A small-angle scattering study on equilibrium clusters in lysozyme solutions

We use small-angle scattering experiments to investigate the structural properties of aqueous lysozyme solutions under conditions where the existence of equilibrium clusters has recently been demonstrated (Nature 2004, 432, 492). We also discuss the possible emergence of a low angle scattering contribution, which recently attracted interest due to its appearance in solutions of various proteins. We demonstrate that in lysozyme solutions under our experimental conditions such rising low q intensities can only be observed under special circumstances and can thus not be attributed to the existence of a universal long-range attraction. We then focus on the structural properties of the equilibrium clusters as a function of protein concentration, temperature, and ionic strength. We show that the experimental structure factors obtained from the scattering measurements exhibit the typical cluster-cluster peak q(c) reflecting the mean distance between charged clusters as well as a monomer-monomer peak q(m), which represents the nearest neighbor shell of monomers within a single cluster. The underlying principle for the formation of these structures is the coexistence of two opposing forces, a short-range attraction and a long-range repulsion due to residual charges. We can quantitatively analyze our scattering data by applying a simple equilibrium cluster model and calculate an average cluster aggregation number, N(c). The thus obtained cluster aggregation number increases linearly with volume fraction. We also observe an increasing N(c) as temperature decreases and as the screening of residual charges increases. We point out the importance of the existence of equilibrium clusters and the universality of this phenomenon for self-assembling processes observed in nature. Finally, we discuss the limitations of our simple globular cluster model in view of recent findings from computer simulations.     

Studying Aggregate in Lysozyme Solution by Atomic Force Microscope

The aggregates in lysozyme solution with different NaCl concentration were investigated by Atomic Force Microscope(AFM).The AFM images show that there exist lysozyme monomers,n-mers and clusters in lysozyme solution when the conditions are not suiable for crystal growth.In favorable conditions for crystal growth,the lysozyme clusters disappear and almost only monomers exist in solution.     

Polyelectrolyte-protein complexes: structure and conformation of each specie revealed by SANS

We report here the structure of complexes made of proteins (lysozyme, positively charged) and polyelectrolytes (PSSNa, negatively charged). We stay in conditions where the volume fractions of the components are of the same order and where PSS concentrations correspond to a semidilute regime. The final complexes structure is determined by SANS. We obtain three main types of structures: (i) For a protein excess and for long polyelectrolyte chains, the network preformed by PSS chains still exists but chains are partially shrunk due to cross-linking by lysozyme. Macroscopically, samples are gelled. (ii) For a protein excess and for short polyelectrolyte chains, PSS chains are locally shrunk and do not form a network anymore. Lysozyme and PSS chains are embedded in dense 3-D aggregates that arrange in a fractal network at a larger scale. Macroscopically, samples are liquid. (iii) For a polyelectrolyte excess and whatever the chain length, the internal structure of the lysozyme changes. After an initial strong electrostatic binding, lysozyme is progressively unfolded thanks to a hydrophobic contact with PSS. The two chainlike objects are finally organized in a homogeneous costructure. Macroscopically, samples are liquids.     

Protein-polyelectrolyte cluster formation and redissolution: a Monte Carlo study

Aqueous solutions of proteins and oppositely charged polyelectrolytes were studied at different polyelectrolyte chain length, ionic strength, and protein-protein interaction potential as a function of the polyelectrolyte concentration. One of the protein models used represented lysozyme in aqueous environment. The model systems were solved by Monte Carlo simulations, and their properties were analyzed in terms of radial distribution functions, structure factors, and cluster composition probabilities. In the system with the strongest electrostatic protein-polyelectrolyte interaction the largest clusters were formed near or at equivalent amount of net protein charge and polyelectrolyte charge, whereas in excess of polyelectrolyte a redissolution appeared. Shorter polyelectrolyte chains and increased ionic strength lead to weaker cluster formation. An inclusion of nonelectrostatic protein-protein attraction promoted the protein-polyelectrolyte cluster formation.     

Online concentration and separation of basic proteins using a cationic polyelectrolyte in the presence of reversed electroosmotic flow

We report an online concentration and separation method for basic proteins using poly(diallyldimethylammonium chloride) (PDDA) solutions in the presence of reversed EOF. Using a capillary dynamically coated with 2% PDDA containing 0.1 M NaCl and filled with 1.2% PDDA under neutral conditions (10 mM phosphate, pH 7.0), we have demonstrated the separation of six basic proteins with peak efficiencies ranging from 175 000 to 616 000 plates/m and RSDs of migration time less than 0.4%. Additionally, high-speed separation of six basic proteins (<7 min) was achieved using a short capillary filled with 0.6% PDDA solutions. Under injection of the large-volume sample (210 nL), the LODs at S/N of 3 for basic proteins are down to nanomolar range. For example, the LOD for lysozyme is 1.2 nM, which is a 260-fold sensitivity enhancement compared with conventional injection method. The proposed method has been applied to the stacking of lysozyme in human saliva samples. Without any pretreatment, we also demonstrated the capability of this method to detect low amounts of peptide samples through the stacking of tryptic peptide of myoglobin. The experimental results indicate that our proposed method has great potential for use in clinical diagnosis and proteomics applications.     

