Lysozyme In Aqueous Solutions Research Articles

Significantly enhanced thermal stability of HMX by phase-transition lysozyme coating

A new robust bio-inspired route by using lysozyme aqueous solution for surface modification on 1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX) was described in this paper. HMX crystals were coated by in situ phase transition of lysozyme (PTL) molecules. The HMX decorated by PTL was characterized by SEM, XRD, FTIR and XPS, demonstrating a dense core-shell coating layer. The coverage of lysozyme on HMX crystal was calculated by the ratio of sulfur content. The surface coverage increased from 60.5% to 93.5% when the content of PTL was changed from 0.5 wt% to 2.0 wt%, indicating efficient coating. The thermal stability of HMX was investigated by in situ XRD and DSC. The thermal phase transition temperature of HMX (β to δ phase) was delayed by 42 °C with 2.0 wt% PTL coating, which prevented HMX from thermal damage and sensitivity by the effect of PTL coating. After heating at 215 °C, large cracks appeared in the naked HMX crystal, while the PTL coated HMX still maintained intact, with the impact energy of HMX dropped dramatically from 5 J to 2 J. However, the impact energy of HMX with 1.0 wt% and 2.0 wt% coating content (HMX@PTL-1.0 and HMX@PTL-2.0) was unchanged (5 J). Present results potentially enable large-scale fabrication of polymorphic energetic materials with outstanding thermal stability by novel lysozyme coating.

Nonthermal acceleration of protein hydration by sub-terahertz irradiation

The collective intermolecular dynamics of protein and water molecules, which overlap in the sub-terahertz (THz) frequency region, are relevant for expressing protein functions but remain largely unknown. This study used dielectric relaxation (DR) measurements to investigate how externally applied sub-THz electromagnetic fields perturb the rapid collective dynamics and influence the considerably slower chemical processes in protein–water systems. We analyzed an aqueous lysozyme solution, whose hydration is not thermally equilibrated. By detecting time-lapse differences in microwave DR, we demonstrated that sub-THz irradiation gradually decreases the dielectric permittivity of the lysozyme solution by reducing the orientational polarization of water molecules. Comprehensive analysis combining THz and nuclear magnetic resonance spectroscopies suggested that the gradual decrease in the dielectric permittivity is not induced by heating but is due to a slow shift toward the hydrophobic hydration structure in lysozyme. Our findings can be used to investigate hydration-mediated protein functions based on sub-THz irradiation.

Multiscale Spectral Analysis on Lysozyme Aqueous Solutions in the Presence of PolyEthyleneGlycol.

Infrared spectroscopy measurements were performed on Lysozyme aqueous solutions also in the presence of PolyEthylene Glycol (PEG 400) as a function of an increasing temperature from T = 27 °C to 90 °C, and, successively in sequence, by decreasing temperatures from T = 90 °C to 27 °C. Data were analyzed by evaluating the spectral difference with respect to the initial spectrum collected at 27 °C. This procedure allows to quantitatively evaluate the thermal restraint related to the thermal scan from T = 27 °C to 90 °C, as well as to introduce a spectral resilience concerning the entire increasing and decreasing thermal paths which allow to highlight the bioprotectant effectiveness of low molecular weight PEG. In particular, the main purpose of the present work is to highlight the effects of a thermal treatment on a mixture of Lysozyme/water and of Lysozyme/water/PEG 400 during an increasing temperature scan, and then after a successive decreasing temperature scan, in order to highlight the bioprotectant role of PEG 400. On that score, an evaluation of the spectral distances of the registered spectra as a function of increasing and decreasing temperatures has been performed and analyzed.

Multi-length scale structural investigation of lysozyme self-assembly

SummaryReactive amyloid oligomers are responsible for cytotoxicity in amyloid pathologies and because of their unstable nature characterizing their behavior is a challenge. The physics governing the self-assembly of proteins in crowded conditions is extremely complex and its comprehension, despite its paramount relevance to understanding molecular mechanisms inside cells and optimizing pharmaceutical processes, remains inconclusive. Here, we focus on the amyloid oligomerization process in self-crowded lysozyme aqueous solutions in acidic conditions. We reveal that the amyloid oligomers form at high protein concentration and low pH. Through multi-length scale spectroscopic investigations, we find that amyloid oligomers can further interconnect with each other by weak and non-specific interactions forming an extended network that leads to the percolation of the whole system. Our multi-length scale structural analysis follows the thermal history of amyloid oligomers from different perspectives and highlights the impact of hierarchical self-assembly of biological macromolecules on functional properties.

