Thermal properties of water in myoglobin crystals and solutions at subzero temperatures.

The water of hydration in myoglobin crystals and solutions was studied at subzero temperatures by calorimetry and infrared spectroscopy (ir). For comparison we also investigated glycine, DL-alanine and DL-valine solutions. The hydration water remains amorphous at low temperatures. We find a broad glass transition between 180 and 270 K depending on the degree of hydration. The ice component shows a noncolligative melting point depression that is attributed to a finite conformational flexibility. The ir spectrum and the specific heat of water in myoglobin crystals was determined for the first time between 180 and 290 K. The glass transition in crystals is qualitatively similar to what is found in amorphous samples at the same water content. These data are compared with M?ssbauer experiments and dielectric relaxation of water in myoglobin crystals. The similar temperature dependencies suggest a cross correlation between structural fluctuations and the thermal motion of crystal water. A hydrogen bond network model is proposed to explain these features. The essential ingredients are cooperativity and a distribution of hydrogen-bonded clusters.     

Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during cryoprotection

Plant shoot tips do not survive exposure to liquid nitrogen temperatures without cryoprotective treatments. Some cryoprotectant solutions, such as plant vitrification solution 2 (PVS2), dehydrate cells and decrease lethal ice formation, but the extent of dehydration and the effect on water freezing properties are not known. We examined the effect of a PVS2 cryoprotection protocol on the water content and phase behavior of mint and garlic shoot tips using differential scanning calorimetry. The temperature and enthalpy of water melting transitions in unprotected and recovering shoot tips were comparable to dilute aqueous solutions. Exposure to PVS2 changed the behavior of water in shoot tips: enthalpy of melting transitions decreased to about 40 J g H2O(-1) (compared to 333 J g H2O(-1) for pure H2O), amount of unfrozen water increased to approximately 0.7 g H2O g dry mass(-1) (compared to approximately 0.4 g H2Og dry mass(-1) for unprotected shoot tips), and a glass transition (T(g)) at -115 degrees C was apparent. Evaporative drying at room temperature was slower in PVS2-treated shoot tips compared to shoot tips receiving no cryoprotection treatments. We quantified the extent that ethylene glycol and dimethyl sulfoxide components permeate into shoot tips and replace some of the water. Since T(g) in PVS2-treated shoot tips occurs at -115 degrees C, mechanisms other than glass formation prevent freezing at temperatures between 0 and -115 degrees C. Protection is likely a result of controlled dehydration or altered thermal properties of intracellular water. A comparison of thermodynamic measurements for cryoprotection solutions in diverse plant systems will identify efficacy among cryopreservation protocols.     

Liquid-like water confined in stacks of biological membranes at 200 k and its relation to protein dynamics

Confined water is of considerable current interest owing to its biophysical importance and relevance to cryopreservation. It can be studied in its amorphous or supercooled state in the "no-man's land", i.e., in the temperature range between 150 and 235 K, in which bulk water is always crystalline. Amorphous deuterium oxide (D(2)O) was obtained in the intermembrane spaces of a stack of purple membranes from Halobacterium salinarum by flash cooling to 77 K. Neutron diffraction showed that upon heating to 200 K the intermembrane water space decreased sharply with an associated strengthening of ice diffraction, indicating that water beyond the first membrane hydration layer flowed out of the intermembrane space to form crystalline ice. It was concluded that the confined water undergoes a glass transition at or below 200 K to adopt an ultraviscous liquid state from which it crystallizes to form ice as soon as it finds itself in an unconfined, bulk-water environment. Our results provide model-free evidence for translational diffusion of confined water in the no-man's land. Potential effects of the confined-water glass transition on nanosecond membrane dynamics were investigated by incoherent elastic neutron scattering experiments. These revealed no differences between flash-cooled and slow-cooled samples (in the latter, the intermembrane space at temperatures <250 K is occupied only by the first membrane hydration layers), with dynamical transitions at 150 and 260 K, but not at 200 K, suggesting that nanosecond membrane dynamics are not sensitive to the state of the water beyond the first hydration shell at cryotemperatures.     

