Adsorption Behavior Of Lysozyme Research Articles

Studies of Protein Wastes Adsorption by Chitosan-Modified Nanofibers Decorated with Dye Wastes in Batch and Continuous Flow Processes: Potential Environmental Applications.

In this study, reactive green 19 dye from wastewater was immobilized on the functionalized chitosan nanofiber membranes to treat soluble microbial proteins in biological wastewater. Polyacrylonitrile nanofiber membrane (PAN) was prepared by the electrospinning technique. After heat treatment, alkaline hydrolysis, and chemically grafted with chitosan to obtain modified chitosan nanofibers (P-COOH-CS), and finally immobilized with RG19 dye, dyed nanofibers were generated (P-COOH-CS-RG19). The synthesis of P-COOH-CS and P-COOH-CS-RG19 are novel materials for protein adsorption that are not deeply investigated currently, with each of the material functions based on their properties in significantly improving the adsorption efficiency. The nanofiber membrane shows good adsorption capacity and great recycling performance, while the application of chitosan and dye acts as the crosslinker in the nanofiber membrane and consists of various functional groups to enhance the adsorption of protein. The dyed nanofibers were applied for the batch adsorption of soluble protein (i.e., lysozyme), and the process parameters including chitosan’s molecular weight, coupling pH, chitosan concentration, dye pH, dye concentration, and lysozyme pH were studied. The results showed that the molecular weight of chitosan was 50 kDa, pH 5, concentration 0.5%, initial concentration of dye at 1 mg/mL dye and pH 12, lysozyme solution at 2 mg/mL at pH 8, and the maximum adsorption capacity was 1293.66 mg/g at a temperature of 318 K. Furthermore, thermodynamic, and kinetic studies suggested that the adsorption behavior of lysozyme followed the Langmuir adsorption isotherm model and the pseudo-second-order kinetic model. The optimal adsorption and desorption conditions based on batch experiments were directly applied to remove lysozyme in a continuous operation. This study demonstrated the potential of dyed nanofibers as an efficient adsorbent to remove approximately 100% of lysozyme from the simulated biological wastewater.

Dielectric Spectroscopy Can Predict the Effect of External AC Fields on the Dynamic Adsorption of Lysozyme.

This report describes the application of dielectric spectroscopy as a simple and fast way to guide protein adsorption experiments. Specifically, the polarization behavior of a layer of adsorbed lysozyme was investigated using a triangular‐wave signal with frequencies varying from 0.5 to 2 Hz. The basic experiment, which can be performed in less than 5 min and with a single sample, not only allowed confirming the susceptibility of the selected protein towards the electric signal but also identified that this protein would respond more efficiently to signals with lower frequencies. To verify the validity of these observations, the adsorption behavior of lysozyme onto optically transparent carbon electrodes was also investigated under the influence of an applied alternating potential. In these experiments, the applied signal was defined by a sinusoidal wave with an amplitude of 100 mV and superimposed to +800 mV (applied as a working potential) and varying the frequency in the 0.1–10000 Hz range. The experimental data showed that the greatest adsorbed amounts of lysozyme were obtained at the lowest tested frequencies (0.1–1.0 Hz), results that are in line with the corresponding dielectric features of the protein.

Correlation between Tribological Properties and the Quantified Structural Changes of Lysozyme on Poly (2-hydroxyethyl methacrylate) Contact Lens.

The ocular discomfort is the leading cause of contact lens wear discontinuation. Although the tear proteins as a lubricant might improve contact lens adaptation, some in vitro studies suggested that the amount of adsorbed proteins could not simply explain the lubricating performance of adsorbed proteins. The purpose of this study was to quantify the structural changes and corresponding ocular lubricating properties of adsorbed protein on a conventional contact lens material, poly (2-hydroxyethyl methacrylate) (pHEMA). The adsorption behaviors of lysozyme on pHEMA were determined by the combined effects of protein–surface and protein–protein interactions. Lysozyme, the most abundant protein in tear, was first adsorbed onto the pHEMA surface under widely varying protein solution concentrations to saturate the surface, with the areal density of the adsorbed protein presenting different protein–protein effects within the layer. These values were correlated with the measured secondary structures, and corresponding friction coefficient of the adsorbed and protein covered lens surface, respectively. The decreased friction coefficient value was an indicator of the lubricated surfaces with improved adaptation. Our results indicate that the protein–protein effects help stabilize the structure of adsorbed lysozyme on pHEMA with the raised friction coefficient measured critical for the innovation of contact lens material designs with improved adaptation.

