Vibrational density of states of hydration water at biomolecular sites: hydrophobicity promotes low density amorphous ice behavior

Inelastic neutron scattering experiments and molecular dynamics simulations have been used to investigate the low frequency modes, in the region between 0 and 100 meV, of hydration water in selected hydrophilic and hydrophobic biomolecules. The results show changes in the plasticity of the hydrogen-bond network of hydration water molecules depending on the biomolecular site. At 200 K, the measured low frequency density of states of hydration water molecules of hydrophilic peptides is remarkably similar to that of high density amorphous ice, whereas, for hydrophobic biomolecules, it is comparable to that of low density amorphous ice behavior. In both hydrophilic and hydrophobic biomolecules, the high frequency modes show a blue shift of the libration mode as compared to the room temperature data. These results can be related to the density of water molecules around the biological interface, suggesting that the apparent local density of water is larger in a hydrophilic environment.     

Temperature and length scale dependence of tetraalkylammonium ion-amide interaction

We have studied the temperature and length scale dependence of the energetics of the pair interaction of well-established hydrophobic solutes tetraalkylammonium bromides with hydrophilic formamide (FA) and hydrophobic hexamethylphosphoric triamide (HMPT). Our results do indicate the anomalous length scale dependence of the tetraalkylammonium cation-amide interaction in water. As the cation size is increased, the unfavorable enthalpy of interaction is increased rather linearly until the maximum is reached, after which there appears to be a reversal of the trend. We believe that this phenomenon arises from the impossibility of water to maintain its H-bond network near large tetraalkylammonium cations that leads to the formation of a somewhat disordered solute hydration shell. The energetic cost for overlapping this shell with the amide hydration shell in water is noticeably smaller than that for tetraalkylammonium cations of a moderated size.     

Electrospray ionization mass spectrometry of terrestrial humic substances and their size fractions

Electrospray ionization mass spectrometry (ESI-MS) was used to evaluate the average molecular mass of terrestrial humic substances, such as humic (HA) and fulvic (FA) acids from a soil, and humic acid from a lignite (NDL). Their ESI mass spectra, by direct infusion, gave average molecular masses comparable to those previously obtained for aquatic humic materials. The soil HA and FA were further separated in size-fractions by preparative high performance size exclusion chromatography (HPSEC) and analyzed with ESI-MS by both direct infusion and a further on-line analytical HPSEC. Unexpectedly, their average molecular mass was only slightly less than for the bulk sample and, despite different nominal molecular size, did not substantially vary among size-fractions. The values increased significantly (up to around 1200 Da) after on-line analytical HPSEC for the HA bulk sample, at both pH 8 and 4, and for the HA size-fractions when pH was reduced from 8 to 4. It was noticed that HA size-fractions at pH 8 were separated by on-line HPSEC in further peaks showing average masses which progressively increased with elution volume. Furthermore, when the HA and NDL bulk samples were sequentially ultracentrifuged at increasing rotational speed, their supernatants showed mass values which were larger than bulk samples and increased with rotational speed. These variations in mass values indicate that the electrospray ionization is dependent on the composition of the humic molecular mixtures and increases when their heterogeneity is progressively reduced. It is suggested that the dominance of hydrophobic compounds in humic supramolecular associations may inhibit the electrospray ionization of hydrophilic components. Our results show that ESI-MS is reasonably applicable to humic substances only after an extensive reduction of their chemical complexity.     

Trace humic and fulvic acid determination in natural water by cloud point extraction/preconcentration using non-ionic and cationic surfactants with FI-UV detection

