A study of factors influencing hydration of sodium hyaluronate from compressibility and high-precision densimetric measurements.

A study of the factors influencing the hydration of the biopolymer hyaluronic acid was made by compressibility and density measurements. The factors investigated were the hydration changes on glycosidic bond formation, and also the influence of counterion type, solution ionic strength and temperature. The results indicate that, with this biopolymer, the hydration of the glucuronate residue is significantly more than that of the N-acetylglucosamine residue, and further that the biopolymer is less hydrated than the sum of its component monosaccharide residues. Change of the counterion salt form of this polyelectrolyte from univalent to bivalent counterion type (Na+ to Ca2+) leads to a small though significant increase in the total hydration sheath surrounding the polymer. An increase in the background ionic strength of the solvent leads to a quantifiable lowering of the hydration of the polymer at physiological ionic strength compared with its value in salt-free aqueous solution. A decrease in hydration with increase in temperature in the range 20-50 degrees C is the opposite of previous reports, and was observed when the polymer was dissolved both in pure water and in 0.15 M-NaCl.     

Volume and sound velocity changes accompanying the -helix to -form and coil to -helix transitions in aqueous solution

The volume increment per amino acid residue for the α-helix to β-form transition of uncharged poly-L -lysine in aqueous solution was 3.8 ml in water and 4.3 ml in 0.2M and 1M NaBr solutions at 26°C, respectively. The sound velocity of the polymer solution was greater with the β-helix than with the β-form, but the difference was less in dilute salt solutions and disappeared in 1 or 2M NaBr solution. Thus, the β poly-L -lysine solution was slightly more compressible than the α-polymer solution, but this difference was diminished with increasing salt concentration. Both the volume change and the change in adiabatic compressibility of the polymer solution suggest that hydrophobic interactions among the lysyl groups in the β-form reduce the amount of “icebergs” surrounding the polymer molecules as compared with the amount originally present with the α-helix. The coil-to-helix transition of poly-L -glutamic acid in aqueous solution was also accompanied by a decrease in sound velocity. This can be attributed to the reduction of the water of hydration which is less compressible than free water.     

Ouabain-insensitive salt and water movements in duck red cells. I. Kinetics of cation transport under hypertonic conditions

Duck red cells in hypertonic media experience rapid osmotic shrinkage followed by gradual reswelling back toward their original volume. This uptake of salt and water is self limiting and demands a specific ionic composition of the external solution. Although ouabain (10(-4)M) alters the pattern of cation accumulation from predominantly potassium to sodium, it does not affect the rate of the reaction, or the total amount of salt or water taken up. To study the response without the complications of active Na-K transport, ouabain was added to most incubations. All water accumulated by the cells can be accounted for by net salt uptake. Specific external cation requirements for reswelling include: sufficient sodium (more than 23 mM), and elevated potassium (more than 7 mM). In the absence of external potassium cells lose potassium without gaining sodium and continue to shrink instead of reswelling. Adding rubidium to the potassium- free solution promotes an even greater loss of cell potassium, yet causes swelling due to a net uptake of sodium and rubidium followed by chloride. The diuretic furosemide (10(-3)M) inhibits net sodium uptake which depends on potassium (or rubidium), as well as inhibits net sodium uptake which depends on sodium. As a result, cell volume is stabilized in the presence of this drug by inhibition of shrinkage, at low, and of swelling at high external potassium. The response has a high apparent energy of activation (15-20 kcal/mol). We propose that net salt and water movements in hypertonic solutions containing ouabain are mediated by direct coupling or cis-interaction, between sodium and potassium so that the uphill movement of one is driven by the downhill movement of the other in the same direction.     

