Structure and phase behavior of a confined nanodroplet composed of the flexible chain molecules

A polymer density functional theory has been employed for investigating the structure and phase behaviors of the chain polymer, which is modelled as the tangentially connected sphere chain with an attractive interaction, inside the nanosized pores. The excess free energy of the chain polymer has been approximated as the modified fundamental measure-theory for the hard spheres, the Wertheim's first-order perturbation for the chain connectivity, and the mean-field approximation for the van der Waals contribution. For the value of the chemical potential corresponding to a stable liquid phase in the bulk system and a metastable vapor phase, the flexible chain molecules undergo the liquid-vapor transition as the pore size is reduced; the vapor is the stable phase at small volume, whereas the liquid is the stable phase at large volume. The wide liquid-vapor coexistence curve, which explains the wide range of metastable liquid-vapor states, is observed at low temperature. The increase of temperature and decrease of pore size result in a narrowing of liquid-vapor coexistence curves. The increase of chain length leads to a shift of the liquid-vapor coexistence curve towards lower values of chemical potential. The coexistence curves for the confined phase diagram are contained within the corresponding bulk liquid-vapor coexistence curve. The equilibrium capillary phase transition occurs at a higher chemical potential than in the bulk phase.     

Influence of protein and stationary phase properties on protein-matrix-interaction in cation exchange chromatography

A large number of different stationary phases for ion-exchange chromatography from different manufacturers are available, which vary significantly in a number of chemical and physical properties. As a consequence, binding mechanisms may be different as well. In the work reported here, the retention data of model proteins (lysozyme, cytochrome c and two monoclonal antibodies) were determined for nine commercially available cation-exchange adsorbents. The linear gradient elution model in combination with a thermodynamic approach was used to analyse the characteristic parameters of the protein-stationary phase-interactions. Based on the pH dependency of the characteristic charge and the equilibrium constant for binding the differences between the standard Gibbs energies in the adsorbed and the solute state for the protein ΔG(P)° and the salt ΔG(S)° were calculated. The characteristic charge B of the proteins strongly depends on the molecular mass of the protein. For small proteins like lysozyme there is almost no influence of the stationary phase chemistry on B, while for the Mabs the surface modification strongly influences the B value. Surface extenders or tentacles usually increase the B values. The variation of the characteristic charge of the MABs is more pronounced the lower the pH value of the mobile phase is, i.e. the higher the negative net charge of the protein is. The standard Gibbs energy changes for the proteins ΔG(P)° are higher for the Mabs compared to lysozyme and more strongly depend on the stationary phase properties. Surface modified resins usually show higher ΔG(P)° and higher B values. A correlation between ΔG(P)° and B is not observed, indicating that non-electrostatic interactions as well as entropic factors are important for ΔG(P)° while for the B values the accessibility of binding sites on the protein surface is most important.     

Investigation of the phase behavior of an embedded charge protein model through molecular simulation

The phase behavior of an embedded-charge model for lysozyme developed by Carlsson and co-workers (J. Phys. Chem. B 2001, 105, 9040) is investigated using grand canonical transition matrix Monte Carlo simulation. Within this model, protein-protein interactions are approximated through a combination of hard-sphere repulsion, isotropic hydrophobic attraction, and screened electrostatic interactions through a series of embedded point charges located at the positions of charged amino acid groups within lysozyme. Liquid-liquid phase diagrams are constructed for a wide range of solution conditions and compared with experimental data. Our results indicate that the model is generally capable of describing qualitative trends in the evolution of protein phase behavior with variation of pH and ionic strength. From a quantitative perspective, model estimates for both the change in critical temperature with variation of the solution conditions and the critical concentration do not agree with experimental results. We find the width of model coexistence curves to be independent of solution conditions and narrow relative to experimentally obtained phase envelopes. Connections between the value of the second virial coefficient evaluated at the critical temperature and the location of the liquid-liquid phase envelope are also examined.     