A preconcentration and determination method for humic and fulvic acids at trace levels in natural water samples was developed. Cloud point extraction was successfully employed for the preconcentration of humic acid (HA) and fulvic acid (FA) prior to the determination by using a flow injection (FI) system coupled to a spectrophotometric UV-Vis detector. The quantitative extraction of HA and FA within the pH range 1-12 was obtained by neutralization of the anionic charge on the humic substances with a cationic surfactant, hexadecyltrimethylammonium bromide (CTAB). This generated a hydrophobic species that was subsequently incorporated (solubilized) into the micelles of a non-ionic surfactant polyethylene glycol, tert-octylphenyl ether (Triton X-114). The FI method for HA and FA determination was developed by injection of 100 microl of the extracted surfactant-rich phase using an HPLC pump with spectrophotometric detection at 350 nm. A 50 ml sample solution preconcentration allowed an enrichment factor of 167. The limit of detection (LOD) obtained under the optimal conditions was 5 microg l(-1). The precision for ten replicate determinations at 0.2 mg l(-1) HA was 3.1% relative standard deviation (RSD), calculated from the peak heights. The calibration using the preconcentration system for HA and FA was linear with a correlation coefficient (r2) of 0.9997 at levels near the detection limits up to at least 1 mg l(-1). The method was successfully applied to the determination of HA and FA in natural water samples (river water).     

Lignite humic acids aggregates studied by high resolution ultrasonic spectroscopy

The thermodynamic stability of lignite humic acids (sodium salt) aggregates was studied by high resolution ultrasonic spectroscopy within the temperature interval from 5 to 90°C. The changes in differential ultrasonic velocity (U12) showed strong differences among humic solutions within the concentration range from 0.005 to 10 g L⁻¹. Measurement revealed several transitions which were attributed to the weakening of humic secondary structure. Concentration around 1 g L⁻¹ seemed to be a limit under which the change of the prevalence and importance of hydration occurred. Above this concentration the difference in U12 decreased following the temperature increase which was explained as a dominance of hydrophilic hydration. In contrast, below this concentration, the temperature dependence of U12 resulted in increasing tendency which was attributed to the prevalence of hydrophobic hydration, i.e. uncovering of apolar groups towards surrounding water. Additional experiments in which the humic sample was modified by hydrochloric acid resulted in a slight structural stabilization which lead to the conclusion that humic micelle-like subaggregates form an open-layer assemblies easily accessible for interaction with an extraneous molecule. That was partly verified by addition of propionic acid which brought about even larger reconformation of humic aggregates and exhibition of polar groups towards hydration water. The reversible changes in humate solutions induced by elevated temperatures provided the evidence about the existence of significant physical interactions among humic molecules resulting in formation of various kinds of aggregates. The nature of aggregates, mainly the stability and conformation, strongly depends on the concentration. Evidently, the changes observed in this work cannot be simply explained as expansions or conformational changes of macromolecular coils.     

Thermodynamical properties of reaction intermediates during apoplastocyanin folding in time domain

Two intermediates observed for the folding process of apoplastocyanin (apoPC) were investigated by using a photoinduced triggering system combined with the transient grating and transient lens methods. The thermodynamic quantities, enthalpy, heat capacity, partial volume, and thermal expansion volume changes during the protein folding reaction were measured in time domain for the first time. An interesting observation is the positive enthalpy changes during the folding process. This positive enthalpy change must be compensated by positive entropy changes, which could be originated from the dehydration effect of hydrophobic residues and/or the translational entropy gain of bulk water molecules. Observed negative heat capacity change was explained by the dehydration effect of hydrophilic residues and/or motional confinement of amino acid side chains and water molecules in apoPC. The signs of the volume change and thermal expansion volume were different for two processes and these changes were interpreted in terms of the different relative contributions of the hydration and the dehydration of the hydrophilic residues. These results indicated two-step hydrophobic collapses in the early stage of the apoPC folding, but the nature of the dynamics was different.     

Effects of acid rain on soil humic compounds

The modifications induced by acid rain on the solubility, molecular configuration and molecular weight distribution of humic (HA) and fulvic (FA) acids were studied. A natural soil was subjected to simulated acid rain until a soil pH of 4 was obtained; HA and FA acids were then extracted and characterised. The results obtained were compared both with those of natural soil and with those of a soil subjected to acid rain. Elute analysis indicates the continuous release of soluble organic compounds as a consequence of acid rain simulation, although no relationship was found with the process of soil acidification. The yields of HA and FA show that HA values are the same while FA amount is higher in the natural soil; in acid soils their water solubility increases. The molecular weight distribution shows that HA consist of a mixture of compounds of different molecular weights; they are molecules for the most part larger than 100 kDa and their distribution is not changed by soil acidification. FA can be considered to form a much more homogeneous system; in natural soil, the molecules are larger than 50 kDa, while in acidified soil they are for the most part smaller than 3 kDa.     