Colloidal aggregation induced by long range attractions

The structure of colloidal clusters formed by long-range attractive interactions under diluted conditions is studied by means of Monte Carlo simulations. For a not-too-long attraction range, clusters show self-similar internal structure with lower density than that typical for diffusive aggregation. For long-range interactions, low kappa, nonfractal clusters are formed (dense at short scales but open at long ones). The dependence on the volume fraction shows that more-compact clusters are grown the higher the colloidal density for diffusive aggregation and attraction-driven aggregation in the fractal regime. The whole trend is explained in terms of the interpenetration among aggregates. In attraction-driven aggregations, the interpenetration of clusters competes with aggregation in the tips of the clusters, causing low-density clusters.     

Effect of the hydrophilic size on the structural phases of aqueous nonionic gemini surfactant solutions

Aggregate structures of aqueous nonionic Gemini surfactant solutions, alpha,alpha'-[2,4,7,9-tetramethyl-5-decyne-4,7-diyl]bis[omega-hydroxyl-polyoxyethylene] with three different length polyoxyethylenes (i.e., 10, 20, and 30 ethylene oxide monomers, denoted from now on as S-10, S-20, and S-30, respectively), are investigated using small angle neutron scattering, dynamic light scattering, and fluorescence spectroscopy. For S-10 at low surfactant concentrations (Cs < 0.9 wt %), large "clusters", with an average hydrodynamic radius (RH) > 40 nm, are found to coexist with monomers. At intermediate Cs (0.9 < Cs < 2 wt %), some clusters break down forming micelles, with an (RH) approximately 2-3 nm, while the remaining clusters coexist with micelles. Increasing Cs further (>2 wt %) results in a pure micellar phase with little or no clusters present. S-20 and S-30 mixtures, on the other hand, differ from S-10 in that irrespective of surfactant concentration, large clusters and small monomers/dimers are found to coexist, while there is no direct evidence for the presence of micelles.     

Study on osmotic pressures for aqueous lysozyme and alpha-chymotrypsin-electrolyte solutions with two Yukawa potentials

The equation of state (EOS) of Duh and Mier-y-Terán for one Yukawa potential is expanded to two Yukawa potentials to describe the nonidealities of the charged lysozyme and alpha-chymotrypsin solutions. Instead of the classical DLVO theory, the attractive dispersion and double-layer repulsion interactions are represented by two Yukawa potentials, respectively. For the aqueous lysozyme solutions, the only adjustable dispersion energy parameter epsilon/k is regressed and the average deviation is 1.76%. For the aqueous alpha-chymotrypsin solutions, two adjustable parameters (the molecular weight and dispersion energy parameter) are regressed and the average deviation is 7.62%. Some correlation and prediction results are discussed.     

Structure, magnetic, and electric properties of bismuth niobates doped with d elements: I. Magnetic behavior of chromium-containing solid solutions of low-and high-temperature bismuth orthoniobate

Magnetic susceptibility of solid solutions of bismuth orthoniobates of low-and high-temperature modifications containing chromium was studied. Parameters of exchange interactions in the dimeric clusters of nickel atoms and also the distribution of monomers and dimers in the solid solutions were calculated. The crystal structure of bismuth orthoniobate of the high-temperature modification is characterized by a greater tolerance to the formation of oxygen vacancies, resulting from heterovalent substitution.     

Structure, stability, and mobility of small Pd clusters on the stoichiometric and defective TiO2 (110) surfaces

We report on the structure and adsorption properties of Pd(n) (n = 1-4) clusters supported on the rutile TiO(2) (110) surfaces with the possible presence of a surface oxygen vacancy or a subsurface Ti-interstitial atom. As predicted by the density functional theory, small Pd clusters prefer to bind to the stoichiometric titania surface or at sites near subsurface Ti-interstitial atoms. The adsorption of Pd clusters changes the electronic structure of the underlying surface. For the surface with an oxygen vacancy, the charge localization and ferromagnetic spin states are found to be largely attenuated owing to the adsorption of Pd clusters. The potential energy surfaces of the Pd monomer on different types of surfaces are also reported. The process of sintering is then simulated via the Metropolis Monte Carlo method. The presence of oxygen vacancy likely leads to the dissociation of Pd clusters. On the stoichiometric surface or surface with Ti-interstitial atom, the Pd monomers tend to sinter into larger clusters, whereas the Pd dimer, trimer, and tetramer appear to be relatively stable below 600 K. This result agrees with the standard sintering model of transition metal clusters and experimental observations.     

