Specific anions effects of on the stability of azurin in ice

This investigation represents a first attempt to gain a quantitative estimate of the effects of the anions sulfate, citrate, acetate, chloride and thiocyanate on the thermodynamic stability (DeltaG degrees) of a model globular protein in ice at -15 degrees C. The method, based on guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, distinguishes between the effects of cooling to subfreezing temperatures from those induced specifically by the formation of a solid ice phase. The results confirm that, both in liquid and frozen states, kosmotropes (sulfate, citrate and acetate) increase significantly protein stability, relative to chloride, whereas the chaotrope thiocyanate decreases it. Throughout, their stabilizing efficacy was found to rank according to the Hofmeister series, sulfate>citrate>acetate>chloride>thiocyanate, although the magnitude of Delta(DeltaG degrees) exhibited a distinct sensitivity among the anions to low temperature and to ice formation. In the liquid state, lowering the temperature from +20 to -15 degreesC weakens considerably the stabilizing efficacy of the organic anions citrate and acetate. Among the anions sulfate stands out as the only strong stabilizer at subfreezing temperatures while SCN- becomes an even stronger denaturant. Freezing of the solution in the presence the "neutral" salt NaCl destabilizes the protein, DeltaG degrees progressively decreasing up to 3-4 kcal/mol as the fraction of liquid water in equilibrium with ice (VL) is reduced to less than 1%. Kosmotropes do attenuate the decrease in protein stability in ice although in the case of citrate and acetate, their efficacy diminishes sharply as the liquid fraction shrinks to below 2.7%. On the contrary, sulfate is remarkable for it maintains constantly high the stability of azurin in liquid and frozen solutions, down to the smallest VL (0.5%) examined. Throughout, the reduction in DeltaG degrees caused by the solidification of water correlates with the decrease in the denaturant m value, an indirect indication that protein-ice interactions generally lead to partial unfolding of the native state. It is proposed that binding of the kosmotropes to the ice interface may inhibit protein adsorption to the solid phase and thereby counter the ice perturbation.     

Effects of polyols and sugars on the structure of water in concentrated gelatin gels as studied by low temperature differential scanning calorimetry

 Phase-transition temperatures, glass-transition temperatures, melting temperatures, gel–sol transition temperatures in differential scanning calorimetry heating curves of gelatin solutions with and without various sugars and polyols quenched by liquid nitrogen were studied. Both sugars and polyols added to concentrated gelatin solutions shifted the glass-transition temperatures to lower temperatures, and it was attributed to the increase of unfreezable water which acts as a plasticizer. The mechanisms of the increase in unfreezable water, however, seem to be different for sugars and polyols; sugars increase unfreezable water by increasing the number of junction zones which hold unfreezable water, while polyols by themselves increase the amount of unfreezable water. Received: 9 January 1997 Accepted: 1 July 1997     

High performance liquid chromatography of raw sugars and polyols using bonded silica gels

Summary Bonded silica columns have been evaluated for their ability to separate carbohydrates and polyols. Mobile phases consisting of dichloromethane/methanol produced the best separations in comparison with the acetonitrile/water mixtures commonly used with amino columns. Of all the bonded phases tested, LiChrospher Diol silica provided the best separations, and selectivities were not very different from those obtained on the most popular system using an amino bonded phase and acetonitrile/water as eluent. In addition, diol columns with a dichloromethane/methanol eluent offer excellent stability with no Schiffs base formation of reducing sugars. Using an evaporative light scattering detector, low limit detection is obtainable (20 ng of glucose from a column) and gradient elution is quite feasible.     