The effect of salt on protein chemical potential determined by ternary diffusion in aqueous solutions

We use accurate thermodynamic derivatives extracted from high-precision measurements of the four volume-fixed diffusion coefficients in ternary solutions of lysozyme chloride in aqueous NaCl, NH4Cl, and KCl at pH 4.5 and 25 degrees C to (a) assess the relative contributions of the common-ion and nonideality effects to the protein chemical potential as a function of salt concentration, (b) compare the behavior of the protein chemical potential for the three salts, which we found to be consistent with the Hofmeister series, and (c) discuss our thermodynamic data in relation to the dependence of the protein solubility on salt concentration. The four diffusion coefficients are reported at 0.6 mM lysozyme chloride and 0.25, 0.5, 0.9, 1.2, and 1.5 M KCl and extend into the protein-supersaturated region. The chemical potential cross-derivatives are extracted from diffusion data using the Onsager reciprocal relation and the equality of molal cross-derivatives of solute chemical potentials. They are compared to those calculated previously from diffusion data for lysozyme in aqueous NaCl and NH4Cl. We estimate the effective charge on the diffusing lysozyme cation at the experimental concentrations. Our diffusion measurements on the three salts allowed us to analyze and interpret the four diffusion coefficients for charged proteins in the presence of 1:1 electrolytes. Our results may provide guidance to the understanding of protein crystallization.     

Effective interactions in lysozyme aqueous solutions: a small-angle neutron scattering and computer simulation study

We report protein-protein structure factors of aqueous lysozyme solutions at different pH and ionic strengths, as determined by small-angle neutron scattering experiments. The observed upturn of the structure factor at small wavevectors, as the pH increases, marks a crossover between two different regimes, one dominated by repulsive forces, and another one where attractive interactions become prominent, with the ensuing development of enhanced density fluctuations. In order to rationalize such experimental outcome from a microscopic viewpoint, we have carried out extensive simulations of different coarse-grained models. We have first studied a model in which macromolecules are described as soft spheres interacting through an attractive r(-6) potential, plus embedded pH-dependent discrete charges; we show that the uprise undergone by the structure factor is qualitatively predicted. We have then studied a Derjaguin-Landau-Verwey-Overbeek (DLVO) model, in which only central interactions are advocated; we demonstrate that this model leads to a protein-rich/protein-poor coexistence curve that agrees quite well with the experimental counterpart; experimental correlations are instead reproduced only at low pH and ionic strengths. We have finally investigated a third, "mixed" model in which the central attractive term of the DLVO potential is imported within the distributed-charge approach; it turns out that the different balance of interactions, with a much shorter-range attractive contribution, leads in this latter case to an improved agreement with the experimental crossover. We discuss the relationship between experimental correlations, phase coexistence, and features of effective interactions, as well as possible paths toward a quantitative prediction of structural properties of real lysozyme solutions.     

Net charge and electrophoretic mobility of lysozyme charge ladders in solutions of nonionic surfactant

We report on the electrophoretic mobility and on the thermal diffusion of lysozyme proteins dissolved in aqueous solutions of a nonionic surfactant (C12E6) at a wide range of concentrations of the surfactant (0-20% by weight). We want to estimate the influence of a dense network of elongated micelles of C12E6 on the effective charge of the proteins as observed in the capillary electrophoresis experiments. The possible mechanism leading to the change in the effective charge of protein could involve the deformation of the cloud of counterions around the protein when it squeezes through the narrow (of the order of a protein diameter) aqueous channels formed in the solution of elongated micelles. The combination of independent measurements of the electrophoretic mobility of a family of modified proteins (lysozyme charge ladder [Colton et al. J. Am. Chem. Soc. 1997, 119, 12701]), of the microviscosity of the solutions of surfactant (obtained via fluorescence correlation spectroscopy), and of the hydrodynamic radius of the proteins (photon correlation spectroscopy) allow us to conclude that the effective charge of the proteins is not affected by the presence of surfactant, even at high concentrations.     

硅钨酸盐的合成及其在醇的四氢吡喃化反应中的催化性能

用硅钨酸分别与硝酸铜、硝酸锌和硝酸铝采用复分解法合成了相应的硅钨酸盐．结合红外光谱、紫外光谱和热重等分析手段对这三种硅钨酸盐结构进行了表征,并考察了三种硅钨酸盐在醇的四氢毗喃化反应中的催化性能．结果表明：用0．7g具有Keggin结构的硅钨酸盐作催化剂,在室温无溶剂条件下,催化二氢吡喃（33mmol）与醇（30mmol）的反应,反应条件温和,工艺简单,催化效果好,无副产物生成．     

Influence of microenvironment and liposomal formulation on secondary structure and bilayer interaction of lysozyme

