Hydration of clupeine in solution

Spin-lattice relaxation times, T1, of H2(17) O at 25 degrees were measured for aqueous solutions of clupeine and its constituent amino acids, which are serine, threonine, proline and arginine. The dynamic hydration numbers, nDHN, of clupeine and amino acids were determined from a concentration dependence of T1. The coordination numbers nh, and the rotational correlation times, tau ch, of water molecules around clupeine and amino acids were estimated and compared with that of pure water. The tau ch/tau co of clupeine was 1.85 and close to that of arginine. The experimental value of nDHN of clupeine was in good agreement with that calculated from the nDHN values of the constituent amino acids. This means that the clupeine molecule has a random conformation in solution.     

Effect of water potential on sol-gel transition and intermolecular interaction of gelatin near the transition temperature

The sol-gel transition of gelatin, measured by thermal analysis and viscosity measurement, was analyzed in terms of the change in hydration state of polymer molecules. A new thermodynamic model was proposed in which the effect of water potential is explicitly taken into account for the evaluation of the free energy change in the sol-gel transition process. Because of the large number of water molecules involved and the small free energy change in the transition process, the contribution of water activity, a(W), was proved to be not negligible in the sol-gel transition process in solutions containing such low-molecular cosolutes as sugars, glycerol, urea, and formamide. The gel-stabilization effect of sugars and glycerol was linear with a(W), which seemed consistent with the contribution of water potential in the proposed model. The different stabilization effect among sugars and glycerol was explained by the difference in solvent ordering, which affects hydrophobic interaction among protein molecules. The gel-destabilization effect of urea and formamide could be explained only by the direct binding of them to protein molecules through hydrogen bonding. On the contrary, the polymer-polymer interaction, measured by the viscosity analysis, in polyethyleneglycol and dextran solutions was not sensitive to the change in a(W), suggesting that no substantial change in hydration state with a(W) occurred in these polymer solutions.     

Hydration state change of proteins upon unfolding in sugar solutions

Change in hydration number of proteins upon unfolding, Deltan, was obtained from the analysis of thermal unfolding behavior of proteins in various sugar solutions with water activity, a(W), varied. By applying the reciprocal form of Wyman-Tanford equation, Deltan was determined to be 133.9, 124.1, and 139.2 per protein molecule for ribonuclease A at pH=5.5, 4.2, and 2.8, respectively, 201.4 for lysozyme at pH=5.5, and 100.1 for alpha-chymotripnogen A at pH=2.0. Among the sugars tested, reducing sugars gave the lower apparent Deltan as compared with nonreducing sugars probably because of the direct interaction of reducing terminal with amino group of proteins at a high temperature. From the knowledge of Deltan, a new thermodynamic model for protein stability was proposed with explicit consideration for hydration state change of protein upon unfolding. From this model, the contribution of a(W) was proven to be always positive for stabilization of proteins and its effect is not negligible depending on Deltan and a(W).     

Study of the Stability Of Vaccinium myrtillus Peroxidase in Reverse Micellar Systems

The stability of a cationic peroxidase isolated and purified from a cell suspension of Vaccinium myrtillus , microencapsulated in reverse micelles of sodium dioctylsulfosuccinate (AOT) was evaluated. By using a central composite design (CCD), some relevant parameters for the enzymatic activity, such as surfactant and water concentration, pH and buffer molarity, were analysed. The response surface curves showed that 50 mM AOT, 500 mM water, 80 mM buffer and pH 7.6 were the best conditions for enzyme stability. The effect of carbohydrates and polyols on enzyme stability was also evaluated. At 20 mM, carbohydrates like arabinose, and trehalose increased the enzymatic stability by a factor of 4.4 and 2.3, respectively, but melezitose had no effect. From the three polyols tested, inositol and sorbitol increased the peroxidase stability by a factor of 3.8 and 1.8, respectively, while mannitol had no effect.     