Enthalpies of solution,partial molal heat capacities and apparent molal volumes of sugars and polyols in water

Integral enthalpies of solution of some sugars and polyols in water at low concentrations have been determined calorimetrically at 25 and 35°C. These data have been used to derive heat capacities of solution C°_p at 30°C. Partial molal heat capacities C°_p,2 have been obtained by combining C°_p with C _p,2 ^* , the heat capacity of pure solid compounds. Apparent molal volumes have been obtained from density data. The sugars as well as polyols show significantly high positive C°_p and C°_p,2 values. The results have been explained in terms of a specific hydration model. The effect of substitution of-OH by glycosidic-OCH₃ and of-CHOH by deoxy-CH₂ are also discussed.     

Formation and stability of cubic ice in water droplets

There is growing evidence that a metastable phase of ice, cubic ice, plays an important role in the Earth's troposphere and stratosphere. Cubic ice may also be important in diverse fields such as cryobiology and planetary sciences. Using X-ray diffraction, we studied the formation of cubic ice in pure water droplets suspended in an oil matrix as a function of droplet size. The results show that droplets of volume median diameter 5.6 microm froze dominantly to cubic ice with stacking faults. These results support previous suggestions that cubic ice is the crystalline phase that nucleates when pure water droplets freeze homogeneously at approximately 235 K. It is also shown that as the size of the water droplets increased from 5.6 to 17.0 microm, the formation of the stable phase of ice, hexagonal ice, was favoured. This size dependence can be rationalised with heat transfer calculations. We also investigated the stability of cubic ice that forms in water droplets suspended in an oil matrix. We observe cubic ice up to 243 K, much higher in temperature than observed in many previous studies. This result adds to the existing literature that shows bulk ice I(c) can persist up to approximately 240 K. The transformation of cubic ice to hexagonal ice also showed a complex time and temperature dependence, proceeding rapidly at first and then slowing down and coming to a halt. These combined results help explain why cubic ice forms in some experiments described in the literature and not others.     

Effects of pi-bonding on stability and reactivity in

Recent synthetic and theoretical investigations of organometallic compounds containing the CpCr(NO) fragment have shown an interdependence of the electronic nature of ancillary ligands and the stability of a given Cr oxidation state. Understanding the correlation between ligand pi-bonding properties and the nature of the metal-based frontier orbitals permits the rationalization of observed reactivity patterns, and the identification and preparation of new classes of target molecules.     

Water effects on electron transfer in azurin dimers

Recent experimental and theoretical analyses indicate that water molecules between or near redox partners can significantly affect their electron-transfer (ET) properties. Here, we study the effects of intervening water molecules on the electron self-exchange reaction of azurin (Az) by using a newly developed ab-initio method to calculate transfer integrals between molecular sites. We show that the insertion of water molecules in the gap between the copper active sites of Az dimers slows down the exponential decay of the ET rates with the copper-to-copper distance. Depending on the distance between the redox sites, water can enhance or suppress the electron-transfer kinetics. We show that this behavior can be ascribed to the simultaneous action of two competing effects: the electrostatic interaction of water with the protein subsystem and its ability to mediate ET coupling pathways.     

Diffusion of polyols in aqueous solution

The diffusion coefficients of aqueous solutions of the polyols CH₂OH-(CHOH)₄-CH₂OH (dulcitol, sorbitol, and myo-inositol) have been determinated using the Gouy interferometric method. Viscosity and density measurements have been carried out at the same temperature and concentration range for these and for mannitol. The concentration dependence of the diffusion coefficients are discussed in terms of solute-solute interactions with the help of thermodynamic literature data. Among the studied polyols, only dulcitol and myo-inositol exhibit a strong evidence for solute-solute interaction, whereas the other shows a preferential interaction with the solvent.     