Unraveling protein stabilization mechanisms: Vitrification and water replacement in a glass transition temperature controlled system

The aim of this study was to elucidate the role of the two main mechanisms used to explain the stabilization of proteins by sugar glasses during drying and subsequent storage: the vitrification and the water replacement theory. Although in literature protein stability is often attributed to either vitrification or water replacement, both mechanisms could play a role and they should be considered simultaneously. A model protein, alkaline phosphatase, was incorporated in either inulin or trehalose by spray drying. To study the storage stability at different glass transition temperatures, a buffer which acts as a plasticizer, ammediol, was incorporated in the sugar glasses. At low glass transition temperatures (< 50 °C), the enzymatic activity of the protein strongly decreased during storage at 60 °C. Protein stability increased when the glass transition temperature was raised considerably above the storage temperature. This increased stability could be attributed to vitrification. A further increase of the glass transition temperature did not further improve stability. In conclusion, vitrification plays a dominant role in stabilization at glass transition temperatures up to 10 to 20 °C above storage temperature, depending on whether trehalose or inulin is used. On the other hand, the water replacement mechanism predominately determines stability at higher glass transition temperatures.     

Glass transition and dynamics in BSA-water mixtures over wide ranges of composition studied by thermal and dielectric techniques

Protein-water dynamics in mixtures of water and a globular protein, bovine serum albumin (BSA), was studied over wide ranges of composition, in the form of solutions or hydrated solid pellets, by differential scanning calorimetry (DSC), thermally stimulated depolarization current technique (TSDC) and dielectric relaxation spectroscopy (DRS). Additionally, water equilibrium sorption isotherm (ESI) measurements were performed at room temperature. The crystallization and melting events were studied by DSC and the amount of uncrystallized water was calculated by the enthalpy of melting during heating. The glass transition of the system was detected by DSC for water contents higher than the critical water content corresponding to the formation of the first sorption layer of water molecules directly bound to primary hydration sites, namely 0.073 (grams of water per grams of dry protein), estimated by ESI. A strong plasticization of the T(g) was observed by DSC for hydration levels lower than those necessary for crystallization of water during cooling, i.e. lower than about 0.3 (grams of water per grams of hydrated protein) followed by a stabilization of T(g) at about -80°C for higher water contents. The α relaxation associated with the glass transition was also observed in dielectric measurements. In TSDC a microphase separation could be detected resulting in double T(g) for some hydration levels. A dielectric relaxation of small polar groups of the protein plasticized by water, overlapped by relaxations of uncrystallized water molecules, and a separate relaxation of water in the crystallized water phase (bulk ice crystals) were also recorded.     

Dynamic mechanical properties of elastin.

The dynamic mechanical properties of water-swollen elastin under physiological conditions have been investigated. When elastin is tested as a colsed, fixed-volume system, mechanical data could be temperature shifted to produce master curves. Master curves for elastin hydrated at 36°C (water content, 0.46 g water/g protein) and 55°C (water content, 0.41 g/g) were constructed, and in both cases elastin goes through a glass transition, with the glass transition temperatures of -46 and -21°C, respectively. Temperature shift data used to construct the master curves follow the WLF equation, and the glass transition appears to be characteristic of an amorphous, random-polymer network. For elastin tested as an open, variable-volume system free to change its swollen volume as temperature is changed, dynamic mechanical properties appear to be virtually independent of temperature. No glass transition is observed because elastin swelling increases with decreased temperature, and the increase in water content shifts elastin away from its glass transition. It is suggested that the hydrophobic character of elastin, which gives rise to the unusual swelling properties of elastin, evolved to provide a temperature-independent elastomer for the cold-blooded, lower vertebrates.     

