Lysozyme In Aqueous Solutions Research Articles

Nonlinear Effect of GdnHCl on Hydration Dynamics of Proteins: A 1H Magnetic Relaxation Dispersion Study

Proton magnetic relaxation dispersion investigations with aqueous solutions of lysozyme and bovine serum albumin (BSA) in the 0-5 M range of guanidine hydrochloride (GdnHCl), pH 4.4, 27 degrees C, were taken up with the objective of examining the hydration dynamics of internal cavity waters as the protein is held under increasingly destabilizing conditions. Field cycling NMR and conventional pulsed NMR techniques were employed to cover a frequency range of 100 kHz to 50 MHz. Analyses of dispersion profiles at different concentrations of GdnHCl were carried out considering the contributions from internal and surface waters. The denaturant-dependent variation of internal water contribution indicates that the reorientational disorder of internal waters decreases with increments of the denaturant up to its subdenaturing limit. For both proteins, the variation of effective correlation time with GdnHCl apparently shows a marginal shrink in hydrodynamic volumes under the subdenaturing condition. These results suggest that subdenaturing amounts of GdnHCl restrict the motional freedom of the internal waters, and can have considerable influence on the surface hydration. On increasing the denaturant concentration further, the dispersion amplitude drops sharply, indicating that the chaotropic action of the denaturant now runs over its own cavity water-ordering effect operative in the subdenaturing limit. The results are fundamentally important for the understanding of the susceptibility of protein structure and hydration to denaturants.

Novel surface modified molecularly imprinted polymer using acryloyl-β-cyclodextrin and acrylamide as monomers for selective recognition of lysozyme in aqueous solution

A novel protein imprinted polymer for selective recognition of lysozyme was obtained. Acryloyl-β-cyclodextrin, which offered hydrophilic exterior and hydrophobic cavity that were allowed to self-assemble with the template protein through hydrogen interaction and hydrophobic interaction, was synthesized and used as the functional monomer. Polymerization was carried out in the presence of acrylamide as an assistant monomer, which resulted in a new type of protein imprinted polymer. Langmuir adsorption model was employed to describe the isotherms, and maximum adsorption capacity was evaluated. The performance of such imprinted polymer was further demonstrated by high-performance liquid chromatography, and the results showed that the column packed with the lysozyme imprinted silica beads could effectively separate lysozyme from the mixture of lysozyme–cytochrome c, lysozyme–bovine serum albumin, lysozyme–avidin or lysozyme–methylated bovine serum albumin, which showed its high selectivity.

Synthesis, characterization and biocompatibility studies on chitosan- graft-poly(EGDMA)

Poly(ethylene glycol dimethacrylate), poly(EGDMA), was grafted onto chitosan by using a redox initiation system. Chitosan- graft-poly(EGDMA) products were characterized by DSC, TGA, FTIR and XRD techniques. Chitosan- graft-poly(EGDMA) was found to be enzymatically degradable in aqueous solutions of lysozyme, lipase and a mixture of α-amylase and protease. The biocompatibility of chitosan- graft-poly(EGDMA) with 871% grafting yield was investigated by studying its cytotoxicity, sensitization, irritation, acute systemic toxicity and hemolytic activity. The results of biocompatibility experiments showed that the product can potentially be used for external intervention devices on bone and other tissue.

Patchy proteins, anions and the Hofmeister series

We investigate specific anion binding to a range of patchy protein models and use ourresults to probe protein–protein interactions for aqueous lysozyme solutions. Our molecularsimulation studies show that the ion–protein interaction mechanism and strength largelydepend on the nature of the interfacial amino acid residues. Via direct ion pairing, smallanions interact with charged side-chains while larger anions are attracted to non-polarresidues due to several solvent assisted mechanisms. Incorporating ion and surfacespecificity into a mesoscopic model for protein–protein interactions we calculate the freeenergy of interaction between lysozyme molecules in aqueous solutions of sodium chlorideand sodium iodide. In agreement with experiment, our finding is that ‘salting out’ followsthe reverse Hofmeister series for pH below the iso-electric point and the direct series for pHabove pI.

