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Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, United Kingdom
Correspondence: Address reprint requests to Dr. Steve G. Ring, Institute of Food Research, Norwich Research Park, Colney Lane, Norwich, NR4 7UA, UK. Tel.: +44-(0)1603-255031; Fax: +44-(0)1603-507723; E-mail: steve.ring{at}bbsrc.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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20 MPa at a mass fraction of 0.62–1.1 GPa at 0.86. The solid was viscoelastic and exhibited stress relaxation with relaxation times increasing from 33 to 610 s over the same concentration range. The concentration dependence of the osmotic pressure was measured, at intermediate concentrations, using an osmotic stress technique and could be described using a hard sphere model, indicating that the intermolecular interactions were predominantly repulsive. In summary, a major structural relaxation results from the collective motion of the globules at the supra-globule length scale and, at 20°C, this is arrested at water contents of 40% w/w. This appears to be analogous to the glass transition in colloidal hard spheres. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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; Debenedetti, 1996), and a change from liquid-like to solid-like behavior. Although this change in behavior can occur over a narrow temperature range, it is often considered to be associated with a glass transition temperature, Tg, where the relaxation time for structural rearrangement of the liquid generally exceeds 100 s. This transition temperature is commonly associated with a sharp change in heat capacity and calorimetry is commonly used in the experimental determination of Tg. The glass transition has a large impact on the properties of low water content biopolymeric materials and is relevant to their usefulness in pharmaceuticals (Franks, 1999), foods (Slade and Levine, 1991), and the preservation of biological materials in the dry state (Crowe et al., 1998). It has been proposed that the reduction in molecular mobility, generally associated with vitrification, has a preservative effect through the slowing of the encounter of reactive species involved in deteriorative reactions (Parker et al., 2002).
Water has a strong plasticizing effect on the glass transition of flexible biopolymers. For example, increasing the water content of a cereal protein, a high molecular weight glutenin, from 3 to 12% w/w, depresses the calorimetric glass transition from 100°C to 25°C (Noel et al., 1995). Both flexible polysaccharides such as starch (Zeleznak and Hoseney, 1987; Orford et al., 1989), and flexible proteins such as gelatin (Yannas, 1972) and elastin (Lillie and Gosline, 1990), show broadly comparable behavior. For globular proteins the situation is less clear. For lysozyme, a marked change in heat capacity from 1.25 Jg-1K-1 to 1.55 Jg-1K-1 is observed on increasing water content from 7% to 20% w/w at room temperature (Rupley and Careri, 1991). As the heat capacity change is associated with the onset of enzyme activity and therefore sufficient mobility for enzyme-catalyzed reaction, this transition can be likened to a glass transition. For a concentrated globular protein-water mixture the observed features in heat capacity as a function of temperature are relatively weak, span a comparatively wide temperature range, and are difficult to attribute to a calorimetric glass transition (Sartor et al., 1994). It is generally concluded that associated with the complex tertiary structure of proteins is a correspondingly complex dynamics (McCammon and Harvey, 1987), which cannot be adequately characterized through a single relaxation process. On the denaturation of globular proteins, with the consequent loss of tertiary structure, the calorimetric features associated with the glass transition become more evident and directly comparable with the behavior of flexible proteins (Sochava and Smirnova, 1993; Belopolskaya et al., 2000; Tsereteli et al., 2000).
In addition to consideration of the chain dynamics of the individual protein globule, it is also important to consider the dynamics of a collection of globules, which will have an impact on mechanical and transport behavior. In principle these systems should be analogous to concentrated colloidal suspensions (Trappe et al., 2001; Mason et al., 1997; Mason and Weitz, 1995; Segre et al., 2001) and granular powders that form glassy, "jammed" structures at high volume fractions (Liu and Nagel, 1998; Jaeger and Nagel, 1997).
The viscosity of colloidal suspensions progressively increases with the increasing volume fraction, 
, of particles (Phan et al., 1996; Krieger, 1972). At high volume fractions there is a sufficient slowing of particle dynamics that liquid-like configurations cannot be explored over practical timescales (Mason et al., 1997; Mason and Weitz, 1995). For small applied stresses the material has the solid-like characteristics of a glass with the particles forming jammed structures that are stress bearing. For a random packing of noninteracting monodisperse hard spheres, these structures may form at volume fractions in the vicinity of 0.6 with a random close packing limit, c, of 
0.644 (Torquato et al., 2000; Rintoul and Torquato, 1998). Although the above phenomena result from repulsive excluded volume interactions when the interaction between particles becomes increasingly attractive (Trappe et al., 2001), three dimensional particle networks, with solid-like characteristics (colloidal gels), will form at lower particle volume fractions (Segre et al., 2001). Recent research has emphasized the similarities between jammed structures that can form with increasing volume fraction of particles, and those, more open structures that form as a result of an increasing attractive interaction between particles (Trappe et al., 2001).