Fluorescence analysis of humic and fulvic acids from two Brazilian oxisols as affected by biosolid amendment

Conventional monodimensional fluorescence spectroscopy in the emission, excitation, and synchronous-scan modes and total luminescence spectroscopy have proven to be sensitive techniques for characterization and differentiation of humic acid (HA) and fulvic acid (FA) fractions isolated from an aerobically and anaerobically digested and limed biosolid, two layers of a sandy and a clayey Brazilian oxisol, and the corresponding biosolid-amended soils. The spectral patterns and the relative fluorescence intensities suggest greater molecular heterogeneity, less aromatic polycondensation, and less humification of biosolid HA and FA compared with soil HA and FA. However, the differences are smaller for the FA fractions than for the HA fractions. Fluorescence properties of soil HA and FA differ slightly as a function of soil type and soil layer. Biosolid application causes a shift to shorter wavelengths of the main fluorescence peaks and marked variation of the relative fluorescence intensities of HA and FA isolated from amended soils. These results suggest that molecular components of relatively small molecular size, with a low level of aromatic polycondensation, and low degree of humification present in biosolid HA and FA are partially and variously incorporated into amended soil HA and FA. In general, these modifications seem to be smaller in HA and FA from the clayey soil layers than in those from the sandy soil layers, possibly because of protective effects exerted by clay minerals of native soil HA and FA against disturbances caused by biosolid application.     

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.     

Potential of capillary zone electrophoresis for estimation of humate acid-base properties

Capillary zone electrophoresis (CZE) has been applied for fractionation and characterization of soil-derived humic acids (HAs). Humic acids from soddy-podzolic (HA(s)) and chernozem (HA(ch)) soils were studied as well as hydrophobic high-molecular-weight (HMW) and hydrophilic low-molecular-weight (LMW) HA(s) fractions obtained by salting-out with ammonium sulfate at a saturation of 0-40% and >70%, respectively. The possibility of CZE partial fractionation of HAs has been demonstrated. The shape of "humic hump" was shown to depend on the pH of running electrolyte. Almost the whole peak overlapping occurred if alkaline solutions were used for fractionation, but the peak resolution was improved at pH 5-7. Under appropriate fractionation conditions (pH 7), at least three humic acid subfractions with different electrophoretic mobilities were distinguished in the electropherograms of initial HA and HA(s) fractions. Such a high peak resolution has never been achieved for humic acids before. The presence of three subfractions in the HA is in agreement with gel-filtration analysis and was confirmed by comparison of the electrophoretic behavior of HA(s) with those of its HMW (hydrophobic) and the LMW (hydrophilic) fractions. The potentiometric titration of HA and its fractions was performed and the pK(a) of the functional groups were calculated. An attempt was made for the first time to relate the variation of electrophoretic mobility values with acid-base properties of humic acids. It was shown that changes in the humate charge resulting from the variation of the ionization degree of its functional groups as a function of pH can be estimated on the basis of electrophoretic mobility values. Potential of CZE in estimation of HA isoelectric point was demonstrated. The pH value corresponding to the lowest absolute electrophoretic mobility value of about 20 x 10(-5) cm(2) V(-1) s(-1) can be used for approximate estimation of HA isoelectric point. The data were discussed and agreement with the random coil structural model has been shown.     