Adsorption Behavior of Lysozyme at Titanium Oxide-Water Interfaces.

We present an in situ X-ray reflectivity study of the adsorption behavior of the protein lysozyme on titanium oxide layers under variation of different thermodynamic parameters, such as temperature, hydrostatic pressure, and pH value. Moreover, by varying the layer thickness of the titanium oxide layer on a silicon wafer, changes in the adsorption behavior of lysozyme were studied. In total, we determined less adsorption on titanium oxide compared with silicon dioxide, while increasing the titanium oxide layer thickness causes stronger adsorption. Furthermore, the variation of temperature from 20 to 80 °C yields an increase in the amount of adsorbed lysozyme at the interface. Additional measurements with variation of the pH value of the system in a region between pH 2 and 12 show that the surface charge of both protein and titanium oxide has a crucial role in the adsorption process. Further pressure-dependent experiments between 50 and 5000 bar show a reduction of the amount of adsorbed lysozyme with increasing pressure.

The adsorption behaviour of lysozyme as a potential antibiofouling agent

Introduction: Biofilms can be problematic or beneficial. Reports on the underlying adsorption in such context-dependent fouling phenomena have been extensive. However, the mechanisms involved is scarce and appears adsorbate or foulant-specific. Methods: In this study, attenuated total reflection infrared spectroscopy was used to probe the solid-liquid interfacial phenomena involving lysozyme for assessing its surface antibacterial actions. Results: Lysozyme preferentially adsorbed more on to the hydrophilic coated ZnSe IR prism than to an uncoated ZnSe IR prism. Data on discrete adsorption rates suggested that lysozyme was adsorbing as multi-layer aggregates on to the ZnSe prism, and showed faster adsorptiondesorption patterns on an uncoated compared to a TiO2-coated ZnSe IR prism. A novel phosphate-modified TiO2 coating material for ZnSe IR prism was prepared as adsorbate which increased the negative charge on the TiO2 surface via P-O-Ti coordinated linkages, increasing the initial rate of lysozyme adsorption. Enhanced adsorption of lysozyme was achieved by increasing both the solution pH and NaCl concentration. Maximum adsorption occurred at pH 11 which corresponded to the protein’s isoelectric pH. Conclusions: ATR-IR spectroscopy has proved to be a useful method to study surface adsorption of antibacterial agents such as lysozyme for further explorations into approaches to control biofouled surfaces.

A study on the nature of interactions of mixed-mode ligands HEA and PPA HyperCel using phenylglyoxal modified lysozyme.

Mixed mode chromatography, or multimodal chromatography, involves the exploitation of combinations of several interactions in a controlled manner, to facilitate the rapid capture of proteins. Mixed-mode ligands like HEA and PPA HyperCel™ facilitate different kinds of interactions (hydrophobic, ionic, etc.) under different conditions. In order to better characterize the nature of this multi-modal interaction, we sought to study a protein, lysozyme, which is normally not retained by these mixed mode resins under normal binding conditions. Lysozyme was modified specifically at Arginine residues by the action of phenylglyoxal, and was extensively studied in this work to better characterize the mixed-mode interactions of HEA HyperCel™ and PPA HyperCel™ chromatographic supports. We show here that the adsorption behaviour of lysozyme on HEA and PPA HyperCel™ mixed mode sorbents varies depending on the degree of charge modification at the surface of the protein. Experiments using conventional cation exchange and hydrophobic interaction chromatography confirm that both charge and hydrophobicity modification occurs at the surface of the protein after lysozyme reaction with phenylglyoxal. The results emanating from this work using HEA and PPA HyperCel sorbents strongly suggest that mixed mode chromatography can efficiently separate closely related proteins of only minor surface charge and/or hydrophobicity differences.