In this article we examine the rheological behavior of concentrated aqueous solutions of the globular protein bovine serum albumin (BSA) to test the proposal that for sufficiently concentrated solutions, glass-like behavior occurs as a consequence of the arrest of particle dynamics. Throughout the composition range examined, the interparticle interaction was probed through the determination of the osmotic pressure as a function of composition.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Preparation of protein solutions and hydrated solid bars
Protein solutions (<30% w/w) were prepared by the slow addition of BSA powder to pH 5.4 acetate buffer containing 100 mM sodium chloride, taking care not to foam the solution during stirring. More concentrated solutions (>30% w/w) were prepared by dialyzing these solutions against buffered PEG solutions containing 100 mM sodium chloride. Hydrated solid bars of BSA were prepared through concentrating BSA, firstly by dialysis against concentrated PEG solutions, as above, and then by equilibration over saturated salt solutions of known water activity. During this process the samples were pressed into 30 mm x 6 mm x 2 mm bars. Each sample was then carefully removed from the dialysis bag and immediately immersed in a silicone oil bath to prevent further drying. The extent of aggregation of BSA was checked by size exclusion chromatography using a 60-cm TSK-gel G2000 SW column (Tosoh-Biosep, Montgomeryville, PA) eluted with 150 mM sodium chloride at a flow rate of 1 ml x min-1. For the BSA solutions the majority of the protein eluted in the main peak accounting for 97% of the material. A minor, faster eluting peak, 3% w/w, indicated a limited aggregation. The extent of aggregation had increased to 7% w/w, after concentration to 60% w/w by osmotic stress and storage at room temperature for 24 h.
Photon correlation spectroscopy
The apparatus employed was an ALV/SP-86 spectrogoniometer (ALV, Langen, Germany) equipped with a Coherent Radiation Innova 100-10 vis Argon Ion laser operating at 0.5 W and wavelength of 514 nm. BSA samples with concentrations <36% w/w were placed into the quartz cuvette and maintained at 25°C. The scattered light intensity was monitored using an ALV/PM-15 ODSIII detection system at a fixed scattering angle of 90°. After amplification and discrimination, signals were directed to an ALV/5000E digital multiple-
correlator and time-intensity correlation functions recorded, typically for 600 s duration. Size distribution functions were computed using the appropriate Windows-based ALV software, which incorporated regularized inverse Laplace transform and ALV-CONTIN packages. Additional analysis was undertaken using Origin V6 (Microcal, Northhampton, MA) proprietary software.
Osmotic stress technique
A buffered 25% w/w BSA solution (2 ml) was dialyzed against buffered PEG solutions at 2°C for 24 h. After further dialysis at 20°C for 24 h the dialysis tube was removed from the PEG solution and the BSA concentration determined in the usual way by spectrophotometry. The osmotic pressure, 
, of a w% w/w PEG solution was calculated from the relationship, log = a + b x (w)c, using published values of the constants a, b, and c (Parsegian et al., 1986). The suitability of this reference data was confirmed by experiment (Ryden et al., 2000).
Rheometry
A Bohlin CS10 controlled stress rheometer, with a double gap concentric cylinder (capacity 35 ml), was used to measure the dependence of BSA solution viscosity on shear rate, in the range 0.001–100 s-1, for 0–40% w/w BSA solutions at 20°C.
The viscoelasticity of concentrated (40–50% w/w) BSA solutions at 20°C was measured using an Instron 3250 mechanical spectrometer (Canton, MA). Oscillatory measurements were undertaken with a parallel plate geometry (15 mm diameter platens, 0.3 mm gap) in the frequency range 0.01 Hz–10 Hz. The sample was transferred directly from the dialysis bag onto the lower platen and upper platen lowered until the required gap was obtained. While manually adjusting the gap the normal force was constantly monitored and not allowed to exceed 0.2 N and indeed was set to within 0.05 N of zero before testing commenced. Surplus material was quickly removed, and the exposed sample edge coated in silicone oil to prevent sample drying. The viscoelastic behavior was measured as a function of strain at a fixed oscillatory frequency of 1 Hz to determine the region of linearity. The frequency-dependent response was then examined at a strain chosen to lie within the linear region. To check for time-dependent effects such as ageing or of a nonlinear viscoelastic nature, small-strain measurements were periodically repeated at 1 Hz.