UV laser induced proton-transfer of protein molecule in the gas phase produced by droplet-beam laser ablation

Droplet-beam laser-ablation mass-spectrometry was applied for a study of the UV-laser induced proton-transfer reaction of protonated lysozyme hydrated clusters in the gas phase. Protonated lysozyme hydrated clusters were produced by irradiation of an IR laser onto a droplet-beam of an aqueous solution of lysozyme and were subsequently irradiated by a UV laser. It is found that H + and H 3O + are produced through photodissociation of protonated lysozyme hydrated clusters. The mechanism of the proton-transfer reaction is discussed.

Configurational Biomimetic Imprinting for Protein Recognition: Structural Characteristics of Recognitive Hydrogels

Using a configurational biomimetic imprinting technique, we prepared a family of molecularly imprinting polyacrylamide polymers with recognition for the template protein chicken egg white lysozyme in aqueous solution. We showed the formation of a network with specific recognitive sites for the template. To determine the overall macroscopic properties of protein-imprinted gel, we utilized a variety of tools. When gels were examined using scanning electron microscopy, definite morphological differences were observed between the CBIP polymers and the controls. Additionally, Fourier transform infrared spectroscopy determined that the presence of template had no effect upon the overall gel composition, whereas differential scanning calorimetry showed no significant change in the molecular weight between cross-links between CBIP polymers and nonimprinted polymers. Using these results, a theory of gel formation was proposed to try to elucidate gel formation in the presence of a macromolecule.

Solubility of Lysozyme in Polyethylene Glycol-Electrolyte Mixtures: The Depletion Interaction and Ion-Specific Effects

The solubility of aqueous solutions of lysozyme in the presence of polyethylene glycol and various alkaline salts was studied experimentally. The protein-electrolyte mixture was titrated with polyethylene glycol, and when precipitation of the protein occurred, a strong increase of the absorbance at 340 nm was observed. The solubility data were obtained as a function of experimental variables such as protein and electrolyte concentrations, electrolyte type, degree of polymerization of polyethylene glycol, and pH of the solution; the last defines the net charge of the lysozyme. The results indicate that the solubility of lysozyme decreases with the addition of polyethylene glycol; the solubility is lower for a polyethylene glycol with a higher degree of polymerization. Further, the logarithm of the protein solubility is a linear function of the polyethylene glycol concentration. The process is reversible and the protein remains in its native form. An increase of the electrolyte (NaCl) concentration decreases the solubility of lysozyme in the presence and absence of polyethylene glycol. The effect can be explained by the screening of the charged amino residues of the protein. The solubility experiments were performed at two different pH values (pH = 4.0 and 6.0), where the lysozyme net charge was +11 and +8, respectively. Ion-specific effects were systematically investigated. Anions such as Br −, Cl −, F −, and H 2 P O 4 − (all in combination with Na +), when acting as counterions to a protein with positive net charge, exhibit a strong effect on the lysozyme solubility. The differences in protein solubility for chloride solutions with different cations Cs +, K +, and Na + (coions) were much smaller. The results at pH = 4.0 show that anions decrease the lysozyme solubility in the order F − < H 2 P O 4 − < C l − < B r − (the inverse Hofmeister series), whereas cations follow the direct Hofmeister series (Cs + < K + < Na +) in this situation.

Trehalose Bioprotective Effects in Lysozyme Aqueous Solution Studied by Brillouin Scattering and Calorimetric Measurements

The bioprotective effect of trehalose in lysozyme aqueous solutions has been investigated by Brillouin scattering and modulated-temperature differential scanning calorimetry (MDSC). MDSC experiments show that the isothermal kinetics of thermally irreversible denaturation can be described by the Arrhenius equation. By the addition of trehalose, the irreversible denaturation of lysozyme is suppressed, and its activation energy is half that of the denaturation without trehalose. The sound velocity of lysozyme–trehalose–water ternary solutions obviously depends on the trehalose concentration. With increasing trehalose concentration, the sound velocity becomes higher because the hydration of trehalose reduces the hydrogen bonds between water molecules. Moreover, hydration around lysozyme molecules increases the sound velocity further. Trehalose molecules tend to aggregate with lysozyme molecules at high trehalose concentrations. The bioprotective effect of trehalose probably originates from the mechanical suppression of conformational fluctuations of lysozyme molecules.