Glass transition temperatures of cassava starch-whey protein concentrate systems at low and intermediate water content

Glass transition temperatures of cassava starch (CS)-whey protein concentrate (WPC) blends were determined by means of differential scanning calorimetry (DSC) in a water content range of 8-20% (dry basis, d.b.). Water equilibration in the samples was carried out by storing them at room temperature (25 °C) during four weeks. Physical aging and phase segregation were observed in some samples after this storage period depending on the water content. Both, first DSC heating scans and tan δ curves of CS-WPC blends with intermediate water content (10-18%), showed two endothermic thermal events. The first one appeared at around 60 °C and was independent of water content. The second one was detected at higher temperatures and moved towards the low-temperature peak as the water content increased. The results can be explained by a phase segregation process that can take place when the samples are conditioned below their glass transition temperatures. The Gordon-Taylor equation described well the plasticizing effect of water on the blends. WPC was also found to decrease the glass transition temperature, at constant water content, an effect attributed to additional water produced during browning reactions in the blends.     

Freezing and melting water in lamellar structures.

The manner in which ice forms in lamellar suspensions of dielaidoylphosphatidylethanolamine, dielaidoylphosphatidylcholine, and dioleoylphosphatidylcholine in water depends strongly on the water fraction. For weight fractions between 15 and 9%, the freezing and melting temperatures are significantly depressed below 0 degree C. The ice exhibits a continuous melting transition spanning as much as 20 degrees C. When the water weight fraction is below 9%, ice never forms at temperatures as low as -40 degrees C. We show that when water contained in a lamellar lipid suspension freezes, the ice is not found between the bilayers; it exists as pools of crystalline ice in equilibrium with the bound water associated with the polar lipid headgroups. We have used this effect, together with the known chemical potential of ice, to measure hydration forces between lipid bilayers. We find exponentially decaying hydration repulsion when the bilayers are less than about 7 A apart. For larger separations, we find significant deviations from single exponential decay.     

Glass Transition and Dynamics in Lysozyme�CWater Mixtures Over Wide Ranges of Composition

Differential scanning calorimetry (DSC) and two dielectric techniques, broadband dielectric relaxation spectroscopy and thermally stimulated depolarization currents (TSDC), were employed to study glass transition and water and protein dynamics in mixtures of water and a globular protein, lysozyme, in wide ranges of water content, both solutions, and hydrated solid samples. In addition, water equilibrium sorption isotherms (ESI) measurements were performed at room temperature. The main objective was to correlate results by different techniques to each other and to determine critical water contents for various processes. From ESI measurements the content of water directly bound to primary hydration sites was determined to 0.088 (grams of water per grams of dry protein), corresponding to 71 water molecules per protein molecule, and that where clustering becomes significant to about 0.25. Crystallization and melting events of water were first observed at water contents 0.270 and 0.218, respectively, and the amount of uncrystallized water was found to increase with increasing water content. Two populations of ice crystals were observed by DSC, primary and bulk ice crystals, which give rise to two separate relaxations in dielectric measurements. In addition, the relaxation of uncrystallized water was observed, superimposed on a local relaxation of polar groups on the protein surface. The glass transition temperature, determined by DSC and TSDC in rather good agreement to each other, was found to decrease significantly with increasing water content and to stabilize at about −90 °C for water contents higher than about 0.25. This is a novel result of this study with potential impact on cryoprotection and pharmaceutics.     

Mechanism of cryoprotection by extracellular polymeric solutes.

To elucidate the means by which polymer solutions protect cells from freezing injury, we cooled human monocytes to -80 degrees C or below in the presence of various polymers. Differential scanning calorimetric studies showed that those polymers which protect cells best have a limiting glass transition temperature (T'g) of approximately -20 degrees C; those with a T'g significantly higher or lower did not protect. Freeze-etch electron micrographs indicated that intracellular ice crystals had formed during this freezing procedure, but remained smaller than approximately 300 nm in the same proportion of cells as survived rapid thawing. We propose that cryoprotection of slowly frozen monocytes by polymers is a consequence of a T'g of -20 degrees C in the extracellular solution. In our hypothesis, the initial concentration and viscosity of protective polymer solutions reduce the extent and rate of cell water loss to extracellular ice and limit the injurious osmotic stress, which cells face during freezing at moderate rates to -20 degrees C. Below -20 degrees C, glass formation prevents further osmotic stress by isolating cells from extracellular ice crystals, virtually eliminating cell water loss at lower temperatures. On the other hand, the protective polymer solutions will allow some diffusion of water away from cells at temperatures above T'g. If conditions are correct, cells will concentrate the cytoplasm sufficiently during the initial cooling to T'g to avoid lethal intracellular freezing between T'g and the intracellular Tg, which has been depressed to low temperatures by that concentration. Thus, when polymers are used as cryoprotective agents, cell survival is contingent upon maintenance of osmotic stress within narrow limits.     

