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J. Phys. Chem. B 2006, 110, 1020-1025
r,r-Trehalose-Water Solutions. VIII. Study of the Diffusive Dynamics of Water by High-Resolution Quasi Elastic Neutron Scattering Salvatore Magazu` ,*,† Federica Migliardo,† and Mark T. F. Telling‡ Dipartimento di Fisica, UniVersita` di Messina, P.O. Box 55, I-98166 Messina, Italy, and ISIS Facility, Rutherford Appleton Laboratory, Chilton, Didcot, UK ReceiVed: July 4, 2005; In Final Form: October 26, 2005
The present paper shows high-resolution quasi-elastic neutron scattering (QENS) findings on homologues disaccharides (i.e. trehalose, maltose, and sucrose)-water mixtures as a function of temperature. The QENS measurements were performed on both partially deuterated disaccharides in D2O and on hydrogenated disaccharides in H2O to separate the solute dynamics from that of the solvent. The results highlight a noticeable disaccharide kosmotrope character, with results more marked for trehalose. Such evidence accounts for its higher bioprotective effectiveness.
I. Introduction An open question in investigating biological systems today is the role played by the solvent in the physical-chemical mechanisms that determine the conformational and dynamical properties of biostructures.1-4 Changes in the structure and internal dynamics of proteins as a function of solvent conditions at physiological temperatures have been found by using several experimental techniques,5-7 demonstrating that an increase of protein conformational flexibility and, hence, of the activity, is related to hydration and that the dynamic behavior of protein solutions follows that of the solvent.8 In particular, the molecules surrounding the protein surface make an environment that may act as either plasticizer or stabilizer by respectively allowing or preventing protein to jump between the conformational substates.9 On the other hand, when the hydration degree increases, not only the protein molecule is able to globally diffuse, but also its internal dynamics appears to be more and more activated.2 A decisively important factor in determining the solute properties is played by the presence of “kosmotrope” and “chaotrope” cosolutes. The terms “kosmotrope” and “chaotrope” originally denoted substances that stabilized, or destabilized, respectively, proteins and membranes.10,11 Later, they referred to the behavior of a transport property such as viscosity. Kosmotrope cosolutes are “order makers”: (i) they impose their own order to the tetrahedral hydrogen-bonded network of water, (ii) they stabilize the biostructures (proteins, membranes), and (iii) they create stronger cosolute-water molecular interactions than among water-water molecules.12 Chaotrope cosolutes are “disorder makers”: (i) they destabilize the biostructures and (ii) create cosolute-water molecular interactions weaker than water-water molecular ones, so avoiding interference with the tetrahedral water’s hydrogen-bonded network.10 Typical kosmotropes are small ions or multiply charged ions, whereas large singly charged ions are classified as chaotropes. The present work is addressed to clarifying the role played by trehalose, maltose, and sucrose in determining the dynamics * Corresponding author. E-mail:
[email protected]. Telephone: +39 0906765025. Fax: +39 090395004. † Dipartimento di Fisica, Universita ` di Messina. ‡ ISIS Facility, Rutherford Appleton Laboratory.