Preparation, characterization, and application of hydrogel derived from chitin with lithium chloride for growth and development of rosemary plants

AbstractIn this study, the N,N‐dimethyl acetamide (DMAc) solution with different lithium chloride concentrations of 3 wt%, 5 wt%, and 7 wt% by phase inversion in ethanol vapor to produce chitin hydrogels for plant growth. The chitin hydrogel prepared with a higher concentration of lithium chloride showed improvements in elastic and mechanical properties. The tensile strength was enhanced from 275.8 to 434 kPa for the chitin hydrogel with 3% and 7% of LiCl. While the reduction in water content suggested forming a denser hydrogel structure at higher LiCl concentrations; the water contact angle (WCA) value indicated that chitin‐derived hydrogels are hydrophilic. Moreover, the bio‐degradation experiment indicated the degradable percent of the chitin hydrogel placed in 10 mg/L of aqueous lysozyme solutions at 37°C increases during 7 to 28 days from 4.72 wt% to 5.45 wt%. The obtained hydrogel was suitable for the growth and development on rosemary plants. The condition CE15 with the hydrogel of 85 wt% chitin‐derived hydrogel and 15 wt% cellulose as best medium planting with the largest effect on the height, the crown's diameters and the number of level‐one branches of the rosemary plants.

Multiscale Water Dynamics on Protein Surfaces: Protein-Specific Response to Surface Ions.

Proteins function in crowded aqueous environments, interacting with a diverse range of compounds, and among them, dissolved ions. These interactions are water-mediated. In the present study, we combine field-dependent NMR relaxation (NMRD) and theory to probe water dynamics on the surface of proteins in concentrated aqueous solutions of hen egg-white lysozyme (LZM) and bovine serum albumin (BSA). The experiments reveal that the presence of salts (NaCl or NaI) leads to an opposite ion-specific response for the two proteins: an addition of salt to LZM solutions increases water relaxation rates with respect to the salt-free case, while for BSA solutions, a decrease is observed. The magnitude of the change depends on the ion identity. The developed model accounts for the non-Lorentzian shape of the NMRD profiles and reproduces the experimental data over four decades in Larmor frequency (10 kHz to 110 MHz). It is applicable up to high protein concentrations. The model incorporates the observed ion-specific effects via changes in the protein surface roughness, represented by the surface fractal dimension, and the accompanying changes in the surface water residence times. The response is protein-specific, linked to geometrical aspects of the individual protein surfaces, and goes beyond protein-independent Hofmeister-style ordering of ions.

Development of low-fouling PVDF membranes blended with poly(2-methoxyethyl acrylate) via NIPS process

PVDF-based membranes with low-fouling and high permeability properties were developed by blending poly(vinylidene fluoride) (PVDF) with poly(2-methoxyethyl acrylate) (PMEA), a low-fouling polymer, via non-solvent induced phase separation (NIPS). Since PMEA suppresses protein adsorption and is insoluble in water, the PMEA homopolymer was used as an additive. Consequently, transparent dope solutions containing 15 wt% PVDF and 0–7 wt% PMEA were prepared using N-methyl-2-pyrrolidone as the solvent. The macrovoids in the blended membranes were larger than those in the pristine PVDF membrane and were probably attributed to the PMEA-promoted solvent exchange during the NIPS process. As a result, the pure water permeabilities of the blended membranes were 5–10 times higher than those of the pristine PVDF membrane. The ATR FT-IR characterization revealed that the PMEA composition of the membrane surfaces increased as the PMEA blend ratio in the dope solution increased. This resulted in the improvement of their low-fouling properties when used in 1000 ppm of bovine serum albumin (BSA) and lysozyme aqueous solutions. Additionally, the PMEA-blended PVDF membrane with the highest PMEA ratio showed the best low-fouling properties.