Biodegradable starch based nanocomposites with low water vapor permeability and high storage modulus

Nanocomposite materials based on a starch matrix reinforced with very small amounts of multi-walled carbon nanotubes (MWCNTs) (from 0.005 wt% to 0.055 wt%) were developed. The material's dynamic-mechanical and water vapor permeability properties were investigated. An increasing trend of storage modulus (E′) and a decreasing trend of water vapor permeability (WVP) with filler content were observed at room temperature. For the composite with 0.055 wt% of filler, E′ value was about 100% higher and WVP value was almost 43% lower than the corresponding matrix values. MWCNTs were wrapped in an aqueous solution of a starch-iodine complex before their incorporation into the matrix, obtaining exceptionally well-dispersed nanotubes and optimizing interfacial adhesion. This excellent filler dispersion leads to the development of an important contact surface area with the matrix material, producing remarkable changes in the starch-rich phase glass transition temperature even in composites with very low filler contents. This transition is shifted towards higher temperatures with increasing content of nanotubes. So at room temperature, some composites are in the rubber zone while others, in the transition zone. Therefore, this change in the material glass transition temperature can be taken as responsible for the important improvements obtained in the composites WVP and E′ values for carbon nanotubes content as low as 0.05 wt%.     

Effect of water on the molecular mobility of elastin

Purified and hydrated elastin is studied by both thermal and dielectric techniques to have insight into the chain dynamics of this protein. By differential scanning calorimetry, the glassy behavior of elastin is highlighted; the glass transition temperature (T(g)) of elastin is found to be widely dependent on hydration, falling from 200 degrees C in the dehydrated state to 30 degrees C for 30% hydration. A limit of T(g) at around 0 degrees C is found when crystallizable water is present in the system, that is, when the formation of ice prevents motions of some 10 nm along the polypeptidic chains. The technique of thermally stimulated currents, carried out in the -180 to 0 degrees C temperature range, is useful to detect localized motions. In this case, too, the localized motions vary considerably according to hydration: a first relaxation mode is observed at -145 degrees C and it is associated with the reorientation of crystallizable water in ice I; a second relaxation mode, more complex and cooperative, occurs at around -80 degrees C and could be attributed to the complex constituted by the dipolar groups of the polypeptidic chain and noncrystallizable water, behaving as a glassy system.     

Reversible self-polymerizing high T(g) lyoprotectants

One mode of action of protectants in the storage of biological materials is by promoting the formation of a vitrified state on cooling or drying. In the case of preservation by drying, the glassy material comprises a low water content mixture of protectant and organic material. The protectant must on drying form a glassy state of glass transition temperature (T(g)) above the desired storage temperature. However, in some applications it must also be easily transported through cell membranes and this restricts the choice to a relatively limited number of small molecules, which typically exhibit very low glass transition temperatures. In this work we describe a self-polymerizing protectant comprising an inorganic salt and a small hydroxy functional molecule such as glycerol. This forms co-ordinate polymer chains of high T(g) on drying but rapidly depolymerizes into the original components on rehydration. The polymerization process is general for polyhydroxy compounds including glucose and related compounds.     

