Adsorption properties of proteins at and near the air/water interface from IRRAS spectra of protein solutions

A detailed study is performed using infrared reflection absorption spectroscopy (IRRAS) to characterize the molecular behaviour of proteins at and near the air/water interface of protein solutions. IRRAS spectra of beta-casein solutions in H2O and D2O show spectral shifts and derivative-like features not commonly observed in monomolecular layer systems. They can be fully understood using optical theory. Fair agreement between experimental and simulated IRRAS spectra over a broad spectral range (4000-1000 cm(-1)) is obtained using a stratified layer model. An attenuated total reflection and transmission spectrum is used to represent the protein extinction coefficient in H2O and D2O, respectively. It is shown that the derivative-like features observed result from the reflective properties of the proteins themselves. Furthermore, both concentration and film thickness could be fitted. At high protein concentrations (100 mg/mL) the spectrum is that of a single homogeneous protein solution. At 0.1 mg/mL, beta-casein is accumulated at the surface in a thin layer of approximately 10 nm thickness, with a concentration about 2500 times higher than in the sub-phase. At an initial concentration of 10 mg/mL, the concentration in the surface layer is about 15 times higher than in the subphase, while the thickness is about 30 nm.     

Effect of weak magnetic fields on fluorescence of water and water-salt solutions. Partial characterization of fluorescing fractions

It has been shown that exposure to weak combined permanent (42 mT) and low-frequency (3-60 Hz) alternating (0.001-0.06 mT) magnetic fields changes the intensity of fluorescence of water and water-salt solutions. The gel filtration of solutions of inorganic salts treated with magnetic fields gives rise to intensively fluorescing fractions. Control solutions not exposed to electromagnetic waves do not exhibit these effects. The results obtained suggest that treatment with weak electromagnetic fields induces structural changes of water solutions, and the manifestations of these changes depend on the conditions of chromatography and chemical composition of solutions under study.     

The study of the role of the water medium in the mechanism of action of peptides in low and ultra low doses

The correlation between the structures and conformations of short peptides KE, EW, AEDG and other, their influence on the dynamic properties of water and dose/biologic effect dependencies in a wide range of concentrations were regarded. Their effects on the dynamic properties of water were studied by temperature dependencies (5-45 degrees C) of infrared spectra of the solutions in the near (5180 cm-1) and far (200 cm-1). In vitro biotesting included the determination of the proliferative activity of thymocytes, a bimodal curve with the second maximum were detected at super-low doses (10(-17)-10(-15) mol/l). Authors propose a hypothesis that for superlow concentrations the formation and distance transmission of a signal from ligand to a target cell without the formation of any ligand-receptor complex take place. An active role in this model belongs to water medium acting according to the solution mechanism.     

Studies on the physical state of water in living cells and model systems. III. The high osmotic activities of aqueous solutions of gelatin, polyvinylpyrrolidone and poly (ethylene oxide) and their relation to the reduced solubility for NA+, sugars, and free amino acids

Very high osmotic activities of concentrated aqueous solutions of gelatin, polyvinylpyrrolidone, and poly(ethylene oxide) were recorded. These observed values are far above those predictable from the molar concentrations of these polymers or those of aqueous solutions of native hemoglobin of equal concentrations. It was shown that these high osmotic activities are closely associated with the ability of the gelatin- or polymer-dominated water to exclude Na+ salts, sucrose, and glycine. Both phenomena are interpreted as reflecting the polarization of multilayers of water by the polymers enhancing the H2O to H2O interaction and also reducing the translational and rotational motional freedom of the water.     