switching off of the solvent, which is a fundamental point for clarifying the bioprotective mechanisms. Disaccharides are, in fact, cryptobiotic substances, i.e., they allow many organisms to enter into a state of “suspended life” (cryptobiosis),13 which includes osmobiosis,13 anoxybiosis,14 anydrobiosis,15,16 and cryobiosis,17,18 under stress environmental conditions, such as lack of oxygen and water, freezing, excessive levels of salinity and pressure, and very high and very low temperatures. The metabolic functions are reactivated at normal levels when the environmental parameters return to favorable conditions. The understanding of the bioprotective mechanisms of disaccharides has evidently relevant biotechnological consequences, allowing optimizing of innovative protocols for stabilization and conservation processes.19,20 Several bioprotection hypotheses have been formulated21-23 to explain the disaccharide bioprotective effectiveness. Green and Angell21 have individuated in the higher value of the glass transition temperature of trehalose the reason it better protects biostructures. Because other similar systems, such as, for exampl,e dextran,24 a linear polysaccharide, present even a higher glass transition temperature value without showing bioprotective functions, it is evident that other factors play a key role in the protection mechanisms. Crowe and co-workers have interpreted the role of trehalose as a “substitute of water”,22 which would be assent in the interaction between the disaccharide and the biostructures. On the other hand, by considering the disaccharide-water interaction, as also stated by Grigera and co-workers,23 no changes in the structural and dynamical properties of water would occur. Previous experimental results obtained by the authors25,26 showed that the structural properties of water are deeply modified by disaccharides and, in particular by trehalose, which, due to strong interactions with water molecules, impose a new order to the tetrahedral network of pure water. The present quasi-elastic neutron scattering study clearly shows that, contrarily to the simulation findings obtained by Grigera,23 also the diffusive dynamics of water is significantly affected by the presence of disaccharides. More specifically, all the investigated disaccharides, and trehalose with a greater extent, slow the dynamics of water, thus showing a high
10.1021/jp0536450 CCC: $33.50 © 2006 American Chemical Society Published on Web 12/22/2005
R,R-Trehalose-Water Solutions “switching off” capability. This circumstance also implies a higher ability to hold volatile substances and results to be more pronounced for trehalose than for the other disaccharides, coherently with its superior bioprotective effectiveness. II. Experimental Section Ultrapure powdered trehalose, maltose, and sucrose, D2O and H2O, purchased from Aldrich-Chemie, were used for the experiment. Measurements were performed in a temperature range of 283-320 K on hydrogenated trehalose, maltose, and sucrose (C12H22O11) in H2O and on partially deuterated trehalose, maltose, and sucrose (C12H14D8O11) in D2O at weight fraction values corresponding to 19 water (H2O and D2O) molecules for each disaccharide molecule. All of the three disaccharides possess hydrogen atoms belonging to the OH groups, which exchange easily with the deuterium atoms of heavy water. To focus the attention on the disaccharide dynamics in D2O, the exchangeable atoms were substituted with deuterium. To obtain partially deuterated samples, the disaccharides were first dissolved in pure D2O at a concentration of ∼40 wt % to exchange the eight labile hydrogen atoms of the disaccharides, and the solutions were, subsequently, lyophilized. The procedure was repeated in order to allow the exchange of all the exchangeable hydrogens. The solution samples were obtained by dissolution of the D2O-exchanged lyophilized disaccharides in pure D2O. In the deuterated solutions (at the investigated concentration), the coherent contribution to the total scattering cross section is ∼5%. In the case of QENS in protonated samples, the attention
J. Phys. Chem. B, Vol. 110, No. 2, 2006 1021 is focused on the incoherent scattering arising from the selfcorrelation function, which involves the motions of protons, the ratio between the incoherent cross-section σi and the scattering cross-section σs being σi/σs ) 0.94. The quasi-elastic neutron scattering experiment was carried out by using the IRIS high-resolution spectrometer at ISIS, the world’s leading pulsed neutron and muon source located at the Rutherford Appleton Laboratory (RAL, UK). IRIS is a highresolution quasi/in-elastic neutron scattering spectrometer with high-resolution, long-wavelength diffraction capabilities. It is an inverted geometry spectrometer such that neutrons scattered by the sample are energy analyzed by means of Bragg scattering from a large-area crystal-analyzer array. The high resolution combined with the most powerful pulsed neutron source of the world make IRIS the best instrument to study the diffusive dynamics.27 We used the high-resolution configuration of IRIS (graphite 002 and mica 006 analyzer reflections) to measure sets of QENS spectra covering a Q,ω domain extending from pω ) -0.3 to 0.6 meV (energy transfer) and Q ) 0.3-1.8 Å-1 (momentum transfer). The detectors used give a mean energy resolution of Γ ) 8 µeV of half width at half-maximum (HWHM), as determined by reference to a standard vanadium plate. The sample was contained in an aluminum cell that allows obtaining liquid samples in the form of slabs of 45 × 35 mm2 and different thickness from 0.2 mm (for hydrogenated samples) to 1 mm (for deuterated samples). By using a sample changer accepting a vertical stack of three precisely aligned cuvettes, it was possible to measure highly reliable spectra and to keep the signal
Figure 1. Best fit of deuterated and hydrogenated aqueous solutions of trehalose for two temperature values. See text for details.