Affinity adsorption of lysozyme with Reactive Red 120 modified magnetic chitosan microspheres

Reactive Red 120 was utilized as an affinity dye–ligand to modify the surface of magnetic chitosan microspheres to improve the adsorption capacity for lysozyme. The impact of pH, ionic strength, adsorption time and initial lysozyme concentration on the adsorption of lysozyme from aqueous solutions was investigated. An increased adsorption amount of 116.9mg/g lysozyme on Reactive Red 120 modified microspheres was achieved in comparison to 24.6mg/g on unmodified magnetic chitosan microspheres. The adsorption behaviour of lysozyme by the Reactive Red 120 modified magnetic chitosan microspheres fitted the pseudo second-order kinetic and the Langmuir thermodynamic model. The desorption ratio was 92.6% using 1M NaCl phosphate buffer (pH 6.0) as the desorption agent. The adsorption capacity of the Reactive Red 120 modified magnetic chitosan microspheres did not decrease significantly after four adsorption–desorption cycles (P>0.05). The as-prepared Reactive Red 120 modified magnetic chitosan microspheres were then used for the extraction of lysozyme from egg white solutions. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) results showed that the purity of the obtained lysozyme was ∼80.7% and that the recovery yield was ∼89.1%.

Filmes ultrafinos de ésteres de celulose: preparo, caracterização e imobilização de proteínas

In this study cellulose acetate butyrate (CAB) and carboxymehtylcellulose acetate butyrate (CMCAB) films adsorbed onto silicon wafers were characterized by means of ellipsometry, atomic force microscopy (AFM), sum frequency generation spectroscopy (SFG) and contact angle measurements. The adsorption behavior of lysozyme (LIS) or bovine serum albumin (BSA) onto CAB and CMCAB films was investigated. The amounts of adsorbed LIS or BSA onto CMCAB films were more pronounced than those onto CAB films due to the presence of carboxymethyl group in the CMCAB structure. Besides, the adsorption of BSA molecules on CMCAB films was more favored than that of LIS molecules. Antimicrobial effect of LIS bound to CAB or CMCAB layers was evaluated using Micrococcus luteus as substrate.

Binding of water-soluble, globular proteins to anionic model membranes

The role of electrostatics is studied in the adsorption of proteins to negatively charged (phosphatidylcholine/phosphatidylglycerol, PC/PG) and neutral (PC) small unilamellar vesicles (SUVs). For model proteins the interaction is monitored vs. pH at low ionic strength. The adsorption behaviour of lysozyme, myoglobin and albumin (isoelectronic point, p I 5–11) is investigated in SUVs, along with changes of the fluorescence emission spectra of the charged proteins, via their adsorption on SUVs. Significant adsorption of the proteins to negatively charged SUVs is found only at pH values, where the number of positive charge moieties exceeds the number of negative charge moieties on the protein, by at least 3 e.u. The fluorescence emission of positively charged proteins increases, after adsorption on negatively charged SUVs. With increasing protein to phospholipid ratio, the increase in the fluorescence emission levels off and reaches a plateau. Neutralization of the SUV charge is the controlling factor in their adsorption. The plateau level depends on the type of protein and pH of the incubation medium. The pH dependency can be ascribed to the mean positive charge of the protein. The effective charge of all proteins is calculated from the charge differences between empty and protein-coated SUVs.