Differential hydration of dA.dT base pairing and dA and dT bulges in deoxyoligonucleotides

The role of water in the formation of stable duplexes of nucleic acids is being studied by determining the concurrent volume change, heats, and counterion uptake that accompany the duplexation process. The variability of the volume contraction that we have observed in the formation of a variety of homoduplexes suggests that sequence and conformation acutely affect the degree of hydration. We have used a combination of densimetric and calorimetric techniques to measure the change in volume and enthalpy resulting from the mixing of two complementary strands to form (a) fully paired duplexes with 10 or 11 base pairs and (b) bulged decameric duplexes with an extra dA or dT unmatched residue. We also monitored absorbance vs temperature profiles as a function of strand and salt concentration for all four duplexes. Relative to the decamer duplex, insertion of an extra dA.dT base pair to form an undecamer duplex results in a favorable enthalpy of -5.6 kcal/mol that is nearly compensated by an unfavorable entropy term of -5.1 kcal/mol. This enthalpy difference correlates with a differential uptake of water molecules, corresponding to an additional hydration of 16 mol of water molecules/mol of base pair. Relative to the fully paired duplexes, both bulged duplexes are 12-16 degrees C less stable and exhibit marginally larger counterion uptake on forming the duplex. The enthalpy change is slightly lower for the T-bulge duplex and less still for the A-bulge duplex. The volume change results indicate that an unmatched residue increases the amount of coulombic and/or structural hydration. The combined results strongly suggest that the destabilizing forces in bulged duplexes are partially compensated by an increase in hydration levels.     

Molecular dynamics simulation of solvated protein at high pressure.

We have completed a molecular dynamics simulation of protein (bovine pancreatic trypsin inhibitor, BPTI) in solution at high pressure (10 kbar). The structural and energetic effects of the application of high pressure to solvated protein are analyzed by comparing the results of the high-pressure simulation with a corresponding simulation at low pressure. The volume of the simulation cell containing one protein molecule plus 2943 water molecules decreases by 24.7% at high pressure. This corresponds to a compressibility for the protein solution of beta = 1.8 x 10(-2) kbar-1. The compressibility of the protein is estimated to be about one-tenth that of bulk water, while the protein hydration layer water is found to have a greater compressibility as compared to the bulk, especially for water associated with hydrophobic groups. The radius of gyration of BPTI decreases by 2% and there is a one third decrease in the protein backbone atomic fluctuations at high pressure. We have analyzed pressure effects on the hydration energy of the protein. The total hydration energy is slightly (4%) more favorable at high pressure even though the surface accessibility of the protein has decreased by a corresponding amount. Large pressure-induced changes in the structure of the hydration shell are observed. Overall, the solvation shell waters appear more ordered at high pressure; the pressure-induced ordering is greatest for nonpolar surface groups. We do not observe evidence of pressure-induced unfolding of the protein over the 100-ps duration of the high-pressure simulation. This is consistent with the results of high-pressure optical experiments on BPTI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Volume changes correlate with entropies and enthalpies in the formation of nucleic acid homoduplexes: Differential hydration of A and B conformations

We have used a combination of densimetric, calorimetric, and uv absorption techniques to obtain a complete thermodynamic characterization for the formation of nucleic acid homoduplexes of known sequence and conformation. The volume change ΔV accompanying the formation of four duplexes was interpreted to reflect changes in hydration based on the electrostriction phenomenon. In 10 mM sodium phosphate buffer at pH 7, the magnitude of the measured ΔV's ranged from ?2.0 to +7.2 ml/mol base pair and followed the order of poly(rA) · poly(dT) ～ poly(dA) · poly(dT) < poly(rA) · poly(dU) ～ poly(rA) · poly(rU). Inclusion of 100 mM NaCl in the same buffer gave the range of ?17.4 to ?2.3 mL/mol base pair and the following order: poly(dA) · poly(dT) < poly(rA) · poly(dT) < poly(rA) · poly(rU) ～ poly(rA) ～ polyr(dU). Standard thermodynamic profiles of forming these duplexes from their corresponding complementary single strands indicated similar free energies that resulted from the compensation of favorable enthalpies with unfavorable entropies along with a similar counterion uptake at both ionic strengths. The differences in these compensating effects of entropy and enthalpy correlated very well with the volume change measurements in a manner suggesting that the homoduplexes in the B conformation are more hydrated than are those in the A conformation. Moreover, the increased thermal stability of these homoduplexes resulted from an increase in the salt concentration corresponding to larger hydration levels as reflected by the ΔV results. © 1993 John Wiley & Sons, Inc.     

