Characterization of the restricted rotation of the dimethyl groups in chemically N-terminal 13C-labeled antifreeze glycoproteins: a temperature-dependent study in water to ice through the supercooled state

Site-specific chemical modification, especially with isotopically enriched groups, allows one to study the structure and dynamics of proteins for which uniform enrichment is difficult. When the N-terminal alanine in antifreeze glycoprotein (AFGP) is replaced with an N,N-dimethyl alanine the methyl groups show signatures of slow rotation about the C-N bond. In order to separate the local dynamics of the N-terminus from the overall protein dynamics, we present a complete characterization of this dynamics. Temperature-dependent nuclear magnetic-resonance experiments from room temperature to subzero temperatures, including the supercooled state and in the presence of ice, are presented. Quantum chemical calculations are also performed on a localized N-terminus of the AFGP. Our results show that in the solution state at room temperature and in the super cooled regime, the dimethyl groups undergo a slow, restricted rotation with an unequal distribution of population between two major conformations. At lower temperatures in the presence of ice, the dynamics become much more complex due to freezing out of several conformational states. Based on these results, we conclude that the segmental dynamics of the N-terminus are local to the first residue and do not affect the overall dynamics of the protein.     

Influence of Sequential Modifications and Carbohydrate Variations in Synthetic AFGP Analogues on Conformation and Antifreeze Activity

Certain Arctic and Antarctic ectotherm species have developed strategies for survival under low temperature conditions that, among others, consist of antifreeze glycopeptides (AFGP). AFGP form a class of biological antifreeze agents that exhibit the ability to inhibit ice growth in vitro and in vivo and, hence, enable life at temperatures below the freezing point. AFGP usually consist of a varying number of (Ala‐Ala‐Thr)_n units (n=4–55) with the disaccharide β‐D ‐galactosyl‐(1→3)‐α‐N‐acetyl‐D ‐galactosamine glycosidically attached to every threonine side chain hydroxyl group. AFGP have been shown to adopt polyproline II helical conformation. Although this pattern is highly conserved among different species, microheterogeneity concerning the amino acid composition usually occurs; for example, alanine is occasionally replaced by proline in smaller AFGP. The influence of minor and major sequence mutations on conformation and antifreeze activity of AFGP analogues was investigated by replacement of alanine by proline and glycosylated threonine by glycosylated hydroxyproline. The target compounds were prepared by using microwave‐enhanced solid phase peptide synthesis. Furthermore, artificial analogues were obtained by copper‐catalyzed azide–alkyne cycloaddition (CuAAC): propargyl glycosides were treated with polyproline helix II‐forming peptides comprising (Pro‐Azp‐Pro)_n units (n=2–4) that contained 4‐azidoproline (Azp). The conformations of all analogues were examined by circular dichroism (CD). In addition, microphysical analysis was performed to provide information on their inhibitory effect on ice recrystallization.     

Ice growth from supercooled aqueous solutions of benzene, naphthalene, and phenanthrene

Classical molecular dynamics (MD) were performed to investigate the growth of ice from supercooled aqueous solutions of benzene, naphthalene, or phenanthrene. The main objective of this study is to explore the fate of those aromatic molecules after freezing of the supercooled aqueous solutions, i.e., if these molecules become trapped inside the ice lattice or if they are displaced to the QLL or to the interface with air. Ice growth from supercooled aqueous solutions of benzene, naphthalene, or phenanthrene result in the formation of quasi-liquid layers (QLLs) at the air/ice interface that are thicker than those observed when pure supercooled water freezes. Naphthalene and phenanthrene molecules in the supercooled aqueous solutions are displaced to the air/ice interface during the freezing process at both 270 and 260 K; no incorporation of these aromatics into the ice lattice is observed throughout the freezing process. Similar trends were observed during freezing of supercooled aqueous solutions of benzene at 270 K. In contrast, a fraction of the benzene molecules become trapped inside the ice lattice during the freezing process at 260 K, with the rest of the benzene molecules being displaced to the air/ice interface. These results suggest that the size of the aromatic molecule in the supercooled aqueous solution is an important parameter in determining whether these molecules become trapped inside the ice crystals. Finally, we also report potential of mean force (PMF) calculations aimed at studying the adsorption of gas-phase benzene and phenanthrene on atmospheric air/ice interfaces. Our PMF calculations indicate the presence of deep free energy minima for both benzene and phenanthrene at the air/ice interface, with these molecules adopting a flat orientation at the air/ice interface.     