Thermoreversible lysozyme hydrogels: properties and an insight into the gelation pathway.

The gelation behaviour of aqueous solutions of hen egg white lysozyme (HEWL) in the presence of 20 mM DTT in the concentration range 0.7 to 4.0 mM has been investigated using microDSC, FTIR, cryoTEM, SANS and oscillatory rheology. The macroscopic critical gelation concentration, Cgel, was found to be ∼ 3.0 mM. The disruption of the disulfide bonds by the DTT and the destabilisation of the protein were found to be a prerequisite for the formation of β-sheet rich fibrils under the mild conditions used in this work. Using our methodology the hydrogels obtained have a pH of 7, hence are suitable for cell culture, and are also thermoreversible. The hydrogel melting temperature was found to increase with increasing concentration and a similar structure was observed across the concentration range investigated. Our results suggest these systems are composed of a well defined regular network where single β-sheet rich fibrils (∼ 3 nm diameter) form initially, then two of these fibrils associate two-by-two to form junctions (∼ 6 nm diameter) and then on cooling further aggregate to form larger bundles of fibres. The network mesh size was found to decrease with increasing concentration. Our results suggest that below Cgel small unconnected gel-like aggregates exist that have a similar structure to the hydrogels obtained above Cgel. Using our data we propose a model for the denaturation and gelation behaviour of our system. During the first heating an α-helix to β-sheet molecular transition for the protein conformation occurs resulting in β-sheet rich fibrils forming through the self-assembly of β-sheet rich denaturated proteins. At high temperature the solution contains β-sheet rich fibrils with dissolved protein. On cooling an increase in the amount of β-sheet was observed via FTIR suggesting that as the temperature is decreased more and more protein forms β-sheet rich fibrils. At the gelation temperature these fibrils associate two-by-two to form the network junctions resulting in the macroscopic gelation of the sample. Our results suggest the network junctions are formed via specific hydrophobic interactions. The hydrogels elastic modulus was found to scale as C2.45 for C > Cgel.

Interplay between Spinodal Decomposition and Glass Formation in Proteins Exhibiting Short-Range Attractions

We investigate the competition between spinodal decomposition and dynamical arrest using aqueous solutions of the globular protein lysozyme as a model system for colloids with short-range attractions. We show that quenches below a temperature Ta lead to gel formation as a result of a local arrest of the protein-dense phase during spinodal decomposition. The rheological properties of these gels allow us to use centrifugation experiments to determine the local densities of both phases and to precisely locate the gel boundary and the attractive glass line close to and within the unstable region of the phase diagram.

Three-Dimensional Distribution Function Theory for the Prediction of Protein−Ligand Binding Sites and Affinities: Application to the Binding of Noble Gases to Hen Egg-White Lysozyme in Aqueous Solution

The three-dimensional distribution function theory of molecular liquids is applied to lysozyme in mixtures of water and noble gases. The results indicate that the theory has the capability of predicting the protein-ligand binding sites and affinities. First, it is shown that the theory successfully reproduces the binding sites of xenon found by X-ray crystallography. Then, the ability of the theory to predict the size selectivity of noble gases is demonstrated. The effect of water on the selectivity is clarified by a theoretical analysis. Finally, it is demonstrated that the dose-response curve, which is employed in experiments for examining the binding affinity, is realized by the theory.