Relaxation of water protons in highly concentrated aqueous protein systems studied by 1H NMR spectroscopy

Concentrated Aqueous Protein Systems, Proton Relaxation Times, Slow Chemical Exchange In this paper we present proton spin-lattice (T1) and spin-spin (T2) relaxation times measured vs. concentration, temperature, pulse interval (tauCPMG) as well as 1H NMR spectral measurements in a wide range of concentrations of bovine serum albumin (BSA) solutions. The anomalous relaxation behaviour of the water protons, similar to that observed in mammalian lenses, was found in the two most concentrated solutions (44% and 46%). The functional dependence of the spin-spin relaxation time vs. tauCPMG pulse interval and the values of the motional activation parameters obtained from the temperature dependencies of spin-lattice relaxation times suggest that the water molecule mobility is reduced in these systems. The slow exchange process on the T2 time scale is proposed to explain the obtained data. The proton spectral measurements support the hypothesis of a slow exchange mechanism in the highest concentrated solutions. From the analysis of the shape of the proton spectra the mean exchange times between bound and bulk water proton groups (tauex) have been estimated for the range of the highest concentrations (30%-46%). The obtained values are of the order of milliseconds assuring that the slow exchange condition is fulfilled in the most concentrated samples.     

The gastrin-secreting effects of the salt components of naftusia water]

Intragastric administration of certain salt components of mineral water naftusia in minimal concentrations (1-5 mmol/l) changes intensity and direction of hypergastrinemic reaction in the rats. The effect is determined by the anionic composition of salts rather than by the cationic one and depends on the initial concentration of gastrin in blood.     

A CD study of the role of metal ions in the conformation of poly(L-lysine)

The effect of LiCl, NaCl, and CsCl as univalent salts, and of CaCl₂, ZnCl₂, and MgCl₂ as divalent salts, on the α and antiparallel β-sheet, and random conformations of poly(L-lysine) (PLL), in water at room temperature were examined by means of CD and compared quantitatively on the basis of elliptical strength at the maximal peak. Changes in the α-helical and antiparallel β-sheet helical conformations of PLL were markedly dependent on the salt concentrations of LiCl, NaCl, and CsCl, which induced decreases in negative intensity in that order. The CD spectrum of the random conformation, the most disordered form, displayed positive cotton effect in concentrations of these salts up to 3.0M and a negative peak in concentrations of 6.0M. The effect of these salts on the random conformation of PLL was stronger than that on the α- and β-conformations in higher concentrations. The CD spectrum of the random conformation in the presence of CaCl₂, ZnCl₂, and MgCl₂, on the other hand, showed negative cotton effect in salt concentrations as low as 3.0M. It was impossible, however, to measure the effect on α- and β-conformations of ZnCl₂ and MgCl₂ above concentrations of 10 mM because of a solubility problem with salts in alkaline solution.     

Surface-enhanced Raman spectroscopy of biopolymers: membrane proteins, bacteriorhodopsin and rhodopsin adsorbed on silver electrodes and silver hydrosols

Surface-enhanced Raman (SER) spectra of purple membranes of Halobacterium halobium and photoreceptor disks of the rod outer segments adsorbed on silver hydrosols were analysed. It has been shown that the intensity of SER spectra of bacterial and visual rhodopsins increases 5 X 10(4) times at adsorption. Concentration relationship of the signal intensity of SER spectra has the maximum at bacteriorhodopsin concentration about 2 X 10(-7) M. It has been shown that adsorption on silver hydrosol leads to fixation of light-induced photochemical transformations in bacterial and visual rhodopsins. Adsorption on the "smooth" electrodes at the potential of the zero charge of silver does not affect the photocycle of bacteriorhodopsin. An increase or decrease of the electrode potential relative to the zero charge point of silver leads to the accumulation of kinetic intermediate K610 and a decrease of the concentration of the form BRh570. It has been shown that on the "smooth" electrode primarily the long-range component of the SER mechanism is realized. Bands corresponding to the vibrations of the atom groups directly contacting with the metal are mainly intensified after redox cycle which increases the concentration of chemosorption centres. A conclusion is drawn that the method of SER spectroscopy of biomolecules adsorbed on "smooth" electrodes, permits obtaining information similar to that obtained from the analysis of Raman spectra of unadsorbed molecules, but at concentrations by two orders less. Adsorption on the electrodes treated with the help of redox cycle permits to obtain highly oriented preparations and to study topography of biopolymers in water solutions and suspensions.     

Effect of salt solutions on the radiosensitivity of mammalian cells as a function of the state of adhesion and the water structure.