Study of the Stability Of Vaccinium myrtillus Peroxidase in Reverse Micellar Systems

The stability of a cationic peroxidase isolated and purified from a cell suspension of Vaccinium myrtillus, microencapsulated in reverse micelles of sodium dioctylsulfosuccinate (AOT) was evaluated. By using a central composite design (CCD), some relevant parameters for the enzymatic activity, such as surfactant and water concentration, pH and buffer molarity, were analysed. The response surface curves showed that 50 mM AOT, 500 mM water, 80 mM buffer and pH 7.6 were the best conditions for enzyme stability. The effect of carbohydrates and polyols on enzyme stability was also evaluated. At 20 mM, carbohydrates like arabinose, and trehalose increased the enzymatic stability by a factor of 4.4 and 2.3, respectively, but melezitose had no effect. From the three polyols tested, inositol and sorbitol increased the peroxidase stability by a factor of 3.8 and 1.8, respectively, while mannitol had no effect.     

Thermal stabilization of antithrombin III by sugars and sugar derivatives and the effects of nonenzymatic glycosylation

A variety of neutral and acidic sugars and related compounds were evaluated in terms of their effect on the midpoint, Td, of the thermal denaturation curve of antithrombin III. The objectives were to determine which structural features of these molecules are responsible for their stabilizing properties and to identify more efficient stabilizers which combine the effects of lyotropic anions such as citrate with those of the polyols in a single molecule. The presence of one or more carboxylate groups in a sugar molecule invariably increased its stabilizing potency, whereas the number and position of hydroxyl groups appeared to have no influence on the molecules' stabilizing ability. Several compounds were shown to be effective in preserving antithrombin III activity during pasteurization for 10 h at 60 degrees C. However, the presence of reducing sugars invariably resulted in a decrease in activity following pasteurization, in spite of their ability to increase Td. In fact, when antithrombin III was pasteurized in the presence of 2 M glucose and 0.5 M citrate, it steadily lost its ability to inhibit thrombin even though Td under these conditions was 10 degrees C higher than in citrate alone where activity was preserved. This effect was shown to be coincident with the covalent incorporation of glucose into the protein molecule.     

Increased thermal stability of proteins in the presence of sugars and polyols.

Sugars and polyols stablize proteins against heat denaturation. Scanning calorimetry was used to obtain a quantitative estimate of the degree of stabilization. Solutions of ovalbumin, lysozyme, conalbumin, and alpha-chymotrypsinogen were heated at a constant rate, and the temperature of the maximum rate of denaturation was estimated (Tm). Addition of a sugar or polyol raised Tm. The magnitude of the stabilizing effect (delta Tm) depended on both the nature of the protein and the nature of the sugar or polyol, ranging from 18.5 degrees C for lysozyme at pH 3 in the presence of 50% (w/w) sorbitol to 0 degrees C for conalbumin at pH 7 in 50% glycerol solution. It is argued that this stablization is due to the effects of sugars and polyols on hydrophobic interactions. The strength of the hydrophobic interaction was measured in model systems in sucrose and glycerol solutions. Sucrose and glycerol strengthened the pairwise hydrophobic interaction between hydrophobic groups; however, they reduced the tendency for complete transfer of hydrophobic groups from an aqueous to a nonpolar environment. The extent of stabliziation by different sugars and polyols is explained by their different influences on the structure of water. The difference between the partial molar volume of the sugar or polyol and its van der Waals volume was used as a rough quantitative measure of the structure-making or structure-breaking effect. There was a linear relationship between this quantity and delta Tm.     

Chemical chaperone-mediated protein folding: stabilization of P22 tailspike folding intermediates by glycerol

Polyol co-solvents such as glycerol increase the thermal stability of proteins. This has been explained by preferential hydration favoring the more compact native over the denatured state. Although polyols are also expected to favor aggregation by the same mechanism, they have been found to increase the folding yields of some large, aggregation-prone proteins. We have used the homotrimeric phage P22 tailspike protein to investigate the origin of this effect. The folding of this protein is temperature-sensitive and limited by the stability of monomeric folding intermediates. At non-permissive temperature (>or=35 degrees C), tailspike refolding yields were increased significantly in the presence of 1-4 m glycerol. At low temperature, tailspike refolding is prevented when folding intermediates are destabilized by the addition of urea. Glycerol could offset the urea effect, suggesting that the polyol acts by stabilizing crucial folding intermediates and not by increasing solvent viscosity. The stabilization effect of glycerol on tailspike folding intermediates was confirmed in experiments using a temperature-sensitive folding mutant protein, by fluorescence measurements of subunit folding kinetics, and by temperature up-shift experiments. Our results suggest that the chemical chaperone effect of polyols observed in the folding of large proteins is due to preferential hydration favoring structure formation in folding intermediates.     