Self-assembly of β-casein and lysozyme

The self-assembly of β-casein and lysozyme, a linear and a globular protein with isoelectric point of pH 5.0 and 10.7, respectively, was studied. Polydisperse electrostatic complex micelles formed when mixing β-casein and lysozyme aqueous solutions. After the micelle solution was heated, lysozyme gelated and β-casein was trapped in the gel, producing narrowly dispersed nanoparticles. The nanoparticles were characterized with laser light scattering, ζ-potential, steady state fluorescence, atomic force microscopy, and transmission electron microscopy. The nanoparticles have spherical shape and their sizes depend on the pH of the heat treatment and the molar ratio of β-casein to lysozyme. The nanoparticles display amphoteric property and are relatively hydrophobic at pH around 5 and around 10. The net charges on the surface stabilize the nanoparticles in the solution.

Time evolution of crystallization phase of lysozyme protein in aqueous salt solution as studied by scattering techniques

Formation of protein crystallization in 1 wt% lysozyme in aqueous solution has been studied as a function of salt (KBr) concentration. The onset and the rate of crystallization in this protein solution strongly depend on the KBr concentration. It is found, while the presence of 0.4 M KBr starts producing crystals in 1 h, it takes about 120 h for 0.2 M KBr. The rate of protein crystallization has been studied by small-angle neutron scattering (SANS) and dynamic light scattering (DLS) in 1 wt% lysozyme solution in presence of three different KBr concentrations 0.3, 0.26 and 0.24 M, which show the start of crystallization at about 1.5, 3 and 5 h, respectively. Measurements have been performed for every half an hour from freshly prepared protein sample up to 72 h. Both time-evolved SANS and DLS data have been used to calculate the fraction of crystallization as a function of time. The variation of crystallization with time shows three distinguished regions: (i) prior to and the beginning of crystallization, which shows no significant change in the crystallization, (ii) growth region showing a significant increase in the crystallization with time and (iii) saturation region. These three regions have been found to be strongly depending on the start of the crystallization as controlled by the salt concentration.

The small-angle and wide-angle X-ray scattering set-up at beamline BL9 of DELTA

The multi-purpose experimental endstation of beamline BL9 at the Dortmund Electron Accelerator (DELTA) is dedicated to diffraction experiments in grazing-incidence geometry, reflectivity and powder diffraction measurements. Moreover, fluorescence analysis and inelastic X-ray scattering experiments can be performed. Recently, a new set-up for small-angle and wide-angle X-ray scattering utilizing detection by means of an image-plate scanner was installed and is described in detail here. First small-angle X-ray scattering experiments on aqueous solutions of lysozyme with different cosolvents and of staphylococcal nuclease are discussed. The application of the set-up for texture analysis is emphasized and a study of the crystallographic texture of natural bio-nanocomposites, using lobster and crab cuticles as model materials, is presented.

Phase equilibria for salt-induced lysozyme precipitation: Effect of salt concentration and pH

The salt-induced precipitation of lysozyme from aqueous solutions was studied at 25 °C and various pH values by cloud-point investigations, precipitation experiments (analysing the compositions of the coexisting phases) and microscopic investigations of the precipitates. Sodium sulphate as well as ammonium sulphate were used to induce the precipitation. The experimental results are discussed and used to develop a scheme of the phase equilibrium in water-rich aqueous solutions of lysozyme and either Na 2SO 4 or (NH 4) 2SO 4.

Effect of DMSO on Denaturation of Hen Egg-white Lysozyme in Aqueous Solution

A study on thermal denaturation of hen egg-white lysozyme(HEWL)was carried out by differential scanning calorimetry(DSC)and modulated temperature differential scanning calorimetry(MDSC).It was found that DSC profiles of HEWL were dependent on heating rates and concentrations of enzyme as well as cosolvent.The enthalpy of denaturation(ΔHm)increased with increasing enzyme concentration in aqueous solution.In aqueous solution containing dimethylsolfoxide(DMSO),denaturation temperature(Tm)shifted toward lower temperature,and the transition peak became shallower and broader with increasing volume proportion of DMSO.When the content of DMSO was more than 70%,only a declining smooth line could be observed instead of a single endothermic transition curve.Interestingly,from MDSC curves of HEWL in aqueous solution without DMSO,a small and symmetrical peak in front of the main transition(I)was observed marginally,which was regarded as a pre-transition(II).Specially,this small and marginal peak could not be found in aqueous solution containing DMSO.In addition,with increasing amount of protein,Tm(II)shifted toward lower temperature,and ΔHm(II)decreased along with the temperature gap between two transitions lengthened.The results were considered to be due to the reversible dissociation of HEWL dimers in aqueous solution.