The conformation of peptide and protein drugs in various microenvironments and the interaction with drug carriers such as liposomes are of considerable interest. In this study the influence of microenvironments such as pH, salt concentration, and surface charge on the secondary structure of a model protein, lysozyme, either in solution or entrapped in liposomes with various molar ratios of phosphatidylcholine (PC):cholesterol (Chol) was investigated. It was found that entrapment efficiency was more pronounced in negatively charged liposomes than in non-charged liposomes, which was independent of Chol content and pH of hydration medium. The occurrence of aggregation, decrease in zeta potential, and alteration of ³¹P NMR chemical shift of negatively charged lysozyme liposomes compared to blank liposomes suggested that the electrostatic interaction plays a major role in protein–lipid binding. Addition of sodium chloride could impair the neutralizing ability of positively charged lysozyme on negatively charged membrane via chloride counterion binding. Neither lysozyme in various buffer solutions with sodium chloride nor that entrapped in liposomes showed any significant change in their secondary structures. However, significant decrease in α-helical content of lysozyme in non-charged liposomes at higher pH and salt concentrations was discovered.     

Liquid-liquid coexistence surface for lysozyme: role of salt type and salt concentration

The liquid-liquid phase separation curves for lysozyme in a salt solution are known to depend on salt type and salt concentration. For the case of monovalent cations, the cloud point temperature typically increases with increasing salt concentration, for fixed lysozyme concentration. For the case of divalent cations, however, a maximum in the cloud point temperature is observed that has been interpreted as being due to ion binding to the protein surface and subsequent water structuring. In this paper, we use a simple square well model due to Grigsby et al. (Biophys. Chem. 2001, 91, 231-243), whose well depth depends on salt type and salt concentration, to determine the phase coexistence surfaces from experimental data. The surfaces are shown as a function of temperature, salt concentration, and protein concentration for two typical salts, NaCl and MgCl2. These surfaces are calculated using the results of a single standard Monte Carlo simulation and a simple scaling argument and are in reasonably good agreement with known experimental results.     

Protein-lipid interactions at the air/water interface

Surface pressure measurements and external reflection FTIR spectroscopy have been used to probe protein-lipid interactions at the air/water interface. Spread monomolecular layers of stearic acid and phosphocholine were prepared and held at different compressed phase states prior to the introduction of protein to the buffered subphase. Contrasting interfacial behaviour of the proteins, albumin and lysozyme, was observed and revealed the role of both electrostatic and hydrophobic interactions in protein adsorption. The rate of adsorption of lysozyme to the air/water interface increased dramatically in the presence of stearic acid, due to strong electrostatic interactions between the negatively charged stearic acid head group and lysozyme, whose net charge at pH 7 is positive. Introduction of albumin to the subphase resulted in solubilisation of the stearic acid via the formation of an albumin-stearic acid complex and subsequent adsorption of albumin. This observation held for both human and bovine serum albumin. Protein adsorption to a PC layer held at low surface pressure revealed adsorption rates similar to adsorption to the bare air/water interface and suggested very little interaction between the protein and the lipid. For PC layers in their compressed phase state some adsorption of protein occurred after long adsorption times. Structural changes of both lysozyme and albumin were observed during adsorption, but these were dramatically reduced in the presence of a lipid layer compared to that of adsorption to the pure air/water interface.     

The evaporation/condensation transition of liquid droplets

The condensation of a supersaturated vapor enclosed in a finite system is considered. A phenomenological analysis reveals that the vapor is found to be stable at densities well above coexistence. The system size at which the supersaturated vapor condenses into a droplet is found to be governed by a typical length scale which depends on the coexistence densities, temperature and surface tension. When fluctuations are neglected, the chemical potential is seen to show a discontinuity at an effective spinodal point, where the inhomogeneous state becomes more stable than the homogeneous state. If fluctuations are taken into account, the transition is rounded, but the slope of the chemical potential versus density isotherm develops a discontinuity in the thermodynamic limit. In order to test the theoretical predictions, we perform a simulation study of droplet condensation for a Lennard-Jones fluid and obtain loops in the chemical potential versus density and pressure. By computing probability distributions for the cluster size, chemical potential, and internal energy, we confirm that the effective spinodal point may be identified with the occurrence of a first order phase transition, resulting in the condensation of a droplet. An accurate equation of state is employed in order to estimate the droplet size and the coexisting vapor density and good quantitative agreement with the simulation data is obtained. The results highlight the need of an accurate equation of state data for the Laplace equation to have predictive power.     