The Young's modulus and stress relaxation behavior of the solid-like BSA-water systems (>60% w/w BSA) were measured using a tensile testing machine (Instron 1122) in a three-point bend test with the bar supports 26 mm apart. A load was applied to the bar at the point midway between the supports while maintaining the temperature constant at 20°C. The maximum deflection of the bar at the midpoint of flexure was achieved quickly (within seconds) at a constant but small strain (0.02) that fell within the linear stress-strain region. Stress relaxation data were recorded over periods of time commensurate with the viscoelastic properties of the bars.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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2.66. The conformation of BSA shows a pH dependence, with the native form being found between pH 4 and 8. In this pH range the equivalent hydrodynamic radius is 3.7 nm. The molecular volume of human serum albumin, calculated from the atomic coordinates of the x-ray structure is 88.25 nm3 (Carter and Ho, 1994; He and Carter, 1992).
Photon correlation spectroscopy was used to characterize both dilute and moderately concentrated solutions of BSA through the determination of a translational diffusion coefficient Dt. For a particle in solution subject to Brownian motion, the translational diffusion coefficient is related to the measured intensity correlation function g(2) by the expression
 | (1) |
 | (2) |
the scattering angle, and 
the wavelength of light (Brown, 1993). For a 1% w/w solution of BSA in 100 mM sodium chloride at pH 5.4 and 20°C, the measured diffusion coefficient was 5.6 x 10-11 m2s-1. With increasing concentration of BSA the measured translational diffusion coefficient decreased from 5.6 x 10-11 m2s-1 at 10% w/w, to 3.4 x 10-11 m2s-1 at 36% w/w (Table 1). For the dilute solutions, the observed intensity correlation functions could be approximated to a single diffusive process. With increasing concentration of BSA, more complex behavior was observed, as indicated by the increasing curvature in plots of -ln g(2) vs. . The measurements of the concentration dependence of Dt are consistent with published data (Phillies et al., 1976), and support the view that there is no simple relationship between Dt, measured by photon correlation spectroscopy, and shear viscosity (Tyrrell and Harris, 1984). The photon correlation data on dilute solutions, and the chromatographic data on both dilute solutions and a concentrated solution (60% w/w) that was subsequently diluted indicate that the protein is largely unaggregated (>93%).
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50% w/w. This concentration-dependent rheological behavior was examined using a range of experimental approaches.
Suspensions of spherical particles such as polymer latices have been the subject of extensive rheological investigation, with the viscosity being determined as a function of particle volume fraction, . At intermediate volume fractions (0.2–0.5) these suspensions show two Newtonian plateaus separated by a shear thinning region (Macosko, 1994). For hard sphere suspensions the low shear viscosity, 
0, includes contributions from hydrodynamic forces associated with minimally perturbed equilibrium structures, whereas for the high shear viscosity, 
, the structure is substantially perturbed. The shear stress, 
, at which the viscosity is intermediate between 0 and , is of the order of
 | (3) |
is 8 x 104 Pa. The dependence of viscosity on shear rate of BSA solutions at concentrations up to 40% w/w was examined using double gap concentric cylinder geometry. Fig. 1 shows a plot of shear viscosity versus shear rate for 30 and 40% w/w BSA at pH 5.4 in 100 mM sodium chloride at 20°C. At these concentrations, Newtonian behavior was observed at shear rates, 
, in the range 10–50 s-1. As the associated shear stress was <<104 Pa we ascribe this behavior to being equivalent to the 0 of hard sphere colloidal suspensions. Time-dependent deviations from Newtonian behavior, indicative of structure development, were observed at lower shear rates (<1 s-1), for concentrations in the range 10–20% w/w (data not shown). These observations are consistent with reports of the solid-like behavior of relatively dilute BSA solutions (Ikeda and Nishinari, 2000), with the suggestion that at low concentrations a lattice-type structure is formed, the packing of which is disrupted on further addition of BSA. This solid-like behavior was particularly apparent at low shear rates, in the region of 10-2 s-1, outside the useful range of the instrument for the viscosities tested. At a shear stress of 10 mPa, time-dependent effects could be avoided by completing the experiment in <103 s. As the time-dependent viscosity effect could be reduced by covering the BSA solution with a layer of silicone oil, it suggests that its cause was a surface drying effect. In this study it was not possible to distinguish between structure development in the bulk, and structure development at the air-liquid interface in the low shear rate (<1 s-1), low concentration (<20% w/w) range.