Observation of unfreezable water in aqueous solution of globule protein by microwave dielectric measurement

A freezing process analyzed by the dielectric method on aqueous solution of albumin has revealed water structure around protein molecule. A relaxation peak due to bound water attached on the protein surface around 100 MHz at room temperature was found. It could be seen commonly in globule proteins. Another peak due to a different kind of unfreezable water was found around 1 GHz at ?6°C. The amount of this water is estimated as 0.36 g water/g protein and in good agreement with that obtained by differential scanning calorimetry and nmr measurements. The water molecules form a shell layer around the protein molecule. © 1995 John Wiley & Sons, Inc.     

Static and dynamic light scattering approach to the hydration of hemoglobin and its supertetramers in the presence of osmolites.

We used static and dynamic light scattering for comparing the mass (MW) and hydrodynamic radius (R(h)) of several hemoglobin systems, namely human hemoglobin, bovine hemoglobin, human hemoglobin cross-linked with a sebacyl residue, and bovine hemoglobin cross-linked with an adipoyl residue. We measured the MW and R(h) of these systems in 0.1M phosphate buffer at pH 7.0 in the absence and in the presence of either betaine or glycerol up to 1.7 molal concentrations. The 90 degrees scattering was measured with a photon counting machine equipped with a diode laser at 783 nm. The Rayleigh ratio [R(theta)] of the instrument was estimated using R(theta) = 7.19E-6 cm(-1) for toluene at 783 nm. The refractive index increment of hemoglobin solutions was measured using a laser beam at 750 nm. We estimated a value dn/dc = 0.210 cm3/g in the absence and dn/dc = 0.170 in the presence of 1.7 molal osmolites. For all systems both in liganded and unliganded form, the static light scattering data showed a 16% mass increase with increasing concentration of osmolites. The hydrodynamic radii of all investigated systems in the presence and absence of osmolites were close to 3.17 nm. Assuming a partial specific volume nu = 0.739 for hemoglobin, and using spherical geometry, the estimated average hydration volume of hemoglobin was 32.6 L/mole in the absence of osmolites. It decreased to 23.5 L/mole in the presence of 1.7 molal osmolites. Assuming that the density of water in the hydration volume is D = 1.0 g/cm3, the hydration of Hb was 0.51 gH2O/gHb, with a surface density of 0.20 molH2O/A2. The hydration decreased to 0.33 gH2O/gHb and 0.14 molH2O/A2 in the presence of 1.7 molal osmolites. The decreased hydration was compensated by the increased mass (i.e., decreased surface area per unit volume) so that the thickness of the water shell around these proteins remained close to a single layer of water molecules. These findings indicate that the combination of static and dynamic light scattering offer unique means for investigating the relevance of water activity on the structure and function of biological macromolecules. In the case of hemoglobin, the data suggest that the decreased oxygen affinity in the presence of osmolites reported by Colombo et al. (M. F. Colombo, D. C. Rau, and V. A. Parsegian Science, 1992, Vol. 256, pp. 655-659), as due to ligand linked water binding on hemoglobin surface, is part of a complex phenomenon involving the hydration shell of hemoglobin and the formation of low affinity supertetrameric molecules.     

Immunochemistry of kappa-type carrageenans from certain red algae

Carrageenans from female and male gametophytic plants of the alga Rhodo-glossum californicum, female plants of Chondrus crispus and Gigartina pistillata, and male plants of Iridaea cordata and a Gigartina species from San Francisco Bay were fractionated into potassium chloride-soluble and -insoluble components and were analysed chemically. An anti-K-carrageenan, the reactivity of which is directed to K-type structures (i.e., 3-linked d-galactose 4-sulphate and 4-linked 3,6-anhydro-D-galactose residues) was used to analyse these carrageenans immunochemically. The potassium chloride-insoluble carrageenans from these species were found to be highly reactive K-type carrageenans. The potassium chloride-soluble carrageenans were less reactive to anti-K-carrageenan and, in addition, showed reactivity to an anti-λ-carrageenan preparation. The chemical and immunochemical data suggest that the potassium chloride-soluble carrageenans contain either λ- or μ-carrageenan, as a high proportion of the precursors to the 3,6-anhydro-D-galactose are 4-linked D-galactose 2,6-disulphate residues, and no increase in immunological reactivity to anti K-carrageenan was observed upon alkali treatment.     