Hydration and drying of various polysaccharides studied using DSC

The hydration of cellulose, chitosan, schizophyllan, hyaluronan, and carboxymethyl cellulose was studied using differential scanning calorimetry (DSC). In the first part, the classical freezing/thawing approach was used to determine the amount of non-freezing water. The inconsistency in enthalpies obtained during crystallization and melting of freezable water was discussed with respect to the DSC experimental conditions. Our interpretation questions the recent conclusions about competitive processes occurring during melting which are hypothesized to influence the determined melting enthalpy. In the second part, the hydration and drying were studied using the evaporation enthalpy of water. The dry mass normalized dependency of vaporization enthalpy on water content confirmed an abrupt break at low water content in hyaluronan sample which was attributed to the sudden appearance of a parallel process taking part during the drying. The rest of polysaccharide samples showed only a linear decrease in evaporation enthalpy. The renormalization of enthalpies by the water content revealed the increase in evaporation enthalpy with decreasing water content in most samples which was ascribed to the strong interaction between polysaccharide and water. The exceptions were carboxymethyl cellulose which showed a decrease in evaporation enthalpy. This indicates the existence of a simultaneous process occurring during drying, but unlike in hyaluronan, the processes do not appear abruptly but accompany the evaporation in the wide concentration range. Comparison of determined hydration numbers showed that part of non-freezing water in hyaluronan is not bound to sorption sites but occurs presumably in small temporary pores. In contrast, water-soluble schizophyllan forms temporary pores as well but presumably with higher dimension and the non-freezing water is formed mostly by water molecules interacting with sorption sites.     

Hydration of Cl<Superscript>–</Superscript> ion in a planar nanopore with hydrophilic walls. 2. Thermodynamic stability

The Monte Carlo bicanonical statistical ensemble method has been employed to calculate the dependences of the Gibbs free energy, formation work, and entropy on the size of a hydration shell grown from water vapor on single-charged chlorine anion in a model planar nanopore with hydrophilic structureless walls at 298 K. A refined model comprising many-particle polarization interactions and calibrated with respect to experimental data on the free energy and enthalpy of the initial reactions of attachment of water molecules to the ion has been used. It has been found that a weak hydrophilicity of pore walls leads to destabilization of the hydration shell, while a strong one, on the contrary, causes its stabilization. The physical reason for the instability in the field of hydrophilic walls qualitatively differs from that under the conditions of hydration in bulk water vapor.     

Effect of natural organic matter on aggregation behavior of C60 fullerene in water

The stability of C(60) fullerene particles in water affects its mobility, bioavailability, and toxicity to organisms. Natural organic matters (NOMs) have pronounced effects on the aggregation behavior of C(60) fullerene. This study was to examine the effects of NOM structural properties on the aggregation behavior of fullerene water suspension (FWS). Fulvic acid (FA), tannic acid (TA), and two structurally different humic acids (HA1 and HA7) were studied. HA1 and HA7 were sequentially extracted HAs, where HA7 was more hydrophobic than HA1 and had a higher molecular weight. Aggregation was induced by addition of varying amounts of Ca(2+) to the FWS with 2 mg/L of each NOM. The absolute value of zeta potential |ζ| of pure FWS increased after addition of any type of NOM. Addition of Ca(2+) to the FWS+NOM system decreased |ζ| of fullerene almost uniformly for all types of NOM. FWS critical coagulation concentration (CCC) was equal to 14.5, 6.5, 5.4, and 3.7 mM Ca(2+) for HA7, HA1, FA, and TA, respectively. The order of increasing CCCs was positively correlated to the NOMs molecular weight and negatively to their polarity. A nearly constant ζ for FWS+NOM system at a wide range of Ca(2+) concentrations suggested the steric stability rather than electrostatic one. This study highlighted the role of NOM in the fate of manufactured nanoparticles in the environment and linked the structural properties of NOM to their interaction with manufactured nanoparticles.     

