.3545 79.075−289031.1790 1061.83 1596 1.3605 79.424−273051.4702 1073.93 1615 1.3661 79.718−25590

0.4m NaCl0.0000 1013.16 1521 1.3364 — —0.2983 1028.44 1544 1.3434 79.305−297510.5848 1042.22 1565 1.3497 79.706−276600.8560 1054.58 1584 1.3555 79.993−259371.1767 1068.36 1606 1.3619 80.326−243131.4276 1078.56 1622 1.3667 80.572−22924

0.6m NaCl0.0000 1020.96 1534 1.3383 — —0.2946 1035.69 1556 1.3451 80.280−259710.5735 1048.80 1576 1.3512 80.636−243080.8629 1061.70 1596 1.3572 80.884−228351.1723 1074.70 1617 1.3633 81.155−215551.4286 1084.95 1633 1.3681 81.336−20293

0.8m NaCl0.0000 1028.63 1546 1.3401 — —0.2975 1043.14 1568 1.3470 81.200−233490.6244 1058.12 1591 1.3541 81.468−215660.9000 1070.02 1609 1.3596 81.689−199341.2053 1082.47 1629 1.3654 81.935−187531.5064 1094.08 1648 1.3710 82.151−17660

1.0m NaCl0.0000 1036.19 1557 1.3419 — —0.3032 1050.61 1579 1.3488 82.067−206430.6031 1064.05 1600 1.3551 82.268−193730.9025 1076.74 1620 1.3611 82.431−181041.1840 1087.95 1638 1.3665 82.659−169291.5357 1101.21 1660 1.3729 82.871−15768

aUnits of the properties: diglycine concentration (m) ; density (kg-m−3); speedof sound (m-s−1); Vφ (m3-mol−1); Kφ (m3-kPa−1-mol−1).

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Table II. Measured Values of the Density, Speed of Sound, and Refractive Indexand Calculated Values of Apparent Molar Volume and Isentropic Compressibility

in Triglycine+ NaCl+Water Systema

Triglycine Speed of Refractiveconcentration Density sound index 106Vφ 1015Kφ

No NaCl0.0000 997.04 1497 1.3325 — —0.0492 1000.83 1502 1.3343 111.816−454390.1462 1008.10 1512 1.3377 112.385−448720.2489 1015.59 1522 1.3411 112.664−427380.3538 1022.97 1531 1.3445 113.065−39320

0.2m NaCl0.0000 1005.20 1509 1.3345 — —0.0489 1008.87 1514 1.3361 113.500−416480.1509 1016.35 1524 1.3396 113.801−391880.2529 1023.57 1534 1.3431 114.216−379860.3419 1029.71 1542 1.3460 114.453−36059

0.4m NaCl0.0000 1013.16 1521 1.3364 — —0.0493 1016.76 1526 1.3381 115.166−372870.1411 1023.32 1535 1.3412 115.408−354190.2264 1029.28 1543 1.3440 115.511−338300.3447 1037.27 1554 1.3478 115.817−32300

0.6m NaCl0.0000 1020.96 1534 1.3383 — —0.0497 1024.51 1539 1.3399 116.356−335040.1467 1031.25 1548 1.3431 116.816−300000.2460 1038.00 1557 1.3463 116.882−285610.3428 1044.39 1565 1.3495 117.029−26532

0.8m NaCl0.0000 1028.63 1546 1.3401 — —0.0500 1032.12 1551 1.3418 117.537−301370.1485 1038.83 1560 1.3451 117.820−266530.2499 1045.55 1569 1.3484 117.974−230900.3638 1052.88 1578 1.3519 118.121−22611

1.0m NaCl0.0000 1036.19 1557 1.3419 — —0.0505 1039.64 1562 1.3436 118.540−269790.1510 1046.32 1571 1.3469 118.919−232900.2535 1052.95 1580 1.3502 119.060−219330.3561 1059.40 1588 1.3534 119.188−21153

aUnits of the properties: diglycine concentration (m) ; density (kg-m−3); speed ofsound (m-s−1); Vφ (m3-mol−1); Kφ (m3-kPa−1-mol−1).