In Situ Control of the Oxide Layer on Thermally Evaporated Titanium and Lysozyme Adsorption by Means of Electrochemical Quartz Crystal Microbalance with Dissipation

Electrochemical (EC) quartz crystal microbalance with dissipation monitoring (ECQCM-D) is a new and powerful technique for the in situ study of adsorption phenomena, e.g., as a function of the potential of the substrate. When titanium (Ti) is employed as the substrate, its oxidation behavior needs to be taken into account. Ti is always covered with a native oxide layer that can grow by, e.g., thermal oxidation or under anodic polarization. For biomolecular adsorption studies on oxidized Ti under applied potential, a stable oxide layer is desired in order to be able to distinguish the adsorption phenomena and the oxide growth. Therefore, the oxidation of thermally evaporated Ti films was investigated in phosphate-buffered saline by means of ECQCM-D, using a specially designed EC flow cell. Upon stepping the potential applied to Ti up to 2.6 V vs standard hydrogen electrode (SHE), a fast increase of the mass was observed initially for each potential step, evolving slowly to an asymptotic mass change after several hours. The oxide layer thickness increased as a quasi-linear function of the oxidation potential for potentials up to 1.8 V vs SHE. The growth rate of the oxide was around 2.5-3 nm/V. No changes in the dissipation shift were observed for potentials up to 1.8 V vs SHE. The composition of the oxide layer was analyzed by X-ray photoelectron spectroscopy (XPS). It was mainly composed of TiO(2), with a small percentage of suboxides (TiO and Ti(2)O(3)) primarily at the inner metal/oxide interface. The amount of TiO(2) increased, and that of TiO and Ti(2)O(3) decreased, with increasing oxidation potential. For each oxidation potential, the calculated thickness obtained from ECQCM-D correlated well with the thickness obtained by XPS depth profiling. A procedure to prepare Ti samples with a stable oxide layer was successfully established for investigations on the influence of an electric field on the adsorption of biomolecules. As such, the effect of an applied potential on the adsorption behavior of lysozyme on oxidized Ti was investigated. It was observed that the adsorption of lysozyme on oxidized Ti was not influenced by the applied potential.

New α‐amino phenylalanine tetrazole ligand for immobilized metal affinity chromatography of proteins

A new chelating compound has been developed for use in the immobilized metal affinity chromatographic (IMAC) separation of proteins. The bidentate ligand, alpha-amino phenylalanine tetrazole, 4, was synthesized via a five-step synthesis from N-fluorenylmethoxycarbonyl phenylalanine and then immobilized onto silica through the epoxide coupling procedure. The binding behavior of the resulting IMAC sorbent, following chelation with Zn2+ to a density of 183 micromol Zn2+ ions/g silica, was characterized by the retention of proteins in the pH range of 5.0-8.0, and by the adsorption behavior of lysozyme with frontal chromatography at pH 6.0 and 8.0. The prepared column showed the separation ability to four test proteins and the retention time of these proteins increased with an increase in pH. From the derived isotherms, the adsorption capacity, qm, for the binding of lysozyme to immobilized Zn2+-alpha-amino phenylalanine tetrazole-silica was found to be 1.21 micromol/g at pH 6.0 and 1.20 micromol/g sorbent at pH 8.0, respectively, whilst the dissociation constants KD at these pH values were 5.22x10(-6) and 3.49x10(-6) M, respectively, indicating that the lysozyme was retained more stable under alkaline conditions, although the binding capacity in terms of micromole protein per gram sorbent remained essentially unchanged.

Adsorption Behavior of Lysozyme and Tween 80 at Hydrophilic and Hydrophobic Silica–Water Interfaces

Nonionic surfactants such as Tween 80 are used commercially to minimize protein loss through adsorption and aggregation and preserve native structure and activity. However, the specific mechanisms underlying Tween action in this context are not well understood. Here, we describe the interaction of the well-characterized, globular protein lysozyme with Tween 80 at solid-water interfaces. Hydrophilic and silanized, hydrophobic silica surfaces were used as substrates for protein and surfactant adsorption, which was monitored in situ, with ellipsometry. The method of lysozyme and Tween introduction to the surfaces was varied in order to identify the separate roles of protein, surfactant, and the protein-surfactant complex in the observed interfacial behavior. At the hydrophobic surface, the presence of Tween in the protein solution resulted in a reduction in amount of protein adsorbed, while lysozyme adsorption at the hydrophilic surface was entirely unaffected by the presence of Tween. In addition, while a Tween pre-coat prevented lysozyme adsorption on the hydrophobic surface, such a pre-coat was completely ineffective in reducing adsorption on the hydrophilic surface. These observations were attributed to surface-dependent differences in Tween binding strength and emphasize the importance of the direct interaction between surfactant and solid surface relative to surfactant-protein association in solution in the modulation of protein adsorption by Tween 80.