Impact of Borate on Structure of Antifreeze Glycoproteins

Antifreeze glycoproteins (AFGPs) facilitate the survival of various organisms in the polar region by preventing internal ice accumulation via an adsorption-inhibition mechanism. Inhibition of AFGP antifreeze activity by the borate buffers has been widely acknowledged as the direct experimental evidence supporting the hydroxyl, rather than methyl, binding mechanism. On the other hand, perturbation of borate binding on the AFGP configuration, which might have considerable influence on the binding efficiency of not only the hydroxyl but also the methyl groups, has rarely been quantitatively examined. Herein we studied, using molecular dynamics simulations, the perturbation on the configuration of a solvated AFGP8 protein induced by the binding of one single borate anion. Near the freezing point, this binding not only makes the disaccharide groups adjacent to the borate-binding disaccharide close to each other but also affects the entire AFGP8 conformation. The structural changes induced by the binding of borate on different disaccharide sidechains exhibit clear site-specificities and the effect of borate binding on the structural changes is significantly reduced at higher temperatures. Our study is valuable for further understanding the relationship between the structure and antifreeze activity of these antifreeze glycoproteins.     

Glass-liquid transition of carbon dioxide and its effect on water segregation

The interactions between CO(2) and D(2)O molecules have been investigated by using time-of-flight secondary ion mass spectrometry in the temperature rage 13-120 K. The monolayer of CO(2) tends to wet or intermix with the D(2)O film below 40 K and dewets the surface above 60 K. The water nanoclusters deposited on the CO(2) multilayers also start to segregate at 50-60 K and are finally incorporated in the bulk at 85-90 K, where the morphology of the film changes abruptly together with the desorption rate of the CO(2) molecules. The break at 85 K should be caused by the occurrence of the fluidized film whereas the glass-transition temperature of CO(2), as determined from the onset of translational molecular diffusion, is assigned to 50 K. This behavior may be related to the ultraviscous nature of the supercooled liquid, arising from the decoupling between the translational molecular diffusion and viscosity. The He(+) irradiation of the mixed CO(2)-D(2)O ice and the D(2)(+) irradiation of the CO(2) ice at 13 K do not yield any surface residues assignable to H(2)CO(3) and its precursors above 100 K. This result may be related to the segregation between the CO(2) and D(2)O molecules.     

Chain conformations of poly(gamma-benzyl-L-glutamate) pre and post an electrospinning process

In order to reveal mechanisms for the electrospinning of proteins, this study focuses on the polymer chain conformation, which is considered to be a critical factor for successful electrospinning. Poly(gamma-benzyl-L-glutamate) (PBLG) is employed and the relationships between the chain conformations of the pre-spun PBLG molecules and the morphologies of the post-spun PBLG fibers are investigated. By combining viscosity measurements, and circular dichroism and FT-IR spectroscopies, chain conformations of the pre- and post-spun PBLG are characterized. The chain conformations of the pre-spun PBLG changes from an alpha-helix to a random coil upon changing the solvent ratios of dichloromethane (CH(2)Cl(2)) and trifluoroacetic acid (CF(3)COOH) from 100:0 to 0:100. In an alpha-helix conformation, the morphology of PBLG fibers is relatively thick, while that of the random coil is thin and homogenous. The mean fiber diameters decrease when the chain conformations change from an alpha-helix to a random coil. FT-IR spectroscopy and wide-angle X-ray diffraction measurements reveal that electrospinning predominantly induces an alpha-helical conformation in post-spun PBLG fibers, and more highly crystallized fibers are generated as the alpha-helical content in the pre-spun solution increases.     