Optimization of the use of horseradish peroxidase and its antibodies in immunoenzyme analysis

The steady-state kinetics of peroxidation of 8 aromatic amines was studied. p-Phenylenediamine, o-dianisidine (o-DA) and 3,5,3',5'-tetramethylbenzidine were found to be optimal substrates of horse-radish peroxidase. The kinetics of oxidation of these substrates by horseradish peroxidase modified with three molecules of Strophanthin K was studied as well. Within the temperature range from 37 to 53 degrees C the inactivation rate constants were determined for peroxidase and its conjugate with Strophanthin K. The effect of sugars and polyols on thermal stability of the conjugate peroxidase-Strophanthin K was investigated. A comparative kinetic study was performed of oxidation of o-DA and its conjugate with dextran. The results obtained made a basis for an enzyme immunoassay of cardiac glycosides during their isolation from plant raw material.     

Molecular dynamics simulations of trehalose as a 'dynamic reducer' for solvent water molecules in the hydration shell

Systematic computational work for a series of 13 disaccharides was performed to provide an atomic-level insight of unique biochemical role of the alpha,alpha-(1-->1)-linked glucopyranoside dimer over the other glycosidically linked sugars. Superior osmotic and cryoprotective abilities of trehalose were explained on the basis of conformational and hydration characteristics of the trehalose molecule. Analyses of the hydration number and radial distribution function of solvent water molecules showed that there was very little hydration adjacent to the glycosidic oxygen of trehalose and that the dynamic conformation of trehalose was less flexible than any of the other sugars due to this anisotropic hydration. The remarkable conformational rigidity that allowed trehalose to act as a sugar template was required for stable interactions with hydrogen-bonded water molecules. Trehalose made an average of 2.8 long-lived hydrogen bonds per each MD step, which was much larger than the average of 2.1 for the other sugars. The stable hydrogen-bond network is derived from the formation of long-lived water bridges at the expense of decreasing the dynamics of the water molecules. Evidence for this dynamic reduction of water by trehalose was also established based on each of the lowest translational diffusion coefficients and the lowest intermolecular coulombic energy of the water molecules around trehalose. Overall results indicate that trehalose functions as a 'dynamic reducer' for solvent water molecules based on its anisotropic hydration and conformational rigidity, suggesting that macroscopic solvent properties could be modulated by changes in the type of glycosidic linkages in sugar molecules.     

Inhibition by Polyphenolic Phytochemicals and Sulfurous Compounds of the Formation of 8-Chloroguanosine Mediated by Hypochlorous Acid,Human Myeloperoxidase,and Activated Human Neutrophils

The thermal denaturation temperature of a soy protein isolate was increased, but its gel-melting temperature was decreased by the addition of polyols with increasing concentration and number of hydroxyl groups of the polyols. This inverse stabilizing effect of polyols on the protein structure and gel is discussed in terms of the competing solvent effects on intra- and intermolecular hydrophobic interactions and on the peptide-peptide hydrogen bonds of the protein.     

Role of solvent on protein-matrix coupling in MbCO embedded in water-saccharide systems: a Fourier transform infrared spectroscopy study

Embedding protein in sugar systems of low water content enables one to investigate the protein dynamic-structure function in matrixes whose rigidity is modulated by varying the content of residual water. Accordingly, studying the dynamics and structure thermal evolution of a protein in sugar systems of different hydration constitutes a tool for disentangling solvent rigidity from temperature effects. Furthermore, studies performed using different sugars may give information on how the detailed composition of the surrounding solvent affects the internal protein dynamics and structural evolution. In this work, we compare Fourier transform infrared spectroscopy measurements (300-20 K) on MbCO embedded in trehalose, sucrose, maltose, raffinose, and glucose matrixes of different water content. At all the water contents investigated, the protein-solvent coupling was tighter in trehalose than in the other sugars, thus suggesting a molecular basis for the trehalose peculiarity. These results are in line with the observation that protein-matrix phase separation takes place in lysozyme-lactose, whereas it is absent in lysozyme-trehalose systems; indeed, these behaviors may respectively be due to the lack or presence of suitable water-mediated hydrogen-bond networks, which match the protein surface to the surroundings. The above processes might be at the basis of pattern recognition in crowded living systems; indeed, hydration shells structural and dynamic matching is first needed for successful come together of interacting biomolecules.     