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Thermodynamics of Diglycine and Triglycine in NaCl Solutions 15

triglycine + NaCl + water systems, respectively. The apparent molar volumesVφ and apparent molar isentropic compressibilitiesKφ of peptides in solutionswith different NaCl concentrations that are presented in the Tables I and II, werecalculated according to the following relations:

Vφ = M

ρ− ρ − ρ0

mPρρ0(1)

Kφ = MκS

ρ− κS0ρ − κSρ0

mPρρ0(2)

whereM denotes the molecular weight of peptide,mP is the molality of peptide,andρ andρ0 are the densities of the solution and the solvent, respectively. Therespective values ofκS andκS0 denote the isentropic compressibility of the solutionand of the solvent that can be related to the density of the solution and the speedof soundu according to the relation

κS = 1

u2ρ(3)

For systems containing NaCl, the mixture of water+ NaCl is considered as thesolvent. The measured experimental data are correlated using the following linearfunctions:

Vφ = V0φ + SVmP (4)

Kφ = K 0φ + SK mP (5)

whereV0φ and K 0

φ are the infinite dilution apparent molar volume and apparentmolar isentropic compressibility of peptide in aqueous NaCl solutions, respec-tively, and SV and SK are the slopes of the lines obtained from fitting Eqs. (4)and (6) to the experimental data. The values of the infinite dilution apparent molarvolume and isentropic compressibility of diglycine and triglycine are presentedin Table III. These properties are important because at infinite dilution the inter-actions between the peptide molecules are negligible and these properties solelyreflect the interactions between the peptide molecules and the mixed solvent.

Figures 1 to 4 depict the experimental results of the effect of the diglycineand triglycine concentrations on their apparent molar volumes and isentropic com-pressibilities at different NaCl molalities. The solid lines are the results of thecorrelation obtained from Eqs. (4) and (5) with the parameters listed in Table III.As can be seen from these figures,Vφ and Kφ increase with increasing peptideconcentration at a fixed NaCl concentration. It can also be seen that at a fixedpeptide concentration the measured properties are larger at higher NaCl concen-trations. This indicates the positive volume transfer to solutions with higher NaClconcentrations and its increasing trend with increasing NaCl concentration. These

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Table III. Calculated Values ofV0φ , K 0

φ , and the Slopes of Eqs. (4) and (5),andSV andSK obtained from the Fits of the Experimental Dataa

NaCl (m) 0.0 0.2 0.4 0.6 0.8 1.0

Diglycine+ NaCl+ water106 × V0

φ 76.627 77.907 79.019 80.062 80.097 81.865106 × SV 1.515 1.266 1.107 0.919 0.790 0.6561015× K 0

φ −38343 −35286 −31296 −27254 −24543 −21783106 × SK 6945.9 6816.1 5961.8 4918.5 4732.1 9336.8

Triglycine+ NaCl+ water106 × V0

φ 111.69 113.33 115.07 116.35 117.49 118.51106 × SV 3.953 3.340 2.128 2.129 1.817 2.0431015× K 0

φ −47136 −42356 −37927 −34131 −30808 −27543106 × SK 20265 18306 16906 22829 23089 22378

aUnits: Vφ (m3-mol−1); SV (m3-mol−2); K 0φ (m3-kPa−1-mol−1); SK (m3-

kPa−1-mol−2).

phenomena can be mainly attributed to the electrostatic interactions between ionsand the doubly charged (zwitterionic) peptide molecules. The zwitterionic natureof peptides is a result of the dissociation of their amino and carboxyl groups. Theinteractions between simple ions (sodium and chloride) and the charged groupsof peptides influence the hydration cosphere of peptides. The term hydration co-sphere refers to the water molecules in the vicinity of the peptide molecules thatdirectly contact the peptide surface. Due to the charged nature of the peptides,

Fig. 1. Effect of diglycine concentration on its apparent molar volumeat different NaCl molalities. Solid lines are obtained from Eq. (4) andsolid circles are the experimental data.

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Fig. 2. Effect of diglycine concentration on its apparent molar isentropiccompressibility at different NaCl molalities. Solid lines are obtained fromEq. (5) and solid circles are the experimental data.

the water molecules in their hydration cosphere exhibit different physicochemicalcharacteristics compared with bulk water. Interaction of the charged groups of thepeptides with the oppositely charged ions leads to an overlap of their hydrationcospheres and, hence, a positive contribution to the volume transfer of peptides

Fig. 3.Effect of triglycine concentration on its apparent molar volume atdifferent NaCl molalities. Solid lines are obtained from Eq. (4) and solidcircles are the experimental data.

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Fig. 4.Effect of triglycine concentration on its apparent molar isentropiccompressibility at different NaCl molalities. Solid lines are obtained fromEq. (5) and solid circles are the experimental data.

as shown by the experimental data reported in this work. This also diminishesthe structure-breaking tendency of the ions and reduces the mobility of the hydra-tion water, known as the electrostriction effect. On the other hand, the interactionbetween the sodium and chloride ions and the nonpolar (−CH2) groups of thepeptide molecule causes a decrease in the volume transfer of the peptides studied.The experimentally observed positive volume transfers of the peptides studied inthis work suggest that in the ternary peptide+NaCl+water solutions, the electro-static charge–charge interactions are the predominant force. Electrostriction alsoreduces the mobility of the hydration water and its contribution to the relaxationpart of the compressibility that has a decreasing effect on the total compressibilityand may explain the negative value of the compressibility of the peptides. It isalso interesting to note that, as shown in Figs. 1–4, the effect of peptide concen-tration on the values ofV0

φ andK 0φ decreases at higher NaCl concentrations. One

explanation is that at higher NaCl concentrations the charged groups of peptidesare surrounded by a large concentration of oppositely charged sodium and chlo-ride ions that reduces the magnitude of the electrostatic interactions between thepeptide molecules and the solvent.