Uptake measurements of ethanol on ice surfaces and on supercooled aqueous solutions doped with nitric acid between 213 and 243 K

Uptake of ethanol either on pure frozen ice surfaces or supercooled solutions doped with HNO3 (0.63 and 2.49 wt %) has been investigated using a coated wall flow tube coupled to a mass spectrometric detection. The experiments were conducted over the temperature range of 213-243 K. Uptake of ethanol on these surfaces was always found to be totally reversible whatever were the experimental conditions. The number of ethanol molecules adsorbed per surface unit was conventionally plotted as a function of ethanol concentration in the gas phase and subsequently analyzed using Langmuir's model. The amount of ethanol molecules taken up on nitric acid doped-ice surfaces was found to increase largely with increasing nitric acid concentrations. For example at 223 K, and for an ethanol gas-phase concentration of 1x10(13) molecules cm3, the number of adsorbed molecules are (in units of molecules cm-2): approximately 1.3x10(14) on pure ice; approximately 1.4x10(15) on ice doped with HNO3 0.63 wt %; approximately 7.5x10(15) on ice doped with HNO3, 2.49 wt %, i.e. 60 times larger than on pure ice. Since, according to the shape of the isotherms, the adsorption did not proceed beyond monolayer coverage, the enormous increase of ethanol uptake was explained by considering its dissolution in either a supercooled liquid layer (T<230 K) or a liquid solution (T>230 K). The formation of both was indeed favored by the presence of the HNO3. Our experimental results suggest that the amount of ethanol dissolved in such supercooled solutions follows Henry's law and that the Henry's law constants at low temperatures, i.e., 223-243 K, can be estimated by extrapolation from higher temperatures. Such supercooled solutions which exist in the troposphere either in deep convective clouds or in mixed clouds for temperature above 233 K, might be responsible for the scavenging of large amounts of soluble species, such as nitric and sulfuric acids, oxygenated VOCs including alcohols, carboxylic acids, and formaldehyde.     

傅立叶变换红外光谱对琥珀酸-2-乙基己基磺酸钠非极性溶液中 反胶束结构的研究

利用衰减全反射傅立叶变换红外光谱(ATR-FTIR), 对琥珀酸-2-乙基己基磺酸钠(AOT)的反胶束结构进行了研究. 通过对红外光谱进行二维相关分析, 可以分辨出AOT分子在CCl4溶液中具有旁式和反式两种结构, 随着温度的升高, 旁式结构向反式结构转变, 反胶束体系能量降低, 38 ℃时, 反式结构所占比例达到最大值, 此时反胶束具有最大尺寸, 并处于最稳定的能量状态. 随着温度的进一步升高, 反式结构开始向旁式结构转变, 反胶束尺寸变小, 直至最后反胶束被破坏.     

Adsorption of naphthalene and ozone on atmospheric air/ice interfaces coated with surfactants: a molecular simulation study

The adsorption of gas-phase naphthalene and ozone molecules onto air/ice interfaces coated with different surfactant species (1-octanol, 1-hexadecanol, or 1-octanal) was investigated using classical molecular dynamics (MD) simulations. Naphthalene and ozone exhibit a strong preference to be adsorbed at the surfactant-coated air/ice interfaces, as opposed to either being dissolved into the bulk of the quasi-liquid layer (QLL) or being incorporated into the ice crystals. The QLL becomes thinner when the air/ice interface is coated with surfactant molecules. The adsorption of both naphthalene and ozone onto surfactant-coated air/ice interfaces is enhanced when compared to bare air/ice interface. Both naphthalene and ozone tend to stay dissolved in the surfactant layer and close to the QLL, rather than adsorbing on top of the surfactant molecules and close to the air region of our systems. Surfactants prefer to orient at a tilted angle with respect to the air/ice interface; the angular distribution and the most preferred angle vary depending on the hydrophilic end group, the length of the hydrophobic tail, and the surfactant concentration at the air/ice interface. Naphthalene prefers to have a flat orientation on the surfactant coated air/ice interface, except at high concentrations of 1-hexadecanol at the air/ice interface; the angular distribution of naphthalene depends on the specific surfactant and its concentration at the air/ice interface. The dynamics of naphthalene molecules at the surfactant-coated air/ice interface slow down as compared to those observed at bare air/ice interfaces. The presence of surfactants does not seem to affect the self-association of naphthalene molecules at the air/ice interface, at least for the specific surfactants and the range of concentrations considered in this study.     