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Figure 2. Best fit of deuterated and hydrogenated aqueous solutions of trehalose, maltose, and sucrose.
from the empty container below 1% of that from samples. The raw spectra were corrected and normalized by using the standard GENIE procedures and the IRIS data analysis package.28
Strans (Q,ω) ) s
III. Results and Discussion Quasi-elastic neutron scattering (QENS) is a particularly powerful tool for investigating the diffusive properties of hydrogenated systems. When the separation of translational, rotational, and vibrational contributions is allowed (decoupling approximation), the total quasi-elastic scattering law can be written as:29,30 2 2 Squasi (Q,ω) ≈ (Strans X Srot s s s ) exp(- Q 〈u 〉)
where
(1)
DtQ2 1 π (D Q2)2 + ω2 t
2 Srot s (Q,ω) ) j0 (QF)δ(ω) +
1
(2) ∞
(2l + 1)jl2 ∑ π l)1 Drl(l + 1) (QF)
[Drl(l + 1)]2 + ω2
(3)
and exp(-Q2〈u2〉), with 〈u2〉 mean square vibration amplitude,
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is the Debye-Waller factor. In eqs 1, 2, and 3, the symbols have the usual meaning. In the analyzed hydrogenated systems, we can distinguish among different proton populations that contribute to the QENS scattering law: (i) protons of the disaccharide molecule and of its hydration shell that follow the same diffusion law; (ii) protons of the water molecules of higher hydration shells, whose diffusion is influenced by the presence of the disaccharide; (iii) protons by bulk water that, in our case, are not present, as shown by ultrasonic, viscosity, and light scattering measurements.31-33 More specifically, ultrasonic velocity measurements clearly indicate that disaccharides show that the disaccharide-water molecule interaction strength is higher with respect to that between the water molecules, and that, in respect to the other disaccharides, the trehalose-water system is characterized, in all the investigated concentration range, by both the highest value of the solute-solvent interaction strength and of the hydration number. For example, at T ) 25 °C, it is nH ) 15.2 for trehalose, nH ) 14.7 for maltose, and nH ) 14.1 for sucrose, and such a value increases by lowering temperature.31,32 The spectra have been analyzed by using the fitting function:
{
[
Γ1(Q) 1 Sinc(Q,ω) ) A(Q) fDisaccharide F(Q) + π Γ 2(Q) + ω2 1 Γ2(Q) Γ3(Q) 1 1 + f (1 - F(Q)) hydr π Γ 2(Q) + ω2 π Γ 2(Q) + ω2 2
]
3
}
(4)
where the first two terms refer to the translational and rotational contribution of hydrated disaccharide (fDisaccharide and fhydr represent fraction factors of the total scattering from disaccharide and its strongly bonded water molecules, respectively), and the third one refers to hydration water (fDisaccharide + fhydr ) 1). Therefore, the dynamical information of the diffusive dynamics of disaccharide can be obtained by the analysis of disaccharide + D2O spectra analysis for which fhydr results are negligible. For trehalose aqueous solutions, the fhydr parameters keep constant, with Q at the values of 0.032, 0.108, 0.223, and 0.328 for T ) 283, 295, 308, and 320 K, respectively, corresponding to 18.0, 15.7, 12.2, and 9.0 water molecules bound to the trehalose molecules, respectively. These hydration number values are in excellent agreement with those obtained by ultrasonic, hypersonic, viscosity, and Raman scattering techniques.31-33 As far as the hydration number values of the three disaccharides is concerned, at T ) 320 K, the fhydr parameters result in 0.328, 0.348, and 0.378 for trehalose, maltose, and sucrose solutions, respectively, corresponding to 9.0, 8.4, and 7.5 water molecules bound to the disaccharide molecules, respectively. In Figure 1, we report the best fit of deuterated and hydrogenated aqueous solutions of trehalose for three temperature values according to the scattering law (eq 4), whereas in Figure 2, the best fit of deuterated and hydrogenated aqueous solutions of trehalose, maltose, and sucrose according to the scattering law (eq 4) is shown. The total fit (black line) of deuterated samples is composed of the two contributions: the translational one (grey line) and the rotational one (light gray line). The total fit (black line) of protonated samples is composed of the two contributions: the disaccharide one (grey line), resulting by the fit of disaccharide-D2O spectra, and the water one (light gray line), when only the translational part is present. The present model allows performing the best fit procedures, in excellent agreement with the experimental data. The line width Γ1 of the translational contribution of disaccharides as a function of Q2 follows a typical random jump
Figure 3. (A) Line width of the translational contribution as a function of Q2 for trehalose aqueous solutions at four temperature values. (B) Line width of the translational contribution as a function of Q2 for trehalose, maltose, and sucrose at T ) 320 K. The solid lines are fits obtained following the RJD model (eq 5).