Effect of pH and Addition of Salt on the Adsorption Behavior of Lysozyme on Gold, Silica, and Titania Surfaces Observed by Quartz Crystal Microbalance with Dissipation Monitoring

The adsorption behaviors of lysozyme on dentally related Au, SiO2, and TiO2 surfaces were investigated by a quartz crystal microbalance with dissipation monitoring (QCM-D) method. Frequency shifts indicated that while lysozyme (pI 11) was fairly adsorbed on the SiO2 (pI 1.9) surface at both pH 3 and 7, it was adsorbed on TiO2 (pI 6.3) surface only at pH 7. However, adsorption was disturbed by 50 mM NaCl. These results strongly suggested an electrostatic nature of the adsorption behavior. Though a large-scale adsorption of the lysozyme on Au sensor was pH-insensitive, softness of the adlayer as seen from the dissipation profile was pH-dependent, indicating an interaction of another type. With all the surfaces, the small dissipation change indicated a stiff lysozyme adlayer. Results of this study revealed that the controlled electrostatic interaction between the material surface and lysozyme might be a useful method for imparting antibacterial property to the dental materials.

Protein separation using plate-and-frame modules with ion-exchange membranes

The separation performance using plate-and-frame modules with ion-exchange membranes was investigated in this study. The proteins employed were lysozyme, BSA and γ-globulin. In the batch adsorption, the adsorption behaviors of lysozyme onto cation-exchange membranes and BSA onto anion-exchange membranes, both showing the heterogeneous adsorption, could be described more appropriately with the Suen model than the Langmuir model. In the flow process using a single plate-and-frame module, operating conditions greatly influenced the separation performance. At the elution stage, a better performance was achieved by using the cross-flow mode. Other operating conditions such as the number of membranes in a stack, the pH value at adsorption, and the mixed mode in the membrane stack also affected the separation performance. As to the two-module experiments, two different operation designs were tested: connection in tandem and connection in parallel. The tandem design resulted in a better protein recovery than the parallel design when only one kind of ion-exchange membrane was adopted. Regarding the mixed-mode effect in the tandem design, when the ion-exchange membranes suitable for the target protein were placed in the second module, the target protein could gain a longer time for adsorption and, hence, a better recovery was accomplished.

Polysaccharide-Modified Poly(Ether Sulfone) Hollow Fibers as Solid Supports for Affinity Adsorption: Equilibrium Adsorption Study

Poly(ether sulfone) hollow fibers modified with various polysaccharides were used to immobilize Cibacron Blue 3GA for affinity adsorption. Characterization of modified hollow fibers with respect to their activation using ethylene glycol diglycidyl ether, conjugation with polysaccharides, and immobilization with Cibacron Blue 3GA indicates that the surface modification was successfully achieved. This work also studied the adsorption behavior of lysozyme onto immobilized Cibacron Blue 3GA using the polysaccharide-modified fibers as solid supports. The optimal adsorption capacity was observed from the hollow fibers conjugated with hydroxyethyl cellulose or dextran derivative and with high density of immobilized Cibacron Blue 3GA. Moreover, the effect of temperature on lysozyme adsorption was investigated. The results show that the lysozyme-ligand binding on the polysaccharide-modified hollow fibers tends to be endothermic.

Analysis of hen egg white lysozyme adsorption on Si(Ti)O 2 ∣ aqueous solution interfaces at low ionic strength: a biphasic reaction related to solution self-association

Adsorption kinetics of hen-egg white lysozyme at concentrations of 10 −7, 10 −6, 10 −4 M and at pH 8.0 have been measured on Si 0.8Ti 0.2O 2 surfaces by means of optical waveguide lightmode spectroscopy and were found to proceed in two distinct kinetic régimes: a fast and quasi linear increase of the surface concentration followed by slower kinetics that could be described qualitatively by the theory of random sequential adsorption. The transition between these two kinetic régimes was correlated with abrupt changes in the values of the mean refractive index and of the thickness of the adsorbed layer as well as in the desorption kinetics performed either before or after the transition from the first régime to the second. Experiments carried out at the same ionic strength but at different pH values showed that the adsorption behaviour of lysozyme is related to its self-association properties in highly concentrated solutions.