Hydrophobic contribution of amino acids in peptides measured by hydrophobic interaction chromatography

The adsorption behaviors of amino acids in short chain peptides were examined. Each amino acid, aliphatic or charged, was inserted between the two tryptophans of a peptide, GWWG. The capacity factors of these peptides on an Ocytl-Sepharose column were measured. The adsorption enthalpies, entropies, and the number of repelled water molecules after adsorption were estimated to analyze the contribution of each different amino acid to its hydrophobic adsorption. The peptides inserted with aliphatic amino acids owned the highest capacity factors but released the least amount of adsorption heat among all the peptides under examination. It was found that the hydrophobic contribution of aliphatic amino acids was derived from the entropy gain by repelling the ordered water surrounding them. The insertion of negatively charged amino acids greatly reduced the capacity factors but still repelled a significant number of water molecules after adsorption. This indicated that the water molecules surrounding ionic amino acids were not orderly aligned. The dehydration cost energy but the water repelling did not offer enough entropy to drive the adsorption. Subsequently, lower retention was obtained from the peptides inserted with negatively charged ionic amino acids. The insertion of lysine increased the adsorption enthalpy but repelled no water molecules after adsorption. It was speculated that the inserted lysine still interacted with hydrophobic ligands but disturbed the interaction between ligands and adjacent tryptophans. Therefore, the adsorption enthalpy increased and the capacity factors decreased. Different amino acids contributed to hydrophobic interaction in different ways. The simultaneous analysis of capacity factor, adsorption enthalpy, adsorption entropy, and the number of repelled water molecules facilitated the understanding of the adsorption processes.     

Water hydrogen bond analysis on hydrophilic and hydrophobic biomolecule sites

Elastic and quasielastic neutron scattering experiments have been used to investigate the hydrogen bonding network dynamics of hydration water on hydrophilic and hydrophobic sites. To this end the evolution of hydration water dynamics of a prototypical hydrophobic amino acid with polar backbone, N-acetyl-leucine-methylamide (NALMA), and hydrophilic amino acid, N-acetyl-glycine-methylamide (NAGMA), has been investigated as a function of the molecular ratio water : peptide. The results suggest that the dynamical contribution of the intrinsic and low hydration molecules of water is characteristic of pure librational/rotational movement. The water molecule remains attached to the hydrophilic site with only the possibility of hindered rotations that eventually break the bond with the peptide and reform it immediately after. A gradual evolution from librational motions to hindered rotations is observed as a function of temperature. When the hydration increases, we observe (together with the hindered rotations of hydrogen bonds) a slow diffusion of water molecules on the surface of the peptides.     

Entrapment of Humic Acid in a Sol-Gel Matrix—A New Tool for Sorption Studies

Humic substances are natural complexed mixtures of organic compounds originated from the decomposition of plant and animal residues. These compounds are ubiquitous in soils, sediments, surface waters and groundwaters. They contain both hydrophobic and hydrophilic moieties, able to interact with hydrophobic organic contaminants and with heavy metals. These sorption interactions play a crucial role in contaminants fate and transport and their understanding and quantification are essential for modeling and predictions. However, sorption analyses frequently suffer from experimental problems. A novel idea presented in this study is to use sol-gel as an inert matrix to immobilize (entrap) specific, well defined, humic molecules which then be used in sorption studies. We developed a successful procedure for the immobilization of humic acid (HA) in a sol-gel matrix. After gelation and drying, the doped gel was crushed and washed several times, yielding a very stable product. It was then used in a series of batch experiments, studying the sorption of several polycyclic aromatic hydrocarbons (PAHs) with Aldrich HA. The sorption coefficients (K _oc) obtained with the immobilized HA were highly correlated with the values expected based on the hydrophobicity of the contaminants. We concluded that the entrapped HA retained its original properties and that it was accessible to the external contaminants through the pore network.     

Limitations of electrospray ionization in the analysis of a heterogeneous mixture of naturally occurring hydrophilic and hydrophobic compounds