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, at a frequency, 
= 1 Hz (2 rad s-1), is shown in Fig. 2 for 47% w/w and 50% w/w BSA solution at 20°C. At small strains, < 0.01, both G' and G'' are independent of . At concentrations <50% w/w G'' > G' i.e., the material is predominantly behaving as a viscous fluid. The small strain behavior was investigated in more detail. The frequency dependence of G'() and G''() is shown in Fig. 3, for 42.5, 47.0, and 50.0% w/w BSA solutions. Over the frequency range examined of 0.01–10 Hz, both G' and G'' increase with increasing frequency with G' showing a less marked increase at the higher concentrations. Ultimately, at the highest concentration of 50% w/w BSA, G' crosses G'' at 10 Hz, indicative of increasing elastic, solid-like behavior. The dynamic viscosity, ', was calculated from the frequency dependence of G'' (' = G''()/) (Macosko, 1994). Over the concentration range examined ' showed a weak dependence on frequency even at 50% w/w BSA. More extensive measurements on the composition dependence of ', were carried out at a frequency of 1 Hz.
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, is shown in Fig. 4, which combines the dynamic and steady shear measurements. With increasing concentration the viscosity increases with increasing rapidity, exceeding 104 Pa s at a concentration of 50% w/w.
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r, on volume fraction is well described by (Phan et al., 1996)
 | (4) |
r is the viscosity of the suspension relative to that of the solvent, and max is the maximum packing fraction, which is 0.63 ± 0.02 for low shear rates and 0.70 ± 0.02 for high shear rates. Relationships of the form of Eq. 4 show a marked increase in viscosity over a very small range of volume fraction as approaches max. The exponent in Eq. 4 can differ from the value of 2, for example, Krieger (1972) proposed the value of 1.85 though with a correspondingly different value of max. As applied above, Eq. 4 describes the experimental data for hard sphere colloidal dispersions for volume fractions up to 50% and relative viscosities up to 50. When particles deviate from spherical shape it is found that the viscosity diverges at a lower maximum packing fraction (Macosko, 1994). Fig. 4 shows the behavior predicted assuming the protein occupies 0.90 ml g-1 (from the osmotic stress results, see later), max = 0.40 and the solvent viscosity = 1 mPa s. The predicted behavior is somewhat similar to the observed viscous behavior of BSA, with the major difference being that the observed increase in viscosity at higher volume fractions is less dramatic, possibly indicating that the BSA particle is soft. This behavior could arise from particle deformability or from the dependence of the interparticle interaction on average particle separation.
At higher concentrations of BSA the material has solid-like characteristics and its mechanical behavior was examined in a three point bend test. For small instantaneous deformations (strain <0.01) there was a linear relationship between stress and strain. This permitted calculation of the tensile modulus, E, the composition dependence of which, over the concentration range 62–86% w/w, is shown in Table 2. Within the solid range the modulus increases by two orders of magnitude from 10 MPa to 1.0 GPa. This higher value is characteristic of glassy polymers (Ollett et al., 1991). These stiff materials show stress relaxation behavior at long timescales. Relaxation times (m) characterizing the stress relaxation were obtained by fitting the data to a stretched exponential function of the form
 | (5) |
). For undercooled molecular liquids the mechanical relaxation time is of the order of 100 s at the calorimetric glass transition, Tg, where the viscosity is typically of the order of 1012 Pa s. The relaxation times obtained for the BSA mixtures, in the region of 100 s, are therefore indicative of glass-like behavior, and the values of the parameter ß obtained, in the range 0.38–0.64, are similar to those found in molecular glass formers (Struik, 1978; Hodge, 1994).
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). Fig. 6 shows a logarithmic plot of the reduced osmotic pressure, / 
kT, ( is the number density) as a function of mass fraction of BSA at pH 5.4 and 25°C throughout the entire composition range. In our experiments, carried out in the presence of 100 mM sodium chloride and 50 mM acetate buffer, the mass fraction ranged from 0.33 to 0.73. Our data are compared with those of Vilker (Vilker et al., 1981) obtained by osmometry, (mass fraction up to 0.4, in 150 mM sodium chloride adjusted to pH 5.4) and Bull (1944) determined by water vapor sorption from atmospheres of known water activity (mass fraction range 0.73–0.97). The agreement between the different methodologies employed is satisfactory with the osmotic pressure increasing from 5.4 kPa at a mass fraction of 0.089–400 MPa at a mass fraction of 0.97.