Influence of monolayer amounts of HNO3 on the evaporation rate of H2O over ice in the range 179 to 208 K: a quartz crystal microbalance study

The evaporation flux J(ev) of H2O from thin H2O ice films containing between 0.5 and 7 monolayers of HNO3 has been measured in the range 179 to 208 K under both molecular and stirred flow conditions in isothermal experiments. FTIR absorption of the HNO3/H2O condensate revealed the formation of metastable alpha-NAT (HNO(3).3H2O) converting to stable beta-NAT at 205 K. After deposition of HNO3 for 16-80 s on a 1 mum thick pure ice film at a deposition rate in the range (6-60) x 10(12) molecules s(-1) the initial evaporative flux J(ev)(H2O) was always that of pure ice. J(ev)(H2O) gradually decreased with the evaporation of H2O and the concomitant increase of the average mole fraction of HNO3, chi(HNO3), indicating the presence of an amorphous mixture of H2O/HNO3 that is called complexed or (c)-ice whose vapor pressure is that of pure ice. The final value of J(ev) was smaller by factors varying from 2.7 to 65 relative to pure ice. Depending on the doping conditions and temperature of the ice film the pure ice thickness d(D) of the ice film for which J(ev) < 0.85J(ev)(pure ice) varied between 130 and 700 nm compared to the 1000 nm thick original ice film at 208 and 191 K, respectively, in what seems to be an inverse temperature dependence. There exist three different types of H2O molecules under the present experimental conditions, namely (a) free H2O corresponding to pure ice, (b) complexed H2O or c-ice, and (c) H2O molecules originating from the breakup of NAT or amorphous H2O/HNO3 mixtures. The significant decrease of J(ev)(H2O) with increasing chi(HNO3) leads to an increase of the evaporative lifetime of atmospheric ice particles in the presence of HNO3 and may help explain the occurrence of persistent and/or large contaminated ice particles at certain atmospheric conditions.     

In situ UV–visible and IR absorption study of the aggregation of silver colloidal particles via chemisorption of octadecanethiol

Aggregation of silver colloidal particles in the hydrosol via chemisorption of octadecanethiol (ODT) molecules was investigated based on in situ UV–visible transmission and infrared attenuated total reflection (ATR) measurements. As‐prepared silver hydrosol exhibits a single absorption peak at 395 nm because of the plasma resonance of the metal particles. Upon addition of ODT‐dissolved ethanol to the hydrosol, the resonance band red‐shifts and broadens, the detailed features of which depend on the ODT concentration and elapsed time. Both the red‐shift and the broadening are attributed to particle aggregation in the solution via ODT chemisorption. Aggregation on a germanium prism surface by the addition of ODT‐dissolved ethanol to the hydrosol was investigated using infrared ATR spectroscopy. At moderate ODT concentrations, intense aggregation via ODT chemisorption occurs strongly on the prism surface, allowing observation of ODT absorption bands. Angle‐dependent infrared ATR measurements for the ethanol and ODT molecules at the prism/solution interface clearly show that aggregation on the prism surface proceeds via the exclusion of ethanol. Copyright © 2004 John Wiley & Sons, Ltd.     