Polyol-induced molten globule of cytochrome c: an evidence for stabilization by hydrophobic interaction.

To address the contribution of hydrophobic interaction to the stability of molten globule (MG) of proteins, the effects of various polyols (ethylene glycol, glycerol, erythritol, xylitol, sorbitol, and inositol) on the structure of acid-unfolded horse cytochrome c were examined at pH 2, by means of circular dichroism (CD), partial specific volume, adiabatic compressibility, and differential scanning calorimetry (DSC). Addition of polyols induced the characteristic CD spectra of MG, the effect being enhanced with an increase in their concentration and chain length (the number of OH groups) of polyols except for ethylene glycol. The free energy change of MG formation by sorbitol was comparable with those for the salt-induced MG formation but the heat capacity change was negligibly small. The partial specific volume did not change within the experimental error but the adiabatic compressibility largely increased by MG formation. The sorbitol-induced MG showed a highly cooperative DSC thermogram with a large heat capacity change in comparison with the salt-induced one. These results demonstrate that polyols can stabilize the MG state of this protein through the enhanced hydrophobic interaction overcoming the electrostatic repulsion between charged residues. The stabilizing mechanism and structure of MG state induced by polyols were discussed in terms of the preferential solvent interactions and osmotic pressure of the medium, in comparison with the salt-induced one.     

Influence of homologous disaccharides on the hydrogen-bond network of water: complementary Raman scattering experiments and molecular dynamics simulations

A comparative investigation of trehalose, sucrose, and maltose in water solution has been performed using Raman scattering experiments and Molecular Dynamics simulations. From the analysis of the O-H stretching region in the [2500,4000] cm(-1) Raman spectral range, which includes for the first time the contribution of 'free' water, and the statistical distribution of water HB probabilities from MD simulations, this study confirms the privileged interaction of trehalose with water above a peculiar threshold weight concentration of about 30%. The role of the hydration number of sugars--found higher for trehalose--on the destructuring effect of the water hydrogen bond network is also addressed. The analysis of the water O-H-O bending spectral range [1500,1800] cm(-1) reveals a change of the homogeneity of water molecules influenced by sugars, but the three investigated sugars are found to behave similarly.     

The hydration of phospholipids and phospholipid-cholesterol complexes

The hydration characteristics of phosphatidylcholines and the effect of cholesterol on these were studied with differential thermal analysis and water vapour adsorption experiments. Also the water adsorption of egg phosphatidylethanolamine and the effect of cholesterol on this was studied and compared with corresponding qualities of phosphatidylcholine.The differential thermal analysis study showed that the monohydrates of egg, dipalmitoyl, and dioleoyl phosphatidylcholine tightly bind ～9 molecules of water per phosphatidylcholine molecule. Cholesterol is proved to somewhat increase the water binding of the phospholipids. Cholesterol is also shown to decrease the heat change of the chain melting transition of dioleoyl phosphatidylcholine, but not to abolish it completely.The water adsorption experiments indicate that the hydration of phosphatidylcholines takes place in two steps; a strong initial water binding and a second phase of weak binding. The adsorption isotherm of egg phosphatidylethanolamine is strikingly different from that of egg phosphatidylcholine. Cholesterol is shown, also by this method, to increase the hydration of phospholipids especially that of dipalmitoylphosphatidylcholine.The results in this study are in good agreement with those presented by many other authors. Starting with the accumulated information of the hydration characteristics of phosphatidylcholines the organization of the bound water around the polar group is discussed and the most probable model is evaluated.     

Geographic differences on accumulation of sugars and polyols in locust eggs in response to cold acclimation

The accumulation of low molecular weight sugars and polyols is one of major mechanisms hypothesized to increase cold tolerance in overwintering insects. But little is known about whether these sugars and polyols are involved in geographic variation of cold tolerance. In this study, we investigated accumulation patterns of eight low molecular weight sugars and polyols of eggs in tropical and temperate populations of the migratory locust, which exhibits between-population variation in cold tolerance, in response to cold acclimation (5, 0 and −5 °C). Excluding erythritol, the other seven carbohydrates were identified as possible cryoprotectants in locust eggs. Basal maximal and minimal concentrations were 45 μg/g wet weight for trehalose and 0.59 μg/g wet weight for glycerol. Most sugars and polyols were elevated after a −5 °C exposure. In a tropical population, fructose, glucose, sorbitol and myo-inositol were significantly accumulated by low temperature treatments, but glycerol was not. In the temperate population, glycerol, glucose, mannitol, sorbitol, myo-inositol were significantly accumulated but trehalose did not increase. Our results suggest different accumulation patterns of these carbohydrates of locust eggs between tropical and temperate populations and highlighted possible roles for them in geographic variation of cold tolerance in the migratory locust.     