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; Minton, 1995) interpreted the variation of osmotic pressure with concentration of BSA as resulting from excluded volume solute-solute interactions and used a hard sphere model to fit the data. More recently, Farrer and Lips (1999) used an adhesive hard sphere model to interpret sodium caseinate osmotic pressure data thus allowing the effects of attractive interactions to be interpreted. The reduced osmotic pressure, /kT, for the present BSA data shows a monotonic increase with concentration indicative of an interaction that is predominantly repulsive. The data only allows a single parameter to be estimated, an apparent volume of the protein. Fig. 6 shows the osmotic pressure calculated using the Percus-Yevick hard sphere equation of state (Eq. 6) (Farrer and Lips, 1999)
 | (6) |
. This volume depends upon the hydrated volume of the protein and on the intermolecular interactions. The apparent protein volume is larger than the value of 0.67 ml g-1 obtained by Minton (1995) from dilute solution osmotic pressure data (Kanal et al., 1994) and is very much lower than would be predicted from the dilute solution diffusion data (2.13 ml g-1). |
GENERAL DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). Using the effective volume of the protein, 0.90 ml g-1, the concentration of 50% w/w (highest viscosity point in Fig. 4) converts to a volume fraction of 0.52. Thus, the divergence occurs at a lower volume fraction than for hard spheres that can be ascribed to BSA's known deviation from spherical shape (Carter and Ho, 1994; He and Carter, 1992). Also, the approach to the divergence is relatively slow, which is indicative of softness in the interaction.
At an ionic strength of 100 mM the Debye-Huckel length is 0.92 nm (Kuehner et al., 1997), which is comparable with the size of the molecule. However, estimates of the magnitude of dispersion forces in these systems (Kuehner et al., 1997) mean that the potential of mean force is attractive at all interparticle separations and so the repulsive electrostatic interaction is not predicted to contribute to the apparent size of the molecule. This conclusion depends upon the value of the Hamaker constant. The value used, from a Lifshitz theory calculation, was 5 kT (Kuehner et al., 1997); if the value is dropped to 3.1 kT the repulsive electrostatic interaction starts to predominate. Attempts to predict osmotic pressure of BSA solutions from interparticle potentials have resulted in poor agreement with experiment even for dilute solutions (Vilker et al., 1981). Although this was, in part, due to the use of a virial expansion, it also highlighted the need for improved potentials of mean force. Despite this uncertainty concerning the detailed form of the interparticle forces, considerations of charge balance indicate that electrostatic forces are highly important in these concentrated protein solutions. Titrations (Scatchard et al., 1950; Fogh-Andersen et al., 1993) show that while the isoionic point of BSA is pH 5.4, chloride ion binding occurs, which, at 100 mM sodium chloride, leads to a net charge of about -7e. Charge balance calculations show that, in dilute solution, the total negative charge carried by the protein is very much less than that carried by the chloride co-ion, however, at higher concentrations ( > 0.35) the situation is reversed and the protein is predicted to carry the majority of the charge. The Donnan effect will potentially exert a large effect in these systems.
The colloid analogy suggests that the main structural relaxation results from collective motion over a supra-globule length scale. On the basis of this analogy we would predict that a secondary, more localized, cage-rattling, relaxation would occur at the globule length scale (Mason and Weitz, 1995). Further relaxations, specific to the molecular details of the globule, would be anticipated at subglobule length scale. The conclusion regarding the main structural relaxation and predictions following from it indicate a need for further investigations of dynamics in these systems, including investigation of the effect of ionic environment on observed behavior.
The present behavior is relevant to globular proteins subjected to osmotic stress in biological situations. Typical average protein concentrations in the cytoplasm range from 20 to 35% w/w. If the cytoplasm is subject to a dehydration stress then the current results would suggest that the system would vitrify when the protein content exceeds 60% w/w. This vitrification would have a dramatic effect on transport processes within the cytoplasm. It has been proposed that vitrification, and the consequent arrest of some diffusive processes, is a mechanism that is useful in the preservation of life in the dry state. This is commonly associated with the biosynthesis of polyols and low molecular weight carbohydrates that have the ability to form a glassy matrix (Crowe et al., 1998). The current research on the vitrification of a globular protein suggests that discussion of the vitrification of biological structures should recognize that vitrification can occur on several length scales and can include the vitrification of low molecular weight solutes and the vitrification of larger particles such as globular proteins, which occur at very different water contents. On decreasing water content, particle arrest and vitrification precedes the vitrification of low molecular weight solutes.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Submitted on February 12, 2003; accepted for publication August 14, 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). Solid line is the prediction using 
); Bull (1944)