The radiation isodose survival curve of attached Chinese hamster (V79) cells, subjected to a wide concentration range of salt or sucrose solutions, is characterized by two maxima separated by a minimum. Cells are radioprotected at the maxima (high and low hypertonic salt concentrations) while they are radiosensitized at the minimum (intermediate hypertonic salt concentrations). Both cations and anions can alter the cellular radiosensitivity above and beyond the (osmotic) effect observed for cells treated with sucrose solutions. However, the basic curve shape, except in the case of sulphate salts, remains the same. When these experiments are repeated with single cells in suspension, the isodose survival curve is quite different in that high salt concentrations (greater than 0.9 M) do not protect cells in suspension unlike the case with attached cells. The curve shape is also altered in that the second maximum is absent with many salt solutions. If multicellular spheroids are used for these experiments, the data resemble those for single cell suspensions rather than for attached cells. The radiation survival data for cells in suspension in salt solutions correlate with water proton spin-lattice relaxation time (T1) and, in hypo- and iso-tonic solutions, with cell volume.     

Heterogeneity in desiccated solutions: implications for biostabilization

Biopreservation processes such as freezing and drying inherently introduce heterogeneity. We focused on exploring the mechanisms responsible for heterogeneity in isothermal, diffusively dried biopreservation solutions that contain a model protein. The biopreservation solutions used contained trehalose (a sugar known for its stabilization effect) and salts (LiCl, NaCl, MgCl₂, and CaCl₂). Performing Fourier transform infrared spectroscopy analysis on the desiccated droplets, spatial distributions of the components within the dried droplet, as well as their specific interactions, were investigated. It was established that the formation of multiple thermodynamic states was induced by the spatial variations in the cosolute concentration gradients, directly affecting the final structure of the preserved protein. The spatial distribution gradients were formed by two competing flows that formed within the drying droplet: a dominant peripheral flow, induced by contact line pinning, and the Marangoni flow, induced by surface tension gradients. It was found that the changes in cosolute concentrations and drying conditions affected the spatial heterogeneity and stability of the product. It was also found that trehalose and salts had a synergistic stabilizing effect on the protein structure, which originated from destructuring of the vicinal water, which in turn mediated the interactions of trehalose with the protein. This interaction was observed by the change in the glycosidic CO, and the CH stretch vibrations of the trehalose molecule.     

Low-frequency Raman spectra in water and in weak aqueous solutions. Spatial inhomogeneity in hydrogen peroxide solution

We describe some examples of the study of intermolecular interactions in water and in weak aqueous solutions using the low-frequency Raman spectra. An improved method of determining parameters of the dynamic susceptibility from the Raman spectra is described. The nine spectral parameters (the intensity, the frequency and the width of the two oscillators, the intensity and the width of the relaxation motion and the intensity of fluorescent background) completely describe the spectrum in the frequency range of 4 to 320 cm^?1. For hydrogen peroxide the dependences of these parameters on the concentration is shown. Concentration dependence of the spectral parameters of the hydrogen peroxide solution in water indicates a change in the structure of the molecular bonds in water near a peroxide molecule at a distance up to 0.7–0.9 nm. The effect of the exciting laser radiation on the parameters of the spectra in the registration of the Raman spectra of water is shown.     

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.     

The solution conformation of poly(L-lysine). A Raman and infrared spectroscopic study.

The Raman and infrared spectra of poly(L -lysine) and poly(DL -lysine) in solution are reported and the effects of various salts are investigated. The results demonstrate that α-helix formation in solution is induced by specific salts and the spectral data support the hypothesis of regions of local order for poly(L -lysine) in aqueous solutions of low ionic strength.     