Protein adsorption and transport in agarose and dextran-grafted agarose media for ion exchange chromatography: Effect of ionic strength and protein characteristics

This work investigates the effects of ionic strength and protein characteristics on adsorption and transport of lysozyme, BSA, and IgG in agarose-based cation exchangers with short ligand chemistry and with charged dextran grafts. In all cases, the adsorption equilibrium capacity decreased with increasing salt. However, the adsorption kinetics was strongly influenced by the adsorbent structure and protein characteristics. For the smaller and positively charged lysozyme, the effective pore diffusivity was only weakly dependent on salt for the dextran-free media, but declined sharply with salt for the dextran-grafted materials. For this protein, the dextran grafts enhanced the adsorption kinetics at low salt, but the enhancement vanished at higher salt concentrations. For BSA, which was near its isoelectric point for the experimental conditions studied, the effective diffusivity was low for all materials and almost independent of salt. Finally, for the larger and positively charged IgG, the effective diffusivity varied with salt, reaching an apparent maximum at intermediate concentrations for both dextran-free and dextran-grafted media with the kinetics substantially enhanced by the dextran grafts for these conditions. Microscopic observations of the particles during protein adsorption at low ionic strengths showed transient patterns characterized by sharp adsorption fronts for all materials. A theory taking into account surface or adsorbed phase diffusion with electrostatic coupling of diffusion fluxes is introduced to explain the mechanism for the enhanced adsorption kinetics observed for the positively charged proteins.     

Structure and magnetic and electric properties of bismuth niobates doped with <Emphasis Type="Italic">d</Emphasis>-elements: III. Magnetic and electric properties of copper-containing solid solutions of bismuth orthoniobate

Magnetic susceptibility and electrophysical properties of copper-containing solid solutions of the low-temperature modification of bismuth orthoniobate were studied. Parameters of exchange interactions in dimeric copper clusters and distribution of monomers and dimers in the solutions were calculated. The exchange parameter of antiferromagnetic interactions in dimeric copper clusters was found to be 170 cm⁻¹. Original Russian Text N.A. Zhuk, I.V. Piir, N.V. Chezhina, 2007, published in Zhurnal Obshchei Khimii, 2007, Vol. 77, No. 2, pp. 240–245. For communication II, see [1].     

Structure, magnetic, and electric properties of bismuth niobates doped with d elements: II. State of nickel atoms in solid solutions of low-and high-temperature bismuth orthoniobate

The magnetic susceptibility of nickel-doped solid solutions of bismuth orthoniobate of the low-and high-temperature modifications was studied. Parameters of exchange interactions in dimeric clusters of nickel atoms and also the distribution of monomers and dimers in the solid solutions were calculated. The nickel-containing solid solutions of low-and high-temperature bismuth orthoniobate are similar in magnetic behavior.     

Aggregation-fragmentation in a model of DNA-mediated colloidal assembly

We present results from an off-lattice Monte Carlo simulation of DNA-mediated colloidal assembly. In this simulation, the aggregation-fragmentation of a binary mixture of DNA-coated colloidal particles is studied through a simplified model of base-pair hybridization. Bonding between monomers is modeled as a simple temperature-sensitive A/B-type interaction, where type A and B monomers can bond to only the opposite type (no A/A or B/B attachments are allowed). The actual chemistry of base-pair hybridization is not included in the model. The morphological structures of the clusters formed as well as the kinetics of growth are analyzed in our 2D simulations. The fractal dimension and kinetic growth exponents for clusters formed near the DNA "melting" temperature agree with those seen previously for 2D diffusion-limited cluster aggregation (DLCA) models. The clusters appear more compact, exhibiting signs of local order at intermediate temperature values. At higher temperatures, the formation of large clusters is not favorable under the action of temperature-dependent fragmentation, and the system eventually reaches a steady state as a collection of small aggregates. The temperature profile for this dissolution of the colloidal assembly is sharp, indicating that the selective hybridization process provides a highly sensitive measurement tool. At high temperatures, we analyze the steady-state behavior of the average cluster size in terms of an aggregation-fragmentation model.     