Role of cofactors in folding of the blue-copper protein azurin

Many proteins in living cells coordinate cofactors, such as metal ions, to attain their activity. Since the cofactors in such cases often can interact with their corresponding unfolded polypeptides in vitro, it is important to unravel how cofactors modulate protein folding. In this review, I will discuss the role of cofactors in folding of the blue-copper protein Pseudomonas aeruginosa azurin. In the case of both copper (Cu(II) and Cu(I)) and zinc (Zn(II)), the metal can bind to unfolded azurin. The residues involved in copper (Cu(II) and Cu(I)) coordination in the unfolded state have been identified as Cys112, His117, and Met121. The affinities of Cu(II), Cu(I), and Zn(II) are all higher for the folded than for the unfolded azurin polypeptide, resulting in metal stabilization of the native state as compared to the stability of apo-azurin. Cu(II), Zn(II), and several apo forms of azurin all fold in two-state kinetic reactions with roughly identical polypeptide-folding speeds. This suggests that the native-state beta-barrel topology, not cofactor interactions or thermodynamic stability, determines azurin's folding barrier. Nonetheless, copper binds much more rapidly (i.e., 4 orders of magnitude) to unfolded azurin than to folded azurin. Therefore, the fastest route to functional azurin is through copper binding before polypeptide folding; this sequence of events may be the relevant biological pathway.     

Spin-density distribution in the copper site of azurin.

A 95 GHz pulsed deuterium ENDOR study has been performed on single crystals of azurin from Pseudomonas aeruginosa selectively deuterated at the C(beta) position of the copper-coordinating cysteine 112. Complete hyperfine tensors of the two deuterium atoms have been obtained, which reveal identical isotropic parts. Analysis of the hyperfine tensors provides insight into the spin-density delocalization over the cysteine ligand. Approximately 45 % of the spin density in the paramagnetic site can be attributed to copper and 30 % to sulfur.     

Influence of hyperbranched polyesters on the surface tension of polyols

The influence of hyperbranched polyesters with different functional end groups on the surface tension of mixtures with an oligo(ester diol) was investigated. The temperature dependence of the surface tension of the pure components and of the mixtures was measured by a modified Wilhelmy balance technique. The results indicate that the surface tension of the pure hyperbranched polyesters strongly depends on the functionality of the end groups. The functionalization of the hydroxyl end groups by short alkyl chains (methyl, tert-butyl) reduced the surface tension depending on the degree of substitution. The surface tension of the mixtures with the hydroxyl-terminated hyperbranched polyester was slightly increased at higher concentrations of the hyperbranched polymer compared to the surface tension of the pure ester diol. On the other hand, the surface tension of mixtures could be considerably decreased using 1% of hyperbranched polyester polyols partially substituted with short alkyl chains. In that case, the modified hyperbranched polyesters act as surface active agents. On the molecular level, the enrichment of the modified hyperbranched polyester in the surface region was proven by X-ray photoelectron spectroscopy measurements.     

Effects of substituents on the stability of phosphoranyl radicals

The effect of substituents on the geometries, apicophilicities, radical stabilization energies, and bond dissociation energies of (*)P(CH(3))(3)X (X = CH(3), SCH(3), OCH(3), OH, CN, CF(3), Ph) were studied via high-level ab initio molecular orbital calculations. Two alternative definitions for the radical stabilization energy (RSE) were considered: the standard RSE, in which radical stability is measured relative to H-P(CH(3))(3)X, and a new definition, the alpha-RSE, which measures stability relative to P(CH(3))(2)X. We show that these alternative definitions yield almost diametrically opposed trends; we argue that alpha-RSE provides a reasonable qualitative measure of relative radical stability, while the standard RSE qualitatively reflects the relative strength of the P-H bonds in the corresponding H-P(CH(3))(3)X phosphines. The (*)P(CH(3))(3)X radicals assume a trigonal-bipyramidal structure, with the X-group occupying an axial position, and the unpaired electron distributed between a 3p(sigma)-type orbital (that occupies the position of the "fifth ligand"), and the sigma orbitals of the axial bonds. Consistent with this picture, the radical is stabilized by resonance (along the axial bonds) with configurations such as X(-) P(*+)(CH(3))(3) and X(*) P(CH(3))(3). As a result, substituents that are strong sigma-acceptors (such as F, OH, or OCH(3)) or have weak P-X bonds (such as SCH(3)) stabilize these configurations, resulting in the largest apicophilicities and alpha-RSEs. Unsaturated pi-acceptor substituents (such as phenyl or CN) are weakly stabilizing and interact with the 3p(sigma)-type orbital via a through-space effect. As part of this work, we challenge the notion that phosphorus-centered radicals are more stable than carbon-centered radicals.     