The Wolf method applied to the liquid-vapor interface of water

The Wolf method for the calculation of electrostatic interactions is applied in a liquid phase and at the liquid-vapor interface of water and its results are compared with those from the Ewald sums method. Molecular dynamics simulations are performed to calculate the radial distribution functions at room temperature. The interface simulations are used to obtain the coexisting densities and surface tension along the coexistence curve. The water model is a flexible version of the extended simple point charge model. The Wolf method gives good structural results, fair coexistence densities, and poor surface tensions as compared with those obtained using the Ewald sums method.     

Grand canonical ensemble molecular dynamics simulation of water solubility in polyamide-6,6

Grand canonical ensemble molecular dynamics simulation is employed to calculate the solubility of water in polyamide-6,6. It is shown that performing two separate simulations, one in the polymeric phase and one in the gaseous phase, is sufficient to find the phase coexistence point. In this method, the chemical potential of water in the polymer phase is expanded as a first-order Taylor series in terms of pressure. Knowing the chemical potential of water in the polymer phase in terms of pressure, another simulation for water in the gaseous phase, in the grand canonical ensemble, is done in which the target chemical potential is set in terms of pressure in the gas phase. The phase coexistence point can easily be calculated from the results of these two independent simulations. Our calculated sorption isotherms and solubility coefficients of water in polyamide-6,6, over a wide range of temperatures and pressures, agree with experimental data.     

Modulation of lysozyme charge influences interaction with phospholipid vesicles

Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between charge state of the protein and its interaction with negatively charged phospholipid membranes chemical modifications of the proteins were carried out. Succinylation and carbodiimide modification was used to shift the isoelectric point of lysozyme to lower and higher pH values, respectively. The binding of the modified lysozyme to phospholipid vesicles prepared from phosphatidic acid (PA) was determined using microelectrophoresis and ultracentrifugation. At acidic pH of the solution all lysozyme species reduced the surface charges of PA vesicles. Succinylated lysozyme (succ lysozyme) reduced the electrophoretic mobility (EPM) to nearly zero, whereas native lysozyme and carboxylated lysozyme (carbo lysozyme) changed the surface charge to positive values. At neutral pH, the reduction of surface charges was less for carbo lysozyme and unmodified lysozyme. Succ lysozyme did not change the EPM. Unmodified and carbo lysozyme decreased the magnitude of EPM, but the whole complex was still negatively charged. The bound fraction of all modified lysozyme to PA vesicles at high lysozyme/PA ratios was nearly constant at acidic pH. At low lysozyme/PA ratios the extent of bound lysozyme is changed in the order carbo>unmodified>succ lysozyme. Increasing the pH, the extent of bound lysozyme to PA large unilamellar vesicles (LUV) is reduced, at pH 9.0 only 35% of carbo lysozyme, 23% of unmodified lysozyme is bound, whereas succ lysozyme does not bind at pH 7.4 and 9.0. At low pH, addition of all lysozyme species resulted in a massive aggregation of PA liposomes, at neutral pH aggregation occurs at much higher lysozyme/PA ratios. Lysozyme binding to PA vesicles is accompanied by the penetration of lysozyme into the phospholipid membrane as measured by monolayer techniques. The penetration of lysozyme into the monolayer was modulated by pH and ionic strengths. The interaction of lysozyme with negatively charged vesicles leads to a decrease of the phospholipid vesicle surface hydration as measured by the shift of the maximum of the fluorescence signal of a headgroup labeled phospholipid. The binding of bis-ANS as an additional indicator for the change of surface hydrophobicity is increased at low pH after addition of lysozyme to the vesicles. More hydrophobic patches of the lysozyme-PA complex are exposed at low pH. At low pH the binding process of lysozyme to PA vesicles is followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the vesicle aqueous content. The extent of lysozyme interaction with PA LUV at neutral and acidic pH is in the order carbo lysozyme>lysozyme>succ lysozyme.     

An electrospray ionization mass spectrometry investigation of 1-anilino-8-naphthalene-sulfonate (ANS) binding to proteins

The binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to various globular proteins at acidic pH has been investigated by electrospray ionization mass spectrometry (ESI-MS). Maximal ANS binding is observed in the pH range 3-5. As many as seven species of dye-bound complexes are detected for myoglobin. Similar studies were carried out with cytochrome c, carbonic anhydrase, triosephosphate isomerase, lysozyme, alpha-lactalbumin, and bovine pancreatic trypsin inhibitor (BPTI). Strong ANS binding was observed wherever molten globule states were postulated in solution. ANS binding is not observed for lysozyme and BPTI, which have tightly folded structures in the native form. Alpha-lactalbumin, which is structurally related to lysozyme but forms a molten globule at acidic pH, exhibited ANS binding. Reduction of disulfide bonds in these proteins leads to the detection of ANS binding even at neutral pH. Binding was suppressed at very low pH (<2.5), presumably due to neutralization of the charge on the sulfonate moiety. The distribution of the relative intensities of the protein bound ANS species varies with the charge state, suggesting heterogeneity of gas phase conformations. The binding strength of these complexes was qualitatively estimated by dissociating them using enhanced nozzle skimmer potentials. The skimmer voltages also affected the lower and higher charge states of these complexes in a different manner.     