Calorimetric and relaxation properties of xylitol-water mixtures

We present the first broadband dielectric spectroscopy (BDS) and differential scanning calorimetry study of supercooled xylitol-water mixtures in the whole concentration range and in wide frequency (10(-2)-10(6) Hz) and temperature (120-365 K) ranges. The calorimetric glass transition, T(g), decreases from 247 K for pure xylitol to about 181 K at a water concentration of approximately 37 wt. %. At water concentrations in the range 29-35 wt. % a plentiful calorimetric behaviour is observed. In addition to the glass transition, almost simultaneous crystallization and melting events occurring around 230-240 K. At higher water concentrations ice is formed during cooling and the glass transition temperature increases to a steady value of about 200 K for all higher water concentrations. This T(g) corresponds to an unfrozen xylitol-water solution containing 20 wt. % water. In addition to the true glass transition we also observed a glass transition-like feature at 220 K for all the ice containing samples. However, this feature is more likely due to ice dissolution [A. Inaba and O. Andersson, Thermochim. Acta, 461, 44 (2007)]. In the case of the BDS measurements the presence of water clearly has an effect on both the cooperative α-relaxation and the secondary β-relaxation. The α-relaxation shows a non-Arrhenius temperature dependence and becomes faster with increasing concentration of water. The fragility of the solutions, determined by the temperature dependence of the α-relaxation close to the dynamic glass transition, decreases with increasing water content up to about 26 wt. % water, where ice starts to form. This decrease in fragility with increasing water content is most likely caused by the increasing density of hydrogen bonds, forming a network-like structure in the deeply supercooled regime. The intensity of the secondary β-relaxation of xylitol decreases noticeably already at a water content of 2 wt. %, and at a water content above 5 wt. % it has been replaced by a considerably stronger water (w) relaxation at about the same frequency. However, the similarities in time scale and activation energy between the w-relaxation and the β-relaxation of xylitol at water contents below 13 wt. % suggest that the w-relaxation is governed, in some way, by the β-relaxation of xylitol, since clusters of water molecules are rare at these water concentrations. At higher water concentrations the intensity and relaxation rate of the w-relaxation increase rapidly with increasing water content (up to the concentration where ice starts to form), most likely due to a rapid increase of small water clusters where an increasing number of water molecules interacting with other water molecules.     

Two liquid phases of water in the deeply supercooled region and their roles in crystallization and formation of LiCl solution

The properties of supercooled liquid water and the mechanism of crystallization in it were investigated using time-of-flight secondary ion mass spectrometry and reflection absorption infrared spectroscopy. The self-diffusion of the water molecules commences at 136 K, and then the liquid-liquid phase transition occurs at 160-165 K. The latter is evidenced not only by the occurrence of fluidity but also by the formation of a LiCl solution. The infrared absorption band also changes drastically above 160 K due to crystallization of water (on the Au film) and the formation of LiCl solution (on the LiCl film). The immediate crystallization and dissolution of LiCl are thought to be characteristic of normal water that is created in a deeply supercooled region, indicating that viscous liquid water (T > 136 K) is transformed into supercooled liquid water at around 160 K. The crystallization kinetics is different between these two phases because the former (latter) involves nuclear growth (spontaneous nucleation). Without nuclei, crystallization is quenched below 160 K in the present experiment. It is suggested that the viscous liquid phase coexists at the surface or grain boundaries of metastable ice Ic.     

Thermodynamic, structural, and dynamic properties of supercooled water confined in mesoporous MCM-41 studied with calorimetric, neutron diffraction, and neutron spin echo measurements

Thermodynamic, structural, and dynamic properties of heavy water (D(2)O) confined in mesoporous silica glass MCM-41 C10, C12, and C14 were investigated by differential scanning calorimetry, neutron diffraction, and neutron spin echo (NSE) measurements, respectively. The DSC data showed that no crystallization of D(2)O confined in C10 occurs in a temperature range between 298 and 180 K, and that crystalline ice is formed at 204 and 221 K for C12 and C14, respectively. For C10, the neutron radial distribution functions of confined D(2)O suggested a structural change in the supercooled state between 223 and 173 K. For C10 sample, it has been found that the tetrahedral-like water structure is partially enhanced in the central part of pores at 173 K. For all the samples, the intermediate scattering functions from the NSE measurements are fitted by the Kohlrausch-Williams-Watts stretched exponential function which implies that confined supercooled D(2)O exhibits a wide distribution of relaxation times. For C10, C12, and C14 samples, between 298 and 240 K, the relaxation times of supercooled D(2)O follow remarkably well the Vogel-Fulcher-Tamman equation; for C10 sample, below 240 K, the relaxation times of nonfreezing D(2)O show an Arrhenius type behavior. From the present experimental results on calorimetric, structural, and dynamic properties, it has been concluded that supercooled D(2)O confined in MCM-41 C10 experiences a transition from high-density to low-density hydrogen-bonded structure at around 229 K.     