Solubilization of a Lipophilic Compound in Highly Concentrated Saccharide Solutions Containing Protein

Solubilization of a lipophilic compound in highly concentrated saccharide solutions containing protein was studied by measuring the amount of the lipophilic compound solubilized, the surface hydrophobicity of protein, the line width of the water signal in ¹H-NMR spectra, and the unfreezable water content using a differential scanning calorimeter (DSC).

The solubilizing ability, which was shown by the amount of solubilized p-dimethylaminoazobenzene (DMAB), increased with increasing saccharide concentration in the aqueous system. The effects of different saccharides on the solubilizing ability of a saccharide and bovine serum albumin (BSA) mixture decreased in the following order: sucrose > maltose > fructose > glucose. The solubilizing ability of a saccharide and BSA mixture was higher about 4–5 times than that of saccharide only.

A good correlation was observed between the solubilizing ability of a saccharide and BSA mixture and the surface hydrophobicity of BSA. The line width of the water signal in ¹H-NMR spectrum and the unfreezable water content using DSC, that is, the bound water content in saccharide solution containing BSA increased with increasing saccharide concentration.

From these results, a large amount of DMAB solubilized in a highly concentrated saccharide solution containing BSA would be attributed to the hydrophobicity interaction between BSA and DMAB due to the surface hydrophobicity of BSA which increased with increasing bound water content.     

Hydration and protein folding in water and in reverse micelles: compressibility and volume changes

The partial specific volume and adiabatic compressibility of proteins reflect the hydration properties of the solvent-exposed protein surface, as well as changes in conformational states. Reverse micelles, or water-in-oil microemulsions, are protein-sized, optically-clear microassemblies in which hydration can be experimentally controlled. We explore, by densimetry and ultrasound velocimetry, three basic proteins: cytochrome c, lysozyme, and myelin basic protein in reverse micelles made of sodium bis (2-ethylhexyl) sulfosuccinate, water, and isooctane and in aqueous solvents. For comparison, we use beta-lactoglobulin (pI = 5.1) as a reference protein. We examine the partial specific volume and adiabatic compressibility of the proteins at increasing levels of micellar hydration. For the lowest water content compatible with complete solubilization, all proteins display their highest compressibility values, independent of their amino acid sequence and charge. These values lie within the range of empirical intrinsic protein compressibility estimates. In addition, we obtain volumetric data for the transition of myelin basic protein from its initially unfolded state in water free of denaturants, to a folded, compact conformation within the water-controlled microenvironment of reverse micelles. These results disclose yet another aspect of the protein structural properties observed in membrane-mimetic molecular assemblies.     

Experimental data and modelling of apparent molar volumes, isentropic compressibilities and refractive indices in aqueous solutions of glycine + NaCl

Experiments have been performed at 298.15 K to measure the density, sound velocity and refractive index of glycine in aqueous solutions of NaCl over a wide range of both glycine and NaCl concentrations. The values of apparent molar volume and isentropic compressibility of glycine were calculated from the measured data. The results show a positive transfer volume of glycine from an NaCl solution to a more concentrated NaCl solution. This indicates that the size of a glycine molecule is larger in a solution with higher NaCl concentration. The negative values of apparent isentropic compressibility imply that the water molecules around the glycine molecules are less compressible than the water molecules in the bulk solution. These effects are attributed to the doubly charged behaviour of glycine and to the formation of physically bonded ion-pairs between the charged groups of glycine and sodium and chloride ions. The formation of ion-pairs, whose extents of binding reactions depend on the concentrations of both NaCl and glycine, alter the hydration number of glycine. This also explains the reason for the increase in the size of glycine with an increase in the NaCl concentration. A model based on the Pitzer formalism has been developed to correlate the activity coefficient, apparent molar volume and isentropic compressibility of glycine in aqueous solutions of NaCl. The results show that the model can accurately correlate the interactions in aqueous solutions of glycine and NaCl.     