The Influence of Protein Size on Adsorption Kinetics and Equilibria in Ion-Exchange Chromatography

The adsorption behavior of lysozyme and γ-globulin on the strong cation-exchanger Streamline SP was studied by determining the equilibrium isotherms with batch experiments. Adsorption isotherms of the binary mixture of both proteins were modeled using the Langmuir parameters derived from single-component systems. Comparison of experimental results with the competitive Langmuir model displayed significant deviations for the adsorption of lysozyme in the mixture. An extended Langmuir model, accounting for the distinct accessibility of the sorbent's surface for the competing proteins, revealed much better consistency with experimental data. Assuming the rate-limiting step of protein uptake is due to mass transport effects, the kinetics were modeled with a rate model. Taking into account diffusion across the li-quid film and within the adsorbent pores, effective pore diffusion coefficients were determined. It was shown that the effective pore diffusion coefficient of both model proteins depends on the total protein concentration and the concentration ratio of the competing proteins in the liquid phase. In the binary mixture the diffusion rate of the faster diffusing protein lysozyme was slowed down by the lower diffusion rate of the larger protein γ-globulin. In packed bed experiments the weaker adsorbed γ-globulin was displaced by lysozyme and eluted earlier as in single-component breakthrough curves whereas the elution time of lysozyme was almost identical. However, the breakthrough curve of lysozyme was flatter in the presence of γ-globulin due to the diffusional hindrance in the mixture.

The sorption of lysozyme and ribonuclease onto ferromagnetic nickel powder 1. Adsorption of single components

The adsorption behavior of lysozyme and ribonuclease on nickel powder was studied to elucidate the effects of hydrophobicity, structural stability, and electrostatic forces. The results indicated that the equilibrium between proteins and nickel can be reached in about 20 min and some “overshoot” phenomena in adsorption were found. Maximal adsorption at the IEP, which could result in multilayer adsorption, was also noticed. A reduction in adsorption due to ionic shielding was observed for Cl− and SCN−, while the addition of ethanol promoted adsorption. FT-IR/ATR spectra were used to monitor the conformational changes during the adsorption process.

Protein adsorption on polystyrene latex particles

AbstractProtein adsorption is the net result of various types of interaction between the different components present in the system, i.e. the sorbent surface, the protein(s), the solvent and any other solutes such as low molecular weight electrolyte. In this paper methods and techniques will be discussed to study the mechanism of protein adsorption on, in particular, colloidal particles. The discussion is based on experimental data obtained with relatively simple systems that contain one well‐defined protein and well‐characterized polystyrene latexes. Thus, the adsorption behavior of lysozyme and α‐lactalbumin are compared, emphasizing the role of structural rearrangements in the protein molecule.

Studies on Functionalized Chitosan Beads II. Adsorption Behavior of Lysozyme onto Carboxylated Chitosan Beads.

Chitosan porous beads were prepared by coagulating chitosan acetate aqueous dope in basic alcohol. The N-succinylation and O-carboxymethylation were achieved by applying succinic anhydride and monochloroacetic acid, respectively, following the partial crosslinking to make acid durable chitosan beads. The degree of succinylation was regulated by applying acetic anhydride to block the amino group following the crosslinking. The NaOH concentration was a regulation factor for the carboxymethylation. The adsorption mechanisms of lysozyme on chemically modified beads were found to be different each other. The elution profile was affected by the pH shift, but not by ionic strength unlikely those of carboxymethyl-cellulose. N-acetylglucosamine (GlcNAc), saccharose and urea were found to inhibit the interaction of lysozyme with carboxylated chitosan beads in the presence of NaCl. The adsorption of lysozyme was achieved derectly from egg white and eluted fraction contained lysozyme of high purity.