Digestive parameters and water turnover of the leopard tortoise

Leopard tortoises (Stigmochelys pardalis) experience wide fluctuations in environmental conditions and unpredictable availability of food and water within the Nama-Karoo biome. It was hypothesised that tortoises fed two diets differing in preformed water and fibre content would have differing food intake, gut transit rate, assimilation efficiency, faecal and urinary water loss, and urine concentrations. It was predicted that tortoises fed these contrasting diets would attempt to maintain energy and water balance by altering their digestive parameters. Leopard tortoises fed lucerne (Medicago sativa) had a low food intake coupled with long gut transit times, which resulted in the lowest amount of faecal energy and faecal water lost. Tortoises fed tomatoes (Solanum lycopersicum) had higher food intake and faster gut transit times, but more energy and water was lost in the faeces. However, daily energy assimilated and assimilation efficiency were comparable between tortoises fed the two diets. Urine osmolality was significantly different between tortoises on the two diets. Results indicate that leopard tortoises can adjust parameters such as transit rate, food intake, water loss and urine osmolality to maintain body mass, water and energy balance in response to a high fibre, low water content and a low fibre, high water content diet. This study suggests that this digestive flexibility allows leopard tortoises in the wild to take advantage of unpredictable food and water resources.     

A circular dichroism study of charged polypeptides interaction with salts.

The effect of salts on the experimental circular dichroism spectra of polypeptides is presented using poly-L-lysine as the main model. Salt effects are analyzed into: (a) shielding at low (less than 0.5 M) concentrations of all salts; (b) binding to positively charged and some neutrally charged side-chains by certain anions (e.g., CCl3COO-, CF3C00-, ClO4-), with induction of helicity; (c) binding of these same anions, at high concentration, to the backbone leading toward random structure; (d) binding of high concentration of denaturing cations (La+3, Ca++, Li+) to the backbone, with La+3 and Ca++ leading to collapsed random structure (R) while Li+ tends to leave the polypeptide somewhat extended; (e) indirect interaction of salting-out salts (NaH2PO4, (NH4)2SO4, NH4F), at high concentration, leading toward complete alpha helicity, probably by competition with the polypeptide and the anion for available water. Effects of changing the temperature from 5 degrees to 50 degrees on the circular dishroism spectra of different polypeptide-salt solutions throughout the region from extended (LES) to alpha helical conformation are analyzed in terms of introduction of randomness (R) at high temperature. Applications to effects of salt on protein structures are considered.     

Changes in rigidity of the polymeric chain of the lambda-phage DNA in aqueous solutions with low ionic strength over the pH range 4-9.5

It was shown that the decrease in the rigidity (persistent length) of phage lambda DNA, revealed previously by laser correlation spectroscopy, occurs in an aqueous solution at concentrations of sodium salts less than 10(-2) M in the pH range 4-9.5. DNA coils of anomalously small size (approximately twofold less than the size reported by other authors) are formed. The formation of these coils is likely to be due to the separation of "normal", i.e., rigid DNA coils into two phases, which occurs as the concentration of sodium salts decreases to 1.5 x 10(-3) M and pH of the solution decreases to 4. The phase of small-size (nonrigid) coils makes the major contribution to the scattering spectra. The phase of large-size coils disappears at pH 9.5. As pH increases, the size of small coils increases. The occurrence of coils of anomalously small size was registered by another method, quick precipitation. It is assumed that the phase separation of coils is related to the structural features of water.     

Single-molecule detection of yeast cytochrome c by Surface-Enhanced Raman Spectroscopy

The giant enhancement of Raman signal near silver colloidal nanoparticles is exploited to study the Raman spectrum of Cytochrome c from Saccharomyces cerevisiae (Yeast Cytochrome c--YCc) in the limit of single-molecule. The investigation is performed on proteins both in solution and immobilised onto a glass slide using a quasi resonant laser line as exciting source with low excitation intensity. In both cases, spectra acquired at different times exhibit dramatic temporal fluctuations in both the total spectrum and in the specific line intensity, even though averaging of several individual spectra reproduces the main Raman features of bulk YCc. Analysis of the spectral intensity fluctuations from solutions reveals a multimodal distribution of some specific Raman lines, consistent with the approaching of single molecule regime. Among other results, the statistical analysis of the spectra from immobilised samples seems to indicate dynamical processes involving the reorientational of the heme with respect to the metal surface.     