Micelle formation of sodium chenodeoxycholate and solubilization into the micelles: comparison with other unconjugated bile salts

Micellization of sodium chenodeoxycholate (NaCDC) was studied for the critical micelle concentration (CMC), the micelle aggregation number, and the degree of counterion binding to micelle at 288.2, 298.2, 308.2, and 318.2 K. They were compared with those of three other unconjugated bile salts; sodium cholate (NaC), sodium deoxycholate (NaDC), and sodium ursodeoxycholate (NaUDC). The I(1)/I(3) ratio of pyrene fluorescence and the solubility dependence of solution pH were employed to determine the CMC values. As the results, a certain concentration range for the CMC and a stepwise molecular aggregation for micellization were found reasonable. Using a stepwise association model of the bile salt anions, the mean aggregation number (n) of NaCDC micelles was found to increase with the total anion concentration, while the n values decreased with increasing temperature; 9.1, 8.1, 7.4, and 6.3 at 288.2, 298.2, 308.2, and 318.2 K, respectively, at 50 mmol dm(-3). The results from four unconjugated bile salts indicate that the number, location, and orientation of hydroxyl groups in the steroid nucleus are quite important for growth of the micelles. Activity of the counterion (Na(+)) was determined by a sodium ion selective electrode in order to confirm the low counterion binding to micelles. The solubilized amount of cholesterol into the aqueous bile salt solutions increased in the order of NaUDC相似文献   

Sodium and potassium uptake of rice panicles as affected by salinity and season in relation to yield and yield components

Salinity is a major yield-reducing stress in many arid and/or coastal irrigation systems for rice. Past studies on salt stress have mainly addressed the vegetative growth stage of rice, and little is known on salt effects on the reproductive organs. Sodium and potassium uptake of panicles was studied for eight rice cultivars in field trials under irrigation with saline and fresh water in the hot dry season and the wet season 1994 at WARDA in Ndiaye, Senegal. Sodium and potassium content was determined at four different stages of panicle development and related to salt treatment effects on yield, yield components and panicle transpiration. Yield and yield components were strongly affected by salinity, the effects being stronger in the HDS than in the WS. The cultivars differed in the amount of salt taken up by the panicle. Tolerant cultivars had lower panicle sodium content at all panicle development stages than susceptible ones. Panicle potassium concentration decreased with panicle development under both treatments in all cultivars, but to a lesser extent in salt treated susceptible cultivars. Grain weight reduction in the early panicle development stages and spikelet sterility increase in the later PDS were highly correlated (p < 0.01) with an increase in panicle sodium concentration in both seasons, whereas reduction in spikelet number was not. The magnitude of salt-induced yield loss could not be explained with increases in sodium uptake to the panicle alone. It is argued that the amount of sodium taken up by the panicle may be determined by two different factors. One factor (before flowering) being the overall control mechanism of sodium uptake through root properties and the subsequent distribution of sodium in the vegetative plant, whereas the other (from flowering onwards) is probably linked to panicle transpiration. This revised version was published online in August 2006 with corrections to the Cover Date.     

Protein structure and dynamics in nonaqueous solvents: insights from molecular dynamics simulation studies

Protein structure and dynamics in nonaqueous solvents are here investigated using molecular dynamics simulation studies, by considering two model proteins (ubiquitin and cutinase) in hexane, under varying hydration conditions. Ionization of the protein groups is treated assuming "pH memory," i.e., using the ionization states characteristic of aqueous solution. Neutralization of charged groups by counterions is done by considering a counterion for each charged group that cannot be made neutral by establishing a salt bridge with another charged group; this treatment is more physically reasonable for the nonaqueous situation, contrasting with the usual procedures. Our studies show that hydration has a profound effect on protein stability and flexibility in nonaqueous solvents. The structure becomes more nativelike with increasing values of hydration, up to a certain point, when further increases render it unstable and unfolding starts to occur. There is an optimal amount of water, approximately 10% (w/w), where the protein structure and flexibility are closer to the ones found in aqueous solution. This behavior can explain the experimentally known bell-shaped dependence of enzyme catalysis on hydration, and the molecular reasons for it are examined here. Water and counterions play a fundamental and dynamic role on protein stabilization, but they also seem to be important for protein unfolding at high percentages of bound water.     