Effect of sugars on the hydration of serum albumin at low temperatures

The influence of sugars (glucose, fructose, and sucrose) on the hydration characteristics of serum albumin was studied using 1H NMR spectroscopy in combination with layer-by-layer freezing-out of bulk and interfacial water. It was found that the presence of sugars in protein solution leads to a considerable decrease in the concentration of bound water at T < 273 K; i. e., sugars cause the dehydration of protein molecules, which may be caused by those alterations in albumin structure that are associated with the formation of more compact globular structures. The most considerable effect was recorded in case of sucrose, which causes a decrease in the dehydration of albumin by at least one order of magnitude. The interfacial energy values for the protein/water system were calculated.     

Use of molybdenum media with glycerin and admixtures of unassimilated polyols for differentiating actinomycetes

Actinomycetes were cultivated in a medium containing from 0.15 to 0.2% of ammonium molybdate, glycerol and from 0.25 to 1% of polyol which was not assimilated by the cultures and inhibited the production of molybdenum blue in many actinomycetes. The cultures differed in their susceptibility to the inhibition by various polyols. There were not two polyols that would produce an identical effect on all of the cultures. Correlations were established in the action of polyols. The differences in the formation of molybdenum blue can be used for the differentiation and identification of actinomycetes to subdivide them into groups according to their sensitivity to inositol, mannitol, D-arabitol, xylitol, sorbitol, L-arabitol and dulcitol and according to their resistance to dulcitol (minimal, average and maximal resistance). The paper presents schemes for subdividing groups into subgroups and for establishing the properties.     

Thermal stability of beta-lactoglobulin in the presence of aqueous solution of alcohols and polyols

A systematic study concerning the effect of aqueous solution of alcohols and polyols with four carbon atoms on β-lactoglobulin stability is presented. The protein was chosen due to its functional properties and applications in food and pharmaceutical industries and because its structure and properties in aqueous solution have been widely described. The alcohols having a four carbon chain were selected to examine the effect of the gradual increase in the number of OH groups on protein stability.

Protein thermal stability in water, buffers and dilute aqueous solutions of 1-butanol, 1,2-butanediol, 1,2,4-butanetriol and 1,2,3,4-butanetetrol was evaluated by fluorescence spectroscopy. The results were used to determine the temperature range in which the unfolding process is reversible and the protein denaturation temperature in acetate buffer pH 5.5 and in the aqueous mixed solvents. Thermodynamic results show that alcohol denaturating effect diminishes gradually as the number of OH groups increase.     

Thermal stabilization of antithrombin III by sugars and sugar derivatives and the effects on nonenzymatic glycosylation

A variety of neutral and acidic sugars and related compounds were evaluated in terms of their effect on the midpoint, T_d, of the thermal denaturation curve of antithrombin III. The objectives were to determine which structural features of these molecules are responsible for their stabilizing properties and to identify more efficient stabilizers which combine the effects of lyotropic anions such as citrate with those of the polyols in a single molecule. The presence of one or more carboxylate groups in a sugar molecule invariably increased its stabilizing potency, whereas the number and position of hydroxyl groups appeared to have no influence on the molecules' stabilizing ability. Several compounds were shown to be effective in preserving antithrombin III activity during pasteurization for 10 h at 60°C. However, the presence of reducing sugars invariably resulted in a decrease in activity following pasteurization, in spite of their ablity to increase T_d. In fact, when antithrombin III was pasteurized in the presence of 2 M glucose and 0.5 M citrate, it steadily losts its ability to inhibit thrombin even though T_d under the conditions was 10°C higher than in citrate alone where activity was preserved. This effect was shown to be coincident with the covalent incorporation of glucose into the protein molecule.