Fast water uptake and outflow and electrophysiological responses of roots induced by changes in salinity,osmotic pressure,and pH of external solution

Fast reversible water losses by roots of intact seedlings of tomato (Lycopersicon esculentum L.) and sunflower (Helianthus annuus L.) in response to changes in composition and concentration of external medium were studied with a highly sensitive fast-response gravimetric method. The dehydrating effect of solutes, applied at low and moderate concentrations (5–100 mM), increased in the following row: ethanol < glycerol < sucrose neutral salts < base < acid. At concentrations below 10 mM, nonelectrolytes did not cause significant water losses from roots. Neutral salts had a characteristic gradual effect in a wide range of concentrations (0.3–500 mM NaCl). Amplitudes of gravimetric responses to treatments with bases and acids were 1.5–2 times higher (NaOH) and more than 3 times higher (HCl) than the response to equimolar concentration (5 mM) of a neutral salt. In all cases the water loss from roots was fast, reversible, and well reproducible. The presence of electrical charge (ions vs. neutral molecules) was crucial for the strength of the solute effect, especially at low concentrations. In parallel experiments with tomato seedlings, fast kinetics of electric potential difference between the root and the hypocotyle (electrophysiological response) was measured after a change in the composition and concentration of external solution. Possible mechanisms of observed phenomena are discussed.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 74–81.Original Russian Text Copyright © 2005 by Zakharin.This paper concludes a series of authors original studies on physiology of water transport in plants.     

Velocity auto-correlation function of ions and water molecules in different concentrations,anions and ion clusters

The characteristics of ion solvation are important for electrochemical and biophysical phenomena because all such phenomena occur under the presence of solvated ions. In this study, we performed an all-atom molecular dynamics simulation of aqueous NaCl ranging from 0.5 to 3.0 M, and aqueous NaF, NaBr and NaI in 2.0 M, to investigate the time-averaged velocity auto-correlation function (TAVAF) of ions and water molecules. By comparing the concentrations and ion pairs, we observed three behaviours: (i) in the case of NaCl, the velocity auto-correlation of Cl^?  becomes weaker as the concentration increases, whereas those of Na⁺ are not clearly different, (ii) the intensity of fluctuations of the TAVAF gradually decreases following the decrease in ionic radius and (iii) every TAVAF of water molecules in ionic solutions is clearly lower than that of bulk because of the cage effect. Furthermore, we observed that the first minimum of the TAVAF in the cluster is smaller than that of the isolated ions. These results indicate that the diffusion of ions and water molecules is affected by cage effect, and that the generation of ion cluster affects the diffusion of ions.     

Immersion in CO<Subscript>2</Subscript>-rich water containing NaCl diminishes blood pressure fluctuation in anesthetized rats

Remarkably, bathing in hot springs containing high concentrations of carbon dioxide (CO₂) influences cardiovascular function more than bathing in fresh water. The CO₂-enriched water in hot springs generally contains many salts, whose interactions remain unknown. We separately evaluated the actions of individual factors in CO₂-enriched water and confirmed that CO₂ and NaCl have combined effects on blood pressure fluctuations in anesthetized rats. Animals equipped with sensor probes to monitor body temperature, skin blood flow and arterial blood pressure were immersed in bathwater (35°C) containing CO₂ with NaCl, KCl or sucrose. The effects of these factors on cardiovascular function were evaluated using power-spectral analysis of fluctuations in blood pressure and heart rate. Compared with immersion in tap water, heart rate and skin vascular resistance were reduced during immersion in CO₂-enriched water irrespective of the presence of other components. In terms of the very low frequency range (0.02–0.195 Hz), the power of blood pressure fluctuation during immersion was significantly reduced when the CO₂-enriched water contained more than 1.5% NaCl but was not influenced by other components of similar osmotic pressure and the same specific gravity. The results indicated that the coexistence of CO₂ and sodium ions in bathwater reduce blood pressure fluctuations, and suggested that this combination effect of CO₂ and salt contributes to the sedative effect on human cardiovascular functions while bathing in CO₂-hot springs.