Effect of a Good buffer on the fate of metastable protein-rich droplets near physiological composition

Metastable protein-rich microdroplets are produced from liquid-liquid phase separation (LLPS) of protein aqueous solutions. These globules can be intermediates for the formation of other protein-rich phases. Lysozyme aqueous solutions undergo LLPS around 0 °C in the presence of NaCl near physiological conditions. Here, it is shown that insertion of small amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES, 0.1 M) as a second additive to lysozyme-NaCl-water solutions near physiological ionic strength (0.2 M) is an essential step for triggering conversion of protein-rich droplets into another phase. Specifically, LLPS induced by cooling reproducibly leads to a rapid and high-yield formation of compact tetragonal crystalline microparticles only in the presence of HEPES. These microcrystals exhibit small size (1-3 μm), narrow size distribution and guest-binding properties. The temperature-concentration phase diagram shows a characteristic topology with LLPS boundary metastable with respect to tetragonal microcrystals, which in turn become less stable than rod-shaped orthorhombic crystals above 40 °C. Interestingly, dynamic light scattering, hydrogen-ion titrations and isothermal titration calorimetry reveal that lysozyme-HEPES interactions were found to be weakly attractive and exothermic. Our findings indicate that additives of salting-in type can represent an important factor controlling the fate of metastable protein-rich microdroplets relevant to drug formulations, femtosecond crystallography, and potential implications in protein-driven cytoplasmic compartmentalization.

All-natural superhydrophobic coating for packaging and blood-repelling materials

The design of a bio-safe superhydrophobic coating to repel water adsorption, food and blood adherence is significantly important to attenuate food spoilage, food waste and thrombi. Herein, we synthesize an all-natural superhydrophobic interfacial material that has strong interfacial stability on a variety of substrates, and has good repellence towater, highly-viscous liquid foods and biofluids (blood, urine, etc.). The base material is simply prepared in an aqueous solution of lysozyme and cysteine, in which the rapid phase transition of lysozyme is triggered by a cysteine-induced thiol-disulfide exchange reaction. The resulting phase-transited lysozyme (PTL) aggregates into microparticles with a nanoscale roughness, which are further packed into a dense layer with a micro-/nano-topography. The PTL is composed of an inner amyloid-like structure and shows strong interfacial adhesion with the underlying substrate due to amyloid-inspired adhesion. The PTL layer is finally converted to a robust coating to effectively repel liquid foods and biofluids by being decorated with a natural hydrophobic carnauba wax. This coating noticeably enhances the water repellence of cellulose-based packaging materials, and the resulting superhydrophobic paper is completely non-wetted by water without weakening the mechanical stability and usability of the modified paper. The coating further resists the adhesion of milk, yogurt, honey and various other beverages and edible liquids, which leaves no residue on the coated surface. This coating also significantly resists wetting by blood and other human biofluids with a noticeable decrease in platelet adhesion on the surface. This work underlines the importance of an amyloid-like protein assembly in the preparation of an all-natural green interfacial coating towards the robust repellence of water, edible liquids and blood.

Mechanistic differences in the effects of sucrose and sucralose on the phase stability of lysozyme solutions

The effect of two disaccharide analogues, sucrose and sucralose, on the phase stability of aqueous lysozyme solutions has been addressed from a mechanistic viewpoint by a combination of experiment and molecular dynamics (MD) simulations. The influence of the added low molecular weight salts (NaBr, NaI and NaNO3) was considered as well. The cloud-point temperature measurements revealed a larger stabilizing effect of sucralose. Upon increasing sugar concentration, the protein solutions became more stable and differences in the effect of sucralose and sucrose amplified. It was confirmed that the addition of either of the two sugars imposed no secondary structure changes of the lysozyme. Enthalpies of lysozyme-sugar mixing were exothermic and a larger effect was recorded for sucralose. MD simulations indicated that acidic, basic and polar amino acid residues play predominant roles in the sugar-protein interactions, mainly through hydrogen bonding. Such sugar mediated protein-protein interactions are thought to be responsible for the biopreserative nature of sugars. Our observations hint at mechanistic differences in sugar-lysozyme interactions: while sucrose does not interact directly with the protein's surface for the most part (in line with the preferential hydration hypothesis), sucralose forms hydrogen bonds with acidic, basic and polar amino acid residues at the lysozyme's surface (in line with the water replacement hypothesis).