The infinite dilution apparent molar volumeV0φ and infinite dilution appar-

ent molar isentropic compressibilityK 0φ presented in Table III also show similar

behavior and trends as at higher concentrations. Since at infinite dilution the non-polar interactions among the peptide molecules can be ignored, the similarity of thetrends obtained further indicates the importance and domination of the electrostaticinteractions between the charged groups of the peptides and the counter ions.

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Fig. 5. Effect of the number of gly units in the structure of peptideson their infinite dilution apparent molar volume at two different NaClmolalities.

Figure 5 and 6 depict the infinite dilution apparent molar volume and isen-tropic compressibility of several glycine peptides as a function of their gly-repeating units at different NaCl concentrations. The experimental data for glycinewere obtained from our previous study(7) and the experimental data for tetraglycine

Fig. 6. Effect of the number of gly units in the structure of peptides ontheir infinite dilution apparent molar isentropic compressibility at threedifferent NaCl molalities.

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and pentaglycine in water were obtained from H¨ackelet al.(5) The results shownin these figures indicate that at constant NaCl concentration both the apparentmolar volume and isentropic compressibility of the peptides vary linearly with thenumber of gly groups in their structure. This linear relationship was observed atall NaCl concentrations studied. These results suggest that the gly groups in thepeptides studied are hydrated independently.

The experimental refractive indicies, presented in Tables I and II, show anincreasing trend with increasing concentrations of NaCl and the peptides. Thisbehavior is consistent with the results shown for the effect of peptide and NaClconcentrations on the apparent molar volume and the isentropic compressibility, in-dicating that the refractive index is directly related to the interactions in the solution.

4. CONCLUSIONS

Experimental data at 25◦C of the density, speed of sound, and refractive in-dex of diglycine and triglycine in aqueous NaCl have been reported. The values ofthe apparent molar volume and isentropic compressibility of these peptides werecalculated from these experimental results. The results indicated that the inter-actions between the peptides and NaCl strongly affect their behavior in aqueoussolutions. Both peptides exhibit a positive volume transfer to solutions with higherNaCl concentrations. Moreover, the apparent volumes of both peptides increasewith increasing NaCl concentration. Similar to simple electrolytes, these peptidesexhibit a negative isentropic compressibility. The negative values ofKφ implythat the water molecules around both diglycine and triglycine molecules are lesscompressible than the water molecules in the bulk solution. These effects wereattributed to the zwitterionic character of peptides and their interactions with otherions. The analysis of the results proved that the electrostatic forces between thecharged groups of both peptides and the counter ions are the dominant interactionsin solution. The linear dependence of the apparent molar volume of peptides onthe number of gly units in their structure suggests that the gly groups are hydratedindependently.

ACKNOWLEDGMENT

This work was partly financed by the Xunta de Galicia (Spain) under ProjectPGIDTOOPXI20902PR.

REFERENCES

1. R. Bhat and J. C. Ahluwalia,J. Phys. Chem.89, 1099 (1985).2. O. V. Kulikov, A. Zielenkiewicz, W. Zielenkiewicz, V. G. Badelin, and G. A. Krestov,J. Solution

Chem.22, 59 (1993).

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3. T. V. Chalikian, A. P. Sarvazyan, and K. J. Breslauer,Biophys. Chem.51, 89 (1994).4. T. V. Chalikian, V. S. Gindikin, and K. J. Breslauer,Biophys. Chem.75, 57 (1998).5. M. Hackel, G. R. Hedwig, and H-J. Hinz,Biophys. Chem.73, 163 (1998).6. M. P. Breil, J. M. Mollerup, E. S. J. Rudolph, M. Ottens, and L. A. M. van der Wielen,Fluid Phase

Equilib. 191, 127 (2001).7. A. Soto, A. Arce, and M. K. Khoshkbarchi,Biophys. Chem.74, 165 (1998).8. H. Rodriguez, A. Soto, A. Arce, and M. K. Khoshkbarchi,J. Solution Chem.32, 53 (2003).

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Thermodynamics of Diglycine and Triglycine in Aqueous NaCl Solutions: Apparent Molar Volume, Isentropic Compressibility, and Refractive Index