A model heterogeneous mixture of a hydrophilic tripeptide (phenylalanine-glycine-glycine, PGG) and hydrophobic organic acids ((12)C- and (13)C-octanoic acid and pentadecanoic acid) was subjected to electrospray ionization mass spectrometry (ESI-MS). The objective was to verify the previously noted inconsistencies in ESI-MS of complex environmental samples such as humic materials from either aquatic or terrestrial origins. The hydrophobic organic acids, either alone or together, reduced significantly the ESI-MS detection of the tripeptide molecular and self-associated ions at a concentration that was an order of magnitude lower than that of PGG. The most intense peaks were invariably those of the octanoic acid as either deprotonated, self-associated, or acetate-clustered molecules. The presence of equimolar amounts of PGG and organic acids yielded similar results, but with a significant increased detection of PDA and a smaller depression of the PGG signals. This behaviour is attributed to a different electrospray ionization of the mixture compounds depending on their most probable positioning at the surface of the evaporating droplet. The most favoured positioning of hydrophobic molecules at the aqueous-gas interphase allows preferential evaporation of hydrophobic ions whereas the hydrophilic molecules are retained in the droplet interior, and, their ESI-MS detection depressed. These findings suggest that the electrospray ionization of different molecules present in complex heterogeneous mixtures of environmental significance such as humic substances is limited by their concentration and reciprocal attracting forces.     

Estimation of the hydration of polar groups of α-amino acids by differential scanning calorimetry

The heat capacity of hydration of zwitterions derived from aliphatic amino acids depends linearly on the surface area of the amino acid side radicals accessible to water molecules with the slopeb = 2.35±0.11 J mol^–1 K^–1 Å^–2 at 298 K. The linear correlation between hydration heat capacities of zwitterions of aliphatic amino acids and the corresponding aliphatic alcohols with a coefficient of approximately unity confirms the assumption that hydrophobic hydration does not depend on the nature of the surrounding groups. Using the assumption that the hydration of hydrocarbon radicals is independent of the neighboring groups, theb value has been used to calculate the contributions of polar groups. The contributions of OH, COON, and CONH groups of the side radicals in polar amino acids in the zwitterion form are close to zero; in the case of organic nonionic molecules, these contributions are negative. The increments for polar groups obtained for the zwitterions can be used for the calculation of the heat capacities of proteins and polypeptides incorporating charged amino acid residues. The difference between hydrophilic and hydrophobic hydration mechanisms is manifested not only as different magnitudes and signs of heat capacities and temperature coefficients but also in the fact that the neighboring polar (charged) groups have an effect on hydrophilic hydration but have no effect on hydrophobic hydration.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 2237–2242, September, 1996.     

The structural composition of humic compounds as indicator of organic carbon sources

Various analytical techniques ((13)C-NMR, FTIR, elemental analysis) have been used to study the structures of humic compounds present in the sediments of Martignano lake located 50 km from Roma city (Italy). The total amount of humic compounds present in the upper layer of sediments is practically constant; instead, considering humic (HA) and fulvic acids (FA) separately, it can be noticed that while HA increase remarkably from A(1) to A(3), FA diminish, probably as a result of the continuous transformation of algal debris, present in large amount in A(1), from compounds which are in an early stage of decomposition to more highly degraded organic matter. In deeper layers the percentage both of HA and FA is constant and slightly lower than that found in the upper layer, except for two sampling points. In these stations the amount of both HA and FA are very high and the carbohydrate peak areas of HA and FA increase along with the depth, reflecting the presence of a well localized source of organic carbon or different environmental conditions.     

A new angle on heat capacity changes in hydrophobic solvation

The differential solubility of polar and apolar groups in water is important for the self-assembly of globular proteins, lipid membranes, nucleic acids, and other specific biological structures through hydrophobic and hydrophilic effects. The increase in water's heat capacity upon hydration of apolar compounds is one signature of the hydrophobic effect and differentiates it from the hydration of polar compounds, which cause a decrease in heat capacity. Water structuring around apolar and polar groups is an important factor in their differential solubility and heat capacity effects. Here, it is shown that joint radial/angular distribution functions of water obtained from simulations reveal quite different hydration structures around polar and apolar groups: polar and apolar groups have a deficit or excess, respectively, of "low angle hydrogen bonds". Low angle hydrogen bonds have a larger energy fluctuation than high angle bonds, and analysis of these differences provides a physical reason for the opposite changes in heat capacity and new insight into water structure around solutes and the hydrophobic effect.