Effect of salts on the properties of aqueous sugar systems, in relation to biomaterial stabilization. 1. Water sorption behavior and ice crystallization/melting

Trehalose and sucrose, two sugars that are involved in the protection of living organisms under extreme conditions, and their mixtures with salts were employed to prepare supercooled or freeze-dried glassy systems. The objective of the present work was to explore the effects of different salts on water sorption, glass transition temperature (T(g)), and formation and melting of ice in aqueous sugar systems. In the sugar-salt mixtures, water adsorption was higher than expected on the basis of the water uptake by each pure component. In systems with a reduced mass fraction of water (w less-than-or-equal 0.4), salts delayed water crystallization, probably due to ion-water interactions. In systems where > 0.6, water crystallization could be explained by the known colligative properties of the solutes. The glass transition temperature of the maximally concentrated matrix (T(g)') was decreased by the presence of salts. However, the actual T(g) values of the systems were not modified. Thus, the effect of salts on sorption behavior and formation of ice may reflect dynamic water-salt-sugar interactions which take place at a molecular level and are related to the charge/mass ratio of the cation present without affecting supramolecular or macroscopic properties.     

Laser Raman investigation of intact single muscle fibers on the state of water in muscle tissue

Laser Raman spectroscopy has been used to investigate the state of water in intact single muscle fibers of the giant barnacle (Balanus nulilus). The spectra in the region of the O-H (or O-²H) stretching modes of water in unfrozen fibers show that there is no appreciable difference between the shape and relative intensity of the Raman bands due to the water molecules located inside a muscle fiber and those of the corresponding bands in the spectrum of pure water. The presence of significant amounts of “structured” intracellular water, greater than approx. 5% of the total water content, in these fibers is thus excluded. The Raman spectra of frozen fibers have also been recorded in order to evaluate the amount of intracellular water which remains unfrozen at temperatures below the normal freezing point of water. We have been able to reproduce these spectra by assuming that the spectrum of a frozen fiber is the sum of the individual spectra of water and ice. To calculate the amount of unfrozen water from these curve fittings, it was also necessary to determine the intensities of the water and ice Raman bands relative to one another. We have found the I(ice)/I(water) ratio is 1.07 ± 0.01 for H₂O and 1.05 ± 0.03 for ²H₂O With these figures, we have calculated that for a fiber with a normal water content of 80%, 20% of the water molecules remain in the supercooled state at ?5°C, which corresponds to 1 g of water per of fiber dry weight. This amount of bound water was also found to be independent of the water content of the fibers.     

Calorimetric study of crystal growth of ice in hydrated methemoglobin and of redistribution of the water clusters formed on melting the ice.

Calorimetric studies of the melting patterns of ice in hydrated methemoglobin powders containing between 0.43 and 0.58 (g water)/(g protein), and of their dependence on annealing at subzero temperatures and on isothermal treatment at ambient temperature are reported. Cooling rates were varied between approximately 1500 and 5 K min-1 and heating rate was 30 K min-1. Recrystallization of ice during annealing is observed at T > 228 K. The melting patterns of annealed samples are characteristically different from those of unannealed samples by the shifting of the melting temperature of the recrystallized ice fraction to higher temperatures toward the value of "bulk" ice. The "large" ice crystals formed during recrystallization melt on heating into "large" clusters of water whose redistribution and apparent equilibration is followed as a function of time and/or temperature by comparison with melting endotherms. We have also studied the effect of cooling rate on the melting pattern of ice with a methemoglobin sample containing 0.50 (g water)/(g protein), and we surmise that for this hydration cooling at rates of > or = approximately 150 K min-1 preserves on the whole the distribution of water molecules present at ambient temperature.     

Impact of water activity, temperature, and physical state on the storage stability of Lactobacillus paracasei ssp. paracasei freeze-dried in a lactose matrix

The aim of this study was to determine whether the combined effect of water activity and temperature on inactivation rates of freeze-dried microorganisms in a lactose matrix could be explained in terms of the glass transition theory. The stabilized glass transition temperature, Tg, of the freeze-dried products was determined by differential scanning calorimetry at two different temperatures, T (20 and 37 degrees C), and different water activities (0.07-0.48). This information served as a basis for defining conditions of T and water activity, which led to storage of the bacteria in the glassy (T < Tg) and nonglassy (T > Tg) states. The rates of inactivation of the dry microorganisms subjected to different storage conditions were determined by plate counts and could be described by first-order kinetics. Rates were analyzed as a function of water activity, storage temperature, and the difference between Tg and T. Inactivation below Tg was low; however, Tg could not be regarded as an absolute threshold of bacteria stability during storage. When the cells were stored in the nonglassy state (T > Tg), inactivation proceeded faster, however, not as rapid as suggested by the temperature dependence of the viscosity above the glass transition temperature. Furthermore, the first-order rate constant, k, was dependent on the storage temperature per se rather than on the temperature difference between the glass transition temperature and the storage temperature (T - Tg).     