Phase behavior of mixtures of oppositely charged nanoparticles: heterogeneous Poisson-Boltzmann cell model applied to lysozyme and succinylated lysozyme

We study the phase behavior of mixtures of oppositely charged nanoparticles, both theoretically and experimentally. As an experimental model system we consider mixtures of lysozyme and lysozyme that has been chemically modified in such a way that its charge is nearly equal in magnitude but opposite in sign to that of unmodified lysozyme. We observe reversible macroscopic phase separation that is sensitive not only to protein concentration and ionic strength, but also to temperature. We introduce a heterogeneous Poisson-Boltzmann cell model that generally applies to mixtures of oppositely charged nanoparticles. To account for the phase behavior of our experimental model system, in addition to steric and electrostatic interactions, we need to include a temperature-dependent short-ranged interaction between the lysozyme molecules, the exact origin of which is unknown. The strength and temperature dependence of the short-ranged attraction is found to be of the same order of magnitude as that between unmodified lysozyme molecules. The presence of a rather strong short-ranged attraction in our model system precludes the formation of colloidal liquid phases (or complex coacervates) such as those typically found in mixtures of globular protein molecules and oppositely charged polyelectrolytes.     

Thermosensitive hard spheres

We demonstrate an extension of a UV-Vis spectroscopy method to determine the phase boundaries for thermosensitive colloids as an alternative to the time-consuming sedimentation method. The Bragg attenuation peak from colloidal crystallites was monitored during the quasi-equilibrium colloidal crystal melting. The melting and freezing boundaries of the coexistence region were determined via a blue shift of Bragg's peak and the disappearance of peak area. We confirm this method using poly(N-isopropylacrylamide) (PNIPAM) particles at different charge densities and temperatures far below the lower critical solution temperature. At low pH, the particles behave as thermosensitive hard spheres.     

Simulation of chemical potentials and phase equilibria in two- and three-dimensional square-well fluids: finite size effects

We study the simulation cell size dependence of chemical potential isotherms in subcritical square-well fluids by means of series of canonical ensemble Monte Carlo simulations with increasing numbers of particles, for both three-dimensional bulk systems and two-dimensional planar layers, using Widom-like particle insertion methods. By estimating the corresponding vapor/liquid coexistence densities using a Maxwell-like equal area rule for the subcritical chemical potential isotherms, we are able to study the influence of system size not only on chemical potentials but also on the coexistence properties. The chemical potential versus density isotherms show van der Waals-like loops in the subcritical vapor/liquid coexistence range that exhibit distinct finite size effects for both two- and three-dimensional fluids. Generally, in agreement with recent findings for related studies of Lennard-Jones fluids, the loops shrink with increasing number of particles. In contrast to the subcritical isotherms themselves, the equilibrium vapor/liquid densities show only a weak system size dependence and agree quantitatively with the best-known literature values for three-dimensional fluids. This allows our approach to be used to accurately predict the phase coexistence properties. Our resulting phase equilibrium results for two-dimensional square-well fluids are new. Knowledge concerning finite size effects of square-well systems is important not only for the simulation of thermodynamic properties of simple fluids, but also for the simulation of models of more complex fluids (such as aqueous or polymer fluids) involving square-well interactions.     

Role of electromechanical and mechanoelectric effects in protein hydration under hydrostatic pressure

Recent measurements of lysozyme hydration water density under non-denaturing pressure show that it is higher than that of bulk water in the same conditions. High protein hydration layer density has earlier been observed at ambient conditions and ascribed to electrostriction. We calculate the pressure-induced protein mean surface charge density increment Δσ. Within the hydration layer, the higher fields due to Δσ lead to an additional water compression via electrostriction. The increment Δσ is considered as due to a mechanoelectric effect in protein molecules. The mean value of the effective mechanoelectric coefficient d is calculated and compared with piezoelectric coefficients of amino acids and their compounds.