diffusion (RJD) model,34 as shown in Figure 3 for trehalose aqueous solutions for different temperature values, and for trehalose, maltose, and sucrose aqueous solutions at T ) 320 K, respectively:
Γ1(Q) ) DsQ2/(1 + DsQ2τ)
(5)
where Ds is the self-diffusion coefficient of the molecule, and τ is the residence time. The jump diffusion model assumes that the diffusive particle remains in a given site for a time τ, where it vibrates around a center of equilibrium. After τ, it moves rapidly to a new position separated by the vector l from its original site. The RJD model furnishes the diffusion coefficient Ds value from the extrapolation to Q f 0 and the residence time τ from the inverse of the asymptotic value at Qf ∞. For trehalose as a function of temperature, the RJD model furnishes for the diffusion coefficient Ds and the residence time τ the values of Ds ) 2.83 × 10-7 cm2/s and τ ) 24.7 ps, Ds ) 3.82 × 10-7 cm2/s and τ ) 20.6 ps, Ds ) 5.35 × 10-7 cm2/s and τ ) 19.1 ps, and Ds ) 8.50 × 10-7 cm2/s and τ ) 18.3 ps for T ) 283, 295, 308, and 320 K, respectively. From the relation 〈l2〉 ) 6Dτ, we obtain the values 〈l2〉1/2 ) 0.64, 0.68, 0.78, and 0.99 Å for T ) 283, 295, 308, and 320 K, respectively. The RJD model furnishes for the diffusion coefficient Ds and the residence time τ the values of Ds ) 8.50 × 10-7 cm2/s and τ ) 18.3 ps for trehalose, Ds ) 1.00 × 10-6 cm2/s and τ ) 14.3 ps for maltose, and Ds ) 1.23 × 10-6 cm2/s and τ ) 13.8
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Figure 5. Diffusion coefficient and residence time for water in trehalose solutions compared with pure water as a function of temperature.
Figure 4. Line width of the translational contribution as a function of Q2 for (A) water in trehalose aqueous solutions at four temperature values, (B) water in trehalose, maltose, and sucrose aqueous solutions at T ) 320 K, and (C) pure water for four temperature values. The solid lines are fits obtained following the RJD model (eq 5).