Thermodynamic Analysis of the Interaction of Heparin with Lysozyme.

Glycosaminoglycan (GAG)-protein binding governs critically important signaling events in living matter. Aiming at a quantitative analysis of the involved processes, we herein present a thermodynamic study of the interaction of the model GAG heparin and lysozyme in aqueous solution. Heparin is a highly charged linear polyelectrolyte with a charge parameter of 2.9 (37 °C). The binding constant Kb was determined by ITC as a function of the temperature and ionic strength adjusted through the concentration cs of added salt. The dependence on salt concentration cs was used to determine the net number of released counterions. Moreover, the binding constant at a reference salt concentration of 1 M Kb(1 M) was determined by extrapolation. The dependence on temperature of Kb was used to dissect the binding free energy ΔGb into the respective enthalpies ΔHb and entropies ΔSb together with the specific heat Δcp. A strong enthalpy-entropy cancelation was found similar to the results for many other systems. The binding free energy ΔGb could furthermore be split up into a part ΔGci due to counterion release and a residual part ΔGres. The latter quantity reflects specific contributions as, e.g., salt bridges, van der Waals interactions, or hydrogen bonds. The entire analysis shows that heparin-lysozyme interactions are mainly caused by counterion release; that is, ca. three counterions are being released upon binding one lysozyme molecule. Our reported approach of quantifying interactions between glycosaminoglycans and proteins is generally applicable and suitable to provide new insights in the physical modulation of biomolecular signals.

Vitamin C stabilized in lysozyme and CTAB aqueous solutions

Vitamin C (VC) is an antioxidant and assists in treating cancer, but it hydrolyses and degrades in aqueous solutions due to oxidation and loses drug efficiency. It is critical to improve the stability of VC. Lysozyme (LYS) is a global protein and an enzyme, which is used as a model protein to probe the interactions of VC and protein relevant to VC degradation. Micellar solutions, formed by cationic surfactant cetyltrimethylammonium bromide (CTAB) and water, act as micro-reactors to stabilize VC. UV–vis and fluorescence spectroscopic methods are applied to investigate the interactions that control the properties of the VC-LYS-CTAB ternary model system, including kinetics of VC degradation and conformation changes of LYS (unfolding and recovery). The results suggest that LYS stabilizes VC by forming a complex and LYS partially unfolds, and CTAB micelles can slow down the rate of VC degradation. VC is the most stable in the VC- LYS-CTAB ternary system among VC-LYS, VC-CTAB aqueous solutions, and pure water. The strength of interactions decreases in the order of: VC degradation, interaction of CTAB micelles and VC, interaction of LYS and VC, and the weak hydrophobic interaction between CTAB and LYS. The former two are the major factors that control the alleviation of VC degradation and the recovery of LYS conformation. This work provides fundamental insights for drug stability and delivery, and personal product field where vitamins, proteins and surfactants are used.

Determination of Protein-Protein Interactions at High Co-Solvent Concentrations Using Static and Dynamic Light Scattering

Protein-protein interactions are commonly measured in terms of the second osmotic virial coefficient, B22 from static light scattering (SLS) or the interaction parameter, kD from dynamic light scattering (DLS). Often these measurements are carried out at high co-solvent compositions, where correction factors are required for the light scattering analysis. For lysozyme in aqueous solutions containing the co-solvents NaCl, arginine chloride, urea, sucrose or guanidine chloride, we show that B22 determination requires using in the light scattering equation the refractive index increment of the protein measured at constant solvent chemical potential. Because the increment decreases with increasing co-solvent composition, using a constant value can lead to mis-interpretation of protein-protein interaction trends deduced from the B22 measurements. Furthermore, there is a contribution to the intensity auto-correlation function measured by dynamic light scattering due to co-solvents. This effect is removed by including longer delay times when fitting the cumulant analysis to determine the diffusion coefficients. We show that an experimentally observed correlation between B22 and kD is recovered once these correction factors have been applied. The findings are particularly relevant to biopharmaceutical industry, where B22 and kD measurements are used for screening excipient effects in liquid formulations.

Trehalose-induced slowdown of lysozyme hydration dynamics probed by EDLS spectroscopy.