Segregation of hydroxide ions to an ice surface

Hydroxide ions that are initially buried within an ice film segregate to the ice film surface at elevated temperatures. This process was observed by conducting experiments with an ice film constructed with a bottom H(2)O layer and an upper D(2)O layer, with an excess of hydroxide ions trapped at the H(2)O/D(2)O interface as they were generated by Na hydrolysis. The transport of hydroxide ions from the interfacial layer to the surface was examined as a function of time using a low energy sputtering method. The progress of the H/D exchange reaction in surface water molecules was also monitored with the Cs(+) reactive ion scattering technique. At 90 K, only a small portion of buried hydroxide ions moved to the surface in the form of OD(-) species. This was due to hydroxide transport via proton hopping through a D(2)O layer, 3 BL thick, in the surface region. At 135 K, at which point water self-diffusion is active in the ice film, the majority of the buried hydroxide ions segregated to the surface after ～1 h. Both OH(-) and OD(-) species were produced at the surface, at an OH(-)/OD(-) population ratio ≥1. Based on kinetic measurements for the transport of OH(-) and OD(-) species and the H/D exchange of surface water molecules, we concluded that the major transport channel for hydroxide ions in this regime is the migration of molecular hydroxide species. H/D exchange reactions also occur between surface hydroxide ions and water molecules. No evidence was observed for the occurrence of the hop-and-turn process at 135 K, although it is known as an important mechanism of proton transport in ice.     

A study of alpha-helix hydration in polypeptides, proteins, and viruses using vibrational raman optical activity

A vibrational Raman optical activity (ROA) study, supplemented by protein X-ray crystal structure data, of alpha-helices in polypeptides, proteins, and viruses has suggested that ROA bands in the extended amide III spectral region may be used to distinguish between two types of right-handed alpha-helix. One type, associated with a positive ROA band at approximately 1300 cm(-1), dominates in hydrophobic environments and appears to be unhydrated; the other, associated with a positive ROA band at approximately 1340 cm(-1), dominates in hydrophilic environments and appears to be hydrated. Evidence is presented to support the hypothesis that unhydrated alpha-helix corresponds to the canonical conformation alpha(c) and hydrated alpha-helix to a more open conformation alpha(o) stabilized by hydrogen bonding of a water molecule or a hydrophilic side chain to the peptide carbonyl. Alpha-helical poly(L-lysine) and poly(L-ornithine) in aqueous solution and poly(L-alanine) in dichloracetic acid display both bands, but alpha-helical poly(l-glutamic acid) in aqueous solution and poly(gamma-benzyl L-glutamate) in CHCl(3) display only the approximately 1340 cm(-1) band and so may exist purely as alpha(o) due to enhanced stabilization of this conformation by particular side chain characteristics. The ROA spectrum of poly(beta-benzyl L-aspartate) in CHCl(3) reveals that it exists in a single left-handed alpha-helical state more analogous to alpha(o) than to alpha(c).     

Pattern formation of antifreeze glycoproteins via solvent evaporation

Surface patterning of antifreeze glycoprotein fraction 8 (AFGP 8) via a solvent evaporation method is reported here. In this process, lines of AFGP 8 particles and gridlike patterns were formed as as result of the receding of the droplet contact line and the accumulation of the solute during evaporation. The solution concentration strongly affects the protein line spacing. The average height of the protein was measured to be 8.1 +/- 2.5 A, which may be attributed to the height of a single molecule.     