ps for sucrose, respectively, at T ) 320 K. Furthermore, from the relation 〈l2〉 ) 6Dτ, we obtain the value 〈l2〉1/2 ) 1.00 Å for the three disaccharides. The line width Γ2 of the rotational contribution of disaccharides has also been evaluated, resulting nearly constant as a function of Q. For trehalose aqueous solutions, we obtain the values of Γ2 ∼ 30 µeV, Γ2 ∼ 41 µeV, Γ2 ∼ 53 µeV, and Γ2 ∼ 59 µeV for T ) 283, 295, 308, and 320 K, respectively, whereas
we obtain Γ2 ∼ 59 µeV for trehalose, Γ2 ∼ 75 µeV for maltose, and Γ2 ∼ 89 µeV for sucrose, respectively. Such dynamics would occur on a time scale of 21.9, 16.1, 12.4, and 11.2 ps for trehalose for T ) 283, 295, 308, and 320 K, respectively, whereas it occurs on a time scale of 11.2 ps for trehalose, 8.8 ps for maltose, and 7.4 ps for sucrose, respectively. The line width Γ3 of the translational contribution of water is reported in Figure 4 as a function of Q2 together with the best fit according to the RJD model34 for water in trehalose solutions as a function of temperature, for water in trehalose, maltose, and sucrose aqueous solutions at T ) 320 K, and for pure water for four temperature values. In Figure 5, the values of diffusion coefficient and residence time of water are shown for trehalose solutions and for pure water. From the relation 〈l2〉 ) 6Dτ, we obtain for pure water the mean jump length 〈l〉 values of 1.35 Å for all of the investigated temperature values. The whole water dynamics in trehalose solutions for T ) 283, 295, 308, and 320 K resembles that of water at ∼256, ∼261, ∼263, and ∼268 K, respectively, indicating that the water has a diffusive behavior strongly triggered by the trehalose molecules and suffers from a noticeable frozen effect. For the diffusion coefficient of water in the three disaccharide aqueous solutions, we obtained at T ) 320 K the value of Dw ) 8.31 × 10-6 cm2/s for trehalose solution, Dw ) 8.46 × 10-6 cm2/s for maltose solution, and Dw ) 8.60 × 10-6 cm2/s for sucrose solution, respectively, with the values of residence times of τ ) 3.7, 3.4, and 3.0 ps for trehalose, maltose, and sucrose solutions, respectively, obtaining for the mean jump length 〈l〉 the value 〈l2〉1/2 ) 1.36, 1.31, and 1.24 Å for trehalose, maltose, and sucrose solutions, respectively. It is interesting to compare the diffusion coefficient values obtained for water in the
R,R-Trehalose-Water Solutions presence of disaccharides with that of pure water at the same temperature, which is Dw ) 3.94 × 10-5 cm2/s. For the investigated systems, the water dynamics resembles that of water at ∼268 K in the case of trehalose solution, at ∼271 K in the case of maltose solution, and at ∼277 K in the case of sucrose solution. Analogously to the trehalose aqueous solutions, all the disaccharides show a slowing down effect of the water dynamics, which is stronger for trehalose than for the other disaccharides. IV. Conclusions The experimental findings reported in the present paper furnish a clear response about the dynamics of disaccharidewater solutions. The diffusive dynamics of both disaccharide and water in the solutions has been characterized by highresolution quasi-elastic neutron scattering. It is clearly pointed out that the disaccharides strongly affect the water dynamics, with a higher effect in the case of trehalose-water solutions. Disaccharides, and trehalose to a greater extent, besides imposing an order on the tetrahedral hydrogen bond network of water, significantly slow the dynamics of water. The higher slowing down effect of the diffusive dynamics observed for trehalose is evidently linked to its extraordinary capability to “switch off” the metabolic functions. Therefore, such a finding, implying for trehalose a higher kosmotrope character, can account for its bioprotective effectiveness. Acknowledgment. F. Migliardo gratefully acknowledges “L’OREÄ AL Italia Per le Donne e la Scienza”. The authors gratefully acknowledge the ISIS facility (Chilton, UK) and Dr. Mark Adam for the dedicated runs at the IRIS spectrometer. References and Notes (1) Frauenfelder, H.; McMahon, B. Proc. Natl. Acad. Sci. U.S.A. 1998, 9995, 4795. (2) Paciaroni, A.; Cinelli, S.; Onori, G. Biophys. J. 2002, 83, 1157. (3) Vitkup, D.; Dagmar, R.; Petsko, G. A.; Karplus, M. Nat. Struct. Biol. 2000, 7, 34. (4) Zaccai, G. Philos. Trans. R. Soc. London, Ser. B 2004, 359, 1269.
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