Bovine pancreatic trypsin inhibitor crystals with different morphologies: a molecular dynamics simulation study

A molecular dynamics simulation study is reported for three polymorphic protein crystals (4PTI, 5PTI and 6PTI) of bovine pancreatic trypsin inhibitor (BPTI). The simulated lattice constants are in good agreement with experimental data, indicating the reliability of force field used. The fluctuation patterns of peptide chains in the three crystals are similar, and the protein structures are fairly well maintained during simulation. We observe that water forms a pronounced hydration layer near the protein surface. The diffusion coefficients of water in the three crystals are smaller than in bulk phase, and thus, the activation energies are higher. The porosity, fluctuation of peptide chains and solvent-accessible surface area as well as the diffusion coefficients of water and counterion in 5PTI are the largest among the three crystals. The diffusion of water and counterion is anisotropic, and the degree of anisotropy increases in the order of 4PTI < 5PTI < 6PTI. Despite a slight difference, the structural and diffusion properties in the three BPTI crystals are generally close. This simulation study reveals that crystal polymorphism does not significantly affect microscopic properties in the BPTI crystals with different morphologies.     

DSC Measurement of Frozen Water in Liquid Foods

We investigated the state of water in frozen liquid foods, supposed to influence the performance of freeze-drying, by DSC. The amount of unfreezable water in milk was constant irrespective of the initial water content. As expected from the phase equilibrium, the water content of the non-ice part of CAS was constant among the samples with different initial water contents. The volume fraction of ice crystals, which should be known in the analysis of the freeze-drying rate, was significantly smaller at the low initial water content than the one usually estimated with the assumption that the water was all freezable.     

Studies on the interaction between alkali metal cations and polyion by sound velocity

The sound velocities in polyelectrolyte solutions were measured at various concentrations of added salts. When aqueous solutions of tetra (n-butyl)ammonium polyacrylate were titrated with concentrated solutions of LiCl, NaCl, KCl or CsCl, the sound velocity, i.e., the adiabatic compressibility of the solution, did not change linearly with added salt concentration, but showed a breaking point. The degrees of counterion binding on polyacrylate ion estimated from the breaking points were 0.25-0.30, independent of cation species. In polystyrenesulfonate, moreover, no Na+ binding was detected from such sound velocity measurements.     

A study of the quadrupolar NMR splittings of 7Li+, 23Na+, and 133Cs+ counterions in macroscopically oriented DNA fibers.

The hydration and temperature dependencies of the 23Na+, 133Cs+, and 7Li+ quadrupolar splitting have been determined in hydrated, macroscopically oriented DNA fibers. At low water contents the quadrupolar splitting is found to decrease as the water content increases, regardless of counterion, while at high water contents the hydration dependence is reversed. The 23Na+ and 133Cs+ quadrupolar splittings decrease as the temperature increases, while the 7Li+ splitting shows the opposite behavior. At high water contents the 23Na+ and 133Cs+ splittings decrease, and then, after passing zero splitting, increase as the temperature increases. The interpretation of the temperature dependence is discussed in terms of a two-site model (free and bound ions) and a three-site model (free ions and specifically or nonspecifically bound ions). It is suggested that a three-site model is more consistent with the data for the present system. At high water contents, the temperature dependence of the 7Li+ splitting vanishes, indicating counterion condensation. The behavior of the 7Li+ splitting is confirmed by measurements on DNA fibers in equilibrium with a C2H5OD-D2O-LiCl solution. The salt dependence in this system is weak. The counterion quadrupolar splitting is seen to be very sensitive to structural transitions in double-helical DNA.