Exploring the interfacial structure of protein adsorbates and the kinetics of protein adsorption: an in situ high-energy X-ray reflectivity study

The high energy X-ray reflectivity technique has been applied to study the interfacial structure of protein adsorbates and protein adsorption kinetics in situ. For this purpose, the adsorption of lysozyme at the hydrophilic silica-water interface has been chosen as a model system. The structure of adsorbed lysozyme layers was probed for various aqueous solution conditions. The effect of solution pH and lysozyme concentration on the interfacial structure was measured. Monolayer formation was observed for all cases except for the highest concentration. The adsorbed protein layers consist of adsorbed lysozyme molecules with side-on or end-on orientation. By means of time-dependent X-ray reflectivity scans, the time-evolution of adsorbed proteins was monitored as well. The results of this study demonstrate the capabilities of in situ X-ray reflectivity experiments on protein adsorbates. The great advantages of this method are the broad wave vector range available and the high time resolution.     

Proteins conjugation with ZnO sol–gel nanopowders

The conjugation between probe biomolecules and inorganic nanoparticles has been studied. Three different and biologically relevant proteins, bovine serum albumin (BSA), lysozyme (LSZ) and Ribonuclease A (RNAseA), have been selected as model systems because of their difference in size and isoelectric point. Zinc oxide nanoparticles, synthesized via sol–gel, have been thoroughly characterized by X-ray Photoelectron Spectroscopy, Scanning Electron Microscopy and X-ray Diffraction, and subsequently used as platforms for immobilization of the biomolecules. The interaction of the three proteins with the ZnO surface was performed in phosphate buffer solutions at pH 7.2 in order to mimic physiological fluids and was investigated through fluorescence experiments. The obtained results indicate that conjugation of BSA, LZS and RNAseA on the oxide nanoparticles was mostly dictated by the overall charge of the different proteins. Electrostatic bonds dominate the formation of the protein/ZnO conjugates, whereas the size of the proteins seems to play a negligible role under the adopted experimental conditions.     

Röntgenfluorimetrische Bestimmung von Sulfidspuren im ppb-Bereich in wäßrigen Lösungen nach Austauschreaktion an dünnen Silberhalogenidschichten

Sulphide concentrations in the ppm and ppb range can be determined by X-ray fluorescence spectrometry in aqueous solutions with a volume up to 3 l. The solution to be analysed is passed through a thin layer of a silver halide precipitate whereby the sulphide ions are held back as silver sulphide. The membrane filter with the silver halide deposit is directly used for measuring the X-ray intensity from sulphur. The experimental conditions used include: 2?=23.20°, KAP, X-ray tube with a chromium anode, 50 kV, 40 mA, and a throughflow proportional counter. Interferences of numerous accompanying components were investigated as well as the stability of solutions with very low sulphide concentrations. These solutions can be stabilized sufficiently with ascorbic acid at pH 12, but in the case of natural water only in presence of zinc ions. The method is applicable to sulphide amounts greater than 0.1 μg. The variation coefficient for sulphide amounts of ≈ 10 μg was found to be 2.3%.     

Dielectric spectroscopy as a probe for the investigation of conformational properties of proteins

In this brief paper, we review recent and significant results obtained in our laboratory by dielectric spectroscopy (DS). This is a multi purpose and very sensitive approach to investigate structural features of biological systems. DS at radiofrequencies is particularly powerful in the study of structural and conformational properties of proteins. We report on results obtained on three well-known proteins: lysozyme, cytochrome-c and metmyoglobin, which represent very useful models for folding/unfolding studies. The influence of pH and temperature as well as presence of trehalose as a co-solvent, was determined by estimation of the effective hydrodynamic radius and electric dipole moment of the protein in solution. In particular, trehalose was shown to affect the alkaline transition of cytochrome. Conformational effects on the three above-mentioned proteins were observed in a temperature range near the physiological ones. Dynamical properties of lysozyme in mixtures water-glycerol are also discussed. Parallel measurements of photon correlation spectroscopy (PCS) and DS indicated that both translational and rotational diffusive behavior are coherent with the Debye-Stokes-Einstein hydrodynamic model.     

Thermal stability limits of proteins in solution and adsorbed on a hydrophobic surface

A coarse-grained Monte Carlo simulation is used to study thermal denaturation of small proteins in an infinitely dilute solution and adsorbed on a flat hydrophobic surface. Intermolecular interactions are modeled using the Miyazawa-Jernigan (MJ) knowledge-based potential for implicit solvent with the BULDG hydrophobicity scale. We analyze the thermal behavior of lysozyme for its prevalence of α-helices, fibronectin for its prevalence of β-sheets, and a short single helical peptide. Protein dimensions and contact maps are studied in detail before and during isothermal adsorption and heating. The MJ potential is shown to correctly predict the native conformation in solution under standard conditions, and the anticipated thermal stabilization of adsorbed proteins is observed when compared with heating in solution. The helix of the peptide is found to be much less stable thermally than the helices of lysozyme, reinforcing the importance of long-range forces in defining the protein structure. Contact map analysis of the adsorbed proteins shows correlation between the hydrophobicity of the secondary structure and their thermal stability on the surface.     

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.