Mechanistic study of proton transfer and HD exchange in ice films at low temperatures (100-140 K)

We have examined the elementary molecular processes responsible for proton transfer and HD exchange in thin ice films for the temperature range of 100-140 K. The ice films are made to have a structure of a bottom D(2)O layer and an upper H(2)O layer, with excess protons generated from HCl ionization trapped at the D(2)OH(2)O interface. The transport behavior of excess protons from the interfacial layer to the ice film surface and the progress of the HD exchange reaction in water molecules are examined with the techniques of low energy sputtering and Cs(+) reactive ion scattering. Three major processes are identified: the proton hopping relay, the hop-and-turn process, and molecular diffusion. The proton hopping relay can occur even at low temperatures (<120 K), and it transports a specific portion of embedded protons to the surface. The hop-and-turn mechanism, which involves the coupling of proton hopping and molecule reorientation, increases the proton transfer rate and causes the HD exchange of water molecules. The hop-and-turn mechanism is activated at temperatures above 125 K in the surface region. Diffusional mixing of H(2)O and D(2)O molecules additionally contributes to the HD exchange reaction at temperatures above 130 K. The hop-and-turn and molecular diffusion processes are activated at higher temperatures in the deeper region of ice films. The relative speeds of these processes are in the following order: hopping relay>hop and turn>molecule diffusion.     

Cooperative hydration of pyruvic acid in ice

About 3.5 +/- 0.3 water molecules are still involved in the exothermic hydration of 2-oxopropanoic acid (PA) into its monohydrate (2,2-dihydroxypropanoic acid, PAH) in ice at 230 K. This is borne out by thermodynamic analysis of the fact that QH(T) = [PAH]/[PA] becomes temperature independent below approximately 250 K (in chemically and thermally equilibrated frozen 0.1 < or = [PA]/M < or = 4.6 solutions in D2O), which requires that the enthalpy of PA hydration (DeltaHH approximately -22 kJ mol(-1)) be balanced by a multiple of the enthalpy of ice melting (DeltaHM = 6.3 kJ mol(-1)). Considering that: (1) thermograms of frozen PA solutions display a single endotherm, at the onset of ice melting, (2) the sum of the integral intensities of the 1deltaPAH and 1deltaPA methyl proton NMR resonances is nearly constant while, (3) line widths increase exponentially with decreasing temperature before diverging below approximately 230 K, we infer that PA in ice remains cooperatively hydrated within interstitial microfluids until they vitrify.     

Vertical diffusion of water molecules near the surface of ice

We studied diffusion of water molecules in the direction perpendicular to the surface of an ice film. Amorphous ice films of H(2)O were deposited on Ru(0001) at temperature of 100-140 K for thickness of 1-5 bilayer (BL) in vacuum, and a fractional coverage of D(2)O was added onto the surface. Vertical migration of surface D(2)O molecules to the underlying H(2)O multilayer and the reverse migration of H(2)O resulted in change of their surface concentrations. Temporal variation of the H(2)O and D(2)O surface concentrations was monitored by the technique of Cs(+) reactive ion scattering to reveal kinetics of the vertical diffusion in depth resolution of 1 BL. The first-order rate coefficient for the migration of surface water molecules ranged from k(1)=5.7(+/-0.6) x 10(-4) s(-1) at T=100 K to k(1)=6.7(+/-2.0) x 10(-2) s(-1) at 140 K, with an activation energy of 13.7+/-1.7 kJ mol(-1). The equivalent surface diffusion coefficients were D(s)=7 x 10(-19) cm(2) s(-1) at 100 K and D(s)=8 x 10(-17) cm(2) s(-1) at 140 K. The measured activation energy was close to interstitial migration energy (15 kJ mol(-1)) and was much lower than diffusion activation energy in bulk ice (52-70 kJ mol(-1)). The result suggested that water molecules diffused via the interstitial mechanism near the surface where defect concentrations were very high.