The effect of water on the glass transition of human hair

The glass transition of human hair and its dependence on water content were determined by means of differential scanning calorimetry (DSC). The relationship between the data is suitably described by the Fox equation, yielding for human hair a glass transition temperature of T(g) = 144 degrees C, which is substantially lower than that for wool (174 degrees C). This effect is attributed to a higher fraction of hydrophobic proteins in the matrix of human hair, which acts as an internal plasticizer. The applicability of the Fox equation for hair as well as for wool implies that water is homogeneously distributed in alpha-keratins, despite their complex morphological, semicrystalline structure. To investigate this aspect, hair was rendered amorphous by thermal denaturation. For the amorphous hair neither the water content nor T(g) were changed compared to the native state. These results provide strong support for the theory of a quasi-homogeneous distribution of water within alpha-keratins.     

Molecular motions of polysaccharides in the solid state: dextran, pullulan and amylose.

Dextran, pullulan and amylose have been investigated by differential scanning calorimetry, thermogravimetric analysis, dynamic mechanical and dielectric spectroscopy over a wide range of temperatures and frequencies. No melting or glass transition is seen below the range of thermal degradation (about 300 degrees C) for either amylose or pullulan; only dextran shows a Tg at 223 degrees C (delta cp = 0.40 J/g deg). The viscoelastic spectrum of the 'dry' polysaccharides is characterized by a low temperature relaxation that occurs at -94, -73 and -59 degrees C, at 1 kHz, (activation energy 32, 39 and 52 kJ/mol) in dextran, pullulan and amylose respectively and is assigned to small entity local motions of the polysaccharide backbone. Absorbed water strongly modifies the relaxation spectrum, inducing a new relaxation below room temperature and dissipation regions associated with water loss above room temperature. The former appears at temperatures higher than the relaxation characteristic of the dry polymer and moves to lower temperature with increasing water content. In normal 'room humidity' conditions (about 10% absorbed water) the water-induced relaxation, attributed to the motion of complex polymer-water relaxing units, is the only observable feature in the dynamic mechanical and dielectric spectrum below room temperature.     

Freezing avoidance by supercooling in Olea europaea cultivars: the role of apoplastic water,solute content and cell wall rigidity

Plants can avoid freezing damage by preventing extracellular ice formation below the equilibrium freezing temperature (supercooling). We used Olea europaea cultivars to assess which traits contribute to avoid ice nucleation at sub‐zero temperatures. Seasonal leaf water relations, non‐structural carbohydrates, nitrogen and tissue damage and ice nucleation temperatures in different plant parts were determined in five cultivars growing in the Patagonian cold desert. Ice seeding in roots occurred at higher temperatures than in stems and leaves. Leaves of cold acclimated cultivars supercooled down to ?13 °C, substantially lower than the minimum air temperatures observed in the study site. During winter, leaf ice nucleation and leaf freezing damage (LT₅₀) occurred at similar temperatures, typical of plant tissues that supercool. Higher leaf density and cell wall rigidity were observed during winter, consistent with a substantial acclimation to sub‐zero temperatures. Larger supercooling capacity and lower LT₅₀ were observed in cold‐acclimated cultivars with higher osmotically active solute content, higher tissue elastic adjustments and lower apoplastic water. Irreversible leaf damage was only observed in laboratory experiments at very low temperatures, but not in the field. A comparative analysis of closely related plants avoids phylogenetic independence bias in a comparative study of adaptations to survive low temperatures.