Study on Thermal Denaturation of Lysozyme by DSC

The thermal denaturation of solid lysozyme and the effects of denaturants and their concentrations on denaturation of lysozyme in aqueous solutions were studied by differential scanning calorimetry (DSC). The results showed that both denaturation temperature and denaturation enthalpy of lysozyme decreased as the existence of water and the addition of urea and guanidine hydrochloride (GuHCl). In addition, denaturation temperature and denaturation enthalpy decreased as the concentrations of urea and GuHCl increased. As a denaturant, GuHCl is more effective than urea due to its added ability of electrostatic interaction.

Structural interactions of globular proteins—Bovine serum albumin, egg albumin, and lysozyme, in aqueous medium, elucidated with molar volumes, viscosities, energy functions, and IR spectra from 293.15 to 303.15 K

AbstractDensities (ρ), apparent molar volumes (Vϕ), viscosities (η), and IR spectra on 0.0010–0.0018% aqueous solutions of bovine serum albumin (BSA), egg albumin (E‐Alb), and lysozyme at an interval of 0.0004% and at temperatures from 293.15, 298.15, and 303.15 K have obtained. The free energy (ΔG), entropy (ΔS), and enthalpy (ΔH) data with compositions and temperatures are calculated from the values of the flow velocity (vf) of viscous flow, which decrease with temperature. The densities decrease with concentrations and temperatures except BSA, and the Vϕ values slightly increase with concentrations for BSA and lysozyme, which depict structural reorientations and transition states of protein molecules with increase in viscosities and decrease in reduce viscosities. The reduce viscosities at 293.15 K for BSA, E‐Alb, and lysozyme are noted positive, and for BSA and lysozyme remain positive at 298.15 and 303.15 K, whereas for E‐Alb it is negative. Activation energies (E*) for lysozyme remain almost constant, and are higher than those of the BSA and E‐Alb, respectively, also slightly higher E* values for the BSA than those of the E‐Alb at 293.15 and 298.15, and lower than of the E‐Alb at 303.15 K, are observed elucidating greater structural interactions for BSA at lower while weaker at temperatures. Stretching frequencies of amide (NHCO), NH, CO, and CH groups of proteins are noted from IR spectra with broader stretching frequencies for NH. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 1420–1429, 2007

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.

Thermoreversible Protein Hydrogel as Cell Scaffold

A thermoreversible fibrillar hydrogel has been formed from an aqueous lysozyme solution in the presence of dithiothreitol (DTT). Its physical properties and potential as a tissue engineering scaffold have been explored. Hydrogels were prepared by dissolving 3 mM protein in a 20 mM DTT/water mixture, heating to 85 degrees C and cooling at room temperature. No gel was observed for the equivalent sample without DTT. The elastic nature of the gel formed was confirmed by rheology, and the storage modulus of our gel was found to be of the same order of magnitude as for other cross-linked biopolymers. Micro differential scanning calorimetry (microDSC) experiments confirmed that the hydrogel was thermally reversible and that gelation and melting occurs through a solid-liquid-like first-order transition. Infrared spectroscopy of the hydrogel and transmission electron microscopy studies of very dilute samples revealed the presence of beta-sheet-rich fibrils that were approximately 4-6 nm in diameter and 1 mum in length. These fibrils are thought to self-assemble along their long axes to form larger fibers that become physically entangled to form the three-dimensional network observed in both cryo-scanning electron microscopy (cryo-SEM) and small-angle neutron scattering (SANS) studies. The hydrogel was subsequently cultured with 3T3 fibroblasts and cells spread extensively after 7 days and stretched actin filaments formed that were roughly parallel to each other, indicating the development of organized actin filaments in the form of stress fibers in cells.