Effects of surfactants on the formation and the stability of interfacial nanobubbles

Contamination has previously been invoked to explain the flat shape and the long lifetimes of interfacial nanobubbles (INBs). In this study, the effects of surfactants on the formation and the stability of INBs were investigated when surfactants were added to the system before, during, and after the standard solvent exchange procedure (SSEP) for the formation of INBs. The solutions of sodium dodecyl sulfate (SDS) above critical micelle concentration were found to have little effect on the bubble stability. Likewise, cleaning of the substrate with a surfactant solution had little effect. In contrast, addition of a water-insoluble surfactant during the formation dramatically reduced the INBs. Finally, repeated application of SSEP to surfactant-coated substrates progressively rinsed the surfactant off the system. Thus, we found no evidence to support the hypothesis that (1) INBs are stabilized by a layer of insoluble organic contaminant or that (2) SSEP introduces surface-active materials to the system that could stabilize INBs.     

Separation behaviour of nitroxylene isomers on polyols and polyethers in gas-liquid chromatography

Of the six nitroxylene isomers, 2,4- and 2,3-nitroxylene are the most difficult to separate by GLC, but they have been effectively separated on various acidic polyols as stationary phases. Although polyethylene glycol could not generally separate the 2,4- and 2,3-nitroxylene isomers, polyethylene glycol 20M could, and polypropylene glycol could separate all the six isomers effectively.     

Effects of operating parameters on countercurrent extraction of hemicellulosic sugars from pretreated softwood

In a previous study using a continuous countercurrent screw extractor for two-stage dilute-acid hydrolysis, which was focused on the effects of liquid-to-insoluble solids (L/IS) ratio, we demonstrated that by using low volumes of wash water soluble sugars can be recovered from first-stage pretreated softwood at high yields and also at high sugar concentrations. In this study, we investigated the effects of important operating parameters other than the L/IS ratio, such as the feed rates of water and pretreated biomass and the extractor inclined angle, on the performance of the extractor using first-stage pretreated softwood. As biomass and water feed rates increased at the same L/IS ratio, the recovery yield of soluble sugars decreased, probably owing to a reduced solids residence time in the extractor, which is related to the solid/liquid contact time. The sugar recovery yield was higher at a higher extractor inclined angle. This may be attributed to the effects of increased back mixing and a longer residence time for solids at a higher extractor angle. Countercurrent extraction was also carried out with other pretreated biomass having smaller particle sizes and poor drainage rates. The countercurrent screw extractor was found to be unsuitable for these fine materials due to the slow liquid drainage rate and filter-clogging problems. In a test for stability of soluble sugars in first-stage softwood hydrolysate, irrespective of the storage temperature and storage form, the sugar concentration slowly decreased with storage time. However, storage in slurry form showed higher sugar stability compared with that in liquor form at the same conditions.

     

Effects of polymer solutions on colloid stability

Some theoretical properties of a semidilute polymer solution (in a good solvent) near a repulsive wall (no adsorption) are discussed. (i) Near a single wall, the existence of a depletion layer (where the concentration is strongly reduced) with thickness equal to the correlation length ξ(c) is predicted. Scaling laws are obtained for the concentration c_s at the first layer as a function of the bulk concentration c, and for the concentration profile c(z) at distance z from the wall. (ii) When two parallel walls are separated by a slab of polymer solution (thickness d), there is an attractive interaction between the plates. The range of the attraction is ca. 3ξ(c) and can be varied by suitable choice of concentration. It may lead to destabilization of certain colloidal systems.