We use extended depolarized light scattering spectroscopy to study the dynamics of water in a lysozyme-trehalose aqueous solution over a broad time scale, from hundreds to fractions of picoseconds. We provide experimental evidence that the sugar, present in the ternary solution in quantity relevant for biopreservation, strongly modifies the solvation properties of the protein. By comparing aqueous solutions of lysozyme with and without trehalose, we show that the combined action of sugar and protein produces an exceptional dynamic slowdown of a fraction of water molecules around the protein, which become more than twice slower than in the absence of trehalose. We speculate that this ultraslow water may be caged between the sugar and protein surface, consistently with a water entrapment scenario. We also demonstrate that the dynamics of these water molecules gets slower and slower upon cooling. On the basis of these findings, we believe such ultraslow water close to the lysozyme is likely to be involved in the mechanism of bioprotection.

Lysozyme Aptamer-Functionalized Magnetic Nanoparticles for the Purification of Lysozyme from Chicken Egg White.

Lysozyme is in high demand due to its many favorable characteristics such as being naturally occurring, non-toxic, and easy to digest and absorb. Recently, superparamagnetic nanoparticles with strong magnetic responsiveness have attracted significant interest for enzyme purification. The aptamer of the enzyme can be chemically synthesized rapidly at a large scale using simple and low-cost preparation methods. Therefore, Fe3O4 nanoparticles (Fe3O4 NPs) were prepared by chemical co-precipitation and were then functionalized with amino groups to produce NH2-Fe3O4 NPs. The specific reaction of aldehyde and amino groups was used to attach lysozyme aptamers with specific sequences to NH2-Fe3O4 NPs to produce Apt-NH2-Fe3O4 NPs. The synthesized materials were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), hysteresis loop analysis, and thermogravimetric analysis (TGA). The optimal experimental conditions for adsorption of lysozyme were investigated. The effects of initial lysozyme concentration, adsorption time, pH, reaction temperature, and ionic strength were determined. The maximum adsorption capacity and relevant activity of Apt-NH2-Fe3O4 NPs was 460 mg·g−1 and 16,412 ± 55 U·mg−1 in an aqueous lysozyme solution. In addition, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis analysis, lysozyme could be separated from crude fresh egg white using Apt-NH2-Fe3O4 NPs with an amount up to 113 ± 4.2 mg·g−1 and an activity up to 16,370 ± 46 U·mg−1.

Heat transfer analysis on progressive freeze concentration of aqueous lysozyme solution

In this study, operating conditions of progressive freeze concentration method were arranged to deal with heat transfer analysis. Progressive freeze concentration is also known as a method to remove water from the solution by freezing process. The aqueous solution of lysozyme needs a water removal technology system as the residual moisture content has a significant impact on the solid-state stability of this biopharmaceutical product. The process was carried out by a newly designed crystallizer called a multiple probe cryo-concentrator (MPCC) which is completely equipped with a controlled stirrer, adjustable cooling gas system, solution tank and a set of cooling probes. The operating condition involved in this system include operation time, t (20 to 60 min), coolant temperature, CT (-6 to -14 ?C) and stirrer speed, SS (200 to 400 rpm) at constant initial concentration of 10mg/ml. The result of overall heat transfer coefficient (Uo) at varied operating condition was analysed and obtained where coolant temperature of -8 °C, operation time of 20 min and stirrer speed of 350 rpm were found to give the highest Uo. All the operating conditions were found to contribute to the progress of ice development and removal of water in the MPCC system resulting in purity of ice and solution concentration accordingly.

Clusters of lysozyme in aqueous solutions

Equilibrium clusters of protein lysozyme are at the center of an ongoing scientific debate. Previous attempts to provide a microscopic description of the clusters that is consistent with all experimental evidence have not been fully successful. The primary reason is the use of model potentials that have a predefined shape. In this paper we derive a model-free interprotein potential directly from experimental structure factor. The derived potential is globally repulsive but has a local minimum at short distances. The minimum is essential for the correct behavior of the structure factor with protein concentration, in particular the shifting pattern of the signature maximum at short wave vectors. Equilibrium clusters are observed throughout the entire range of concentrations, but their nature differs in the low and high concentration limits. At low concentrations, the clusters are extended in shape. As the concentration is increased, small clusters collapse while large clusters are assembled from the small ones. Hydrodynamic interactions drive a kinetic slowdown at high concentrations, where a transition into a fluid of permanent clusters of specific size is observed. In good agreement with the available experimental data, our simulations shed light on the microscopic nature of protein clusters.

Measuring and modeling thermodynamic properties of aqueous lysozyme and BSA solutions

A novel approach towards modeling proteins in aqueous solutions by applying perturbed-chain statistical associating fluid theory (PC-SAFT) is proposed in this work. Lysozyme and bovine serum albumin (BSA) were chosen as model proteins due to a vast database available in literature. However, the huge majority of these literature data had been measured at certain ionic strengths, which is disadvantageous in order to derive pure-component parameters for proteins. Thus, in a first step within this work osmotic coefficients of dialyzed lysozyme and of dialyzed BSA in pure water were measured at 298.15 K. The data were very different for dialyzed and non-dialyzed solutions and further showed a strong dependence on pH and ionic strength. In a second step, the pure-component parameters of lysozyme and BSA were adjusted to the measured osmotic coefficients and to solution densities of dialyzed lysozyme or dialyzed BSA. PC-SAFT explicitly takes into account the complex association behavior between protein and water as well as among the proteins. Therefore, the number of protein association sites of one protein was determined from the sum of association sites of the single amino-acid constituents of a protein available from previous work (Ind. Eng. Chem. Res 50 (2011) 141–151). Similarly, the two PC-SAFT parameters “segment number” and “segment diameter” of a protein were summed or averaged from the single amino-acid constituents. This approach allowed modeling densities and osmotic coefficients of protein solutions with reasonable accuracy compared to experimental data.

Two-step relaxation mode analysis with multiple evolution times applied to all-atom molecular dynamics protein simulation.

Proteins implement their functionalities when folded into specific three-dimensional structures, and their functions are related to the protein structures and dynamics. Previously, we applied a relaxation mode analysis (RMA) method to protein systems; this method approximately estimates the slow relaxation modes and times via simulation and enables investigation of the dynamic properties underlying the protein structural fluctuations. Recently, two-step RMA with multiple evolution times has been proposed and applied to a slightly complex homopolymer system, i.e., a single [n]polycatenane. This method can be applied to more complex heteropolymer systems, i.e., protein systems, to estimate the relaxation modes and times more accurately. In two-step RMA, we first perform RMA and obtain rough estimates of the relaxation modes and times. Then, we apply RMA with multiple evolution times to a small number of the slowest relaxation modes obtained in the previous calculation. Herein, we apply this method to the results of principal component analysis (PCA). First, PCA is applied to a 2-μs molecular dynamics simulation of hen egg-white lysozyme in aqueous solution. Then, the two-step RMA method with multiple evolution times is applied to the obtained principal components. The slow relaxation modes and corresponding relaxation times for the principal components are much improved by the second RMA.

Interaction of proteins with ionic liquid, alcohol and DMSO and in situ generation of gold nano-clusters in a cell.

In this review, we give a brief overview on how the interaction of proteins with ionic liquids, alcohols and dimethyl sulfoxide (DMSO) influences the stability, conformational dynamics and function of proteins/enzymes. We present experimental results obtained from fluorescence correlation spectroscopy on the effect of ionic liquid or alcohol or DMSO on the size (more precisely, the diffusion constant) and conformational dynamics of lysozyme, cytochrome c and human serum albuminin aqueous solution. The interaction of ionic liquid with biomolecules (e.g. protein, DNA etc.) has emerged as a current frontier. We demonstrate that ionic liquids are excellent stabilizers of protein and DNA and, in some cases, cause refolding of a protein already denatured by chemical denaturing agents. We show that in ethanol-water binary mixture, proteins undergo non-monotonic changes in size and dynamics with increasing ethanol content. We also discuss the effect of water-DMSO mixture on the stability of proteins. We demonstrate how large-scale molecular dynamics simulations have revealed the molecular origin of this observed phenomenon and provide a microscopic picture of the immediate environment of the biomolecules. Finally, we describe how favorable interactions of ionic liquids may be utilized for in situ generation of fluorescent gold nano-clusters for imaging a live cell.