Protein Film Formation Research Articles

Film-forming modifications and mechanistic studies of soybean protein isolate by glycerol plasticization and thermal denaturation: A molecular interaction perspective

Plasticizer and thermal denaturation are indeed important factors for soybean protein film formation. The objective of the study was to investigate the effects of glycerol and thermal denaturation on the film-forming performances of soybean protein isolate (SPI) and elucidate the underlying mechanisms. From the results, glycerol had almost no effect on the protein’s secondary and tertiary structures. Indeed, the dispersion of glycerol diminished the intra- or intermolecular hydrogen bonding of SPI and interacted with the amino acids of subunits through hydrogen bonding and van der Waals forces. By interfering with protein network interactions, the glycerol molecule achieved a plasticizing effect on SPI films. The effects of heat treatment on SPI film properties were mainly realized through the changes in molecular conformation caused by protein denaturation, which manifested in the enhancement of light barrier and mechanical capabilities, and markedly altered the distribution of water states within the film network. This study provided valuable insights to clarify the mechanism of protein film formation.

Cysteines in β-lactoglobulin affects its interfacial adsorption and protein film stabilization

HypothesisDisulfide bonds in proteins are strong chemical bonds forming the secondary and tertiary structure like in the dairy protein β-lactoglobulin. We hypothesize that the partial or complete removal of disulfide bonds affects the structural rearrangement of proteins caused by intra- and intermolecular interactions that in turn define the interfacial activity of proteins at oil/water interfaces. The experimental and numerical investigations contribute to the mechanistic understanding of the structure-function relationship, especially for the interfacial adsorption behavior of proteins. Experimental and numericalSystematically, the 5 cysteines of β-lactoglobulin were recombinantly exchanged by alanine. First, the protein structure of the variants in bulk was analyzed with Fourier-transform-infrared-spectroscopy and molecular dynamic simulations. Second, the structural changes after adsorption to the interface have been also analyzed by molecular dynamic simulations. The adsorption behavior was investigated by pendant drop analysis and the interfacial film properties by dilatational rheology. FindingsThe structural flexibility of β-lactoglobulin with no cysteines encourages its unfolding at the interface, and accelerates the interfacial protein film formation that results in more visco-elastic films in comparison to the reference.

Amyloid-Like Assembly to Form Film at Interfaces: Structural Transformation and Application.

Protein-based biomaterials are attracting broad interest for their remarkable structural and functional properties. Disturbing the native protein's three-dimensional structural stability in vitro and controlling subsequent aggregation is an effective strategy to design and construct protein-based biomaterials. One of the recent developments in regulating protein structural transformation to ordered aggregation is amyloid assembly, which generates fibril-based 1D to 3D nanostructures as functional materials. Especially, the amyloid-like assembly to form films at interfaces has been reported, which is induced by the effective reduction of the intramolecular disulfide bond. The main contribution of this amyloid-like assembly is the large-scale formation of protein films at interfaces and excellent adhesion to target substrates. This review presents the research progress of the amyloid-like assembly to form films and related applications and thereby provides a guide to exploiting protein-based biomaterials. This article is protected by copyright. All rights reserved.

Modification of Tubings for Peristaltic Pumping of Biopharmaceutics

Protein particle formation during peristaltic pumping of biopharmaceuticals is due to protein film formation on the inner tubing surface followed by rupture of the film by the roller movement. Protein adsorption can be prevented by addition of surfactants as well as by increasing the hydrophilicity of the inner surface. Attempts based on covalent surface coating were mechanically not stable against the stress of roller movement. We successfully incorporated surface segregating smart polymers based on a polydimethylsiloxane (PDMS) backbone and polyethylene glycol (PEG) side blocks in the tubing wall matrix. For this we applied an easy, reproducible and cost-effective process based on soaking of tubing in toluene containing the PDMS-PEG copolymer. With this tubing modification we could drastically reduce protein particle formation during peristaltic pumping of a monoclonal antibody and human growth hormone (HGH) formulation in silicone and thermoplastic elastomer-based tubing. The modification did not impact the tubing integrity during pumping while hydrophilicity was increased and protein adsorption was prevented. Free PDMS-PEG copolymer might have an additional stabilizing effect, but less than 50 ppm of the PDMS-PEG copolymer leached from the modified tubing during 1 h of pumping in the experimental setup. In summary, we present a new method for the modification of tubings which reduces protein adsorption and particle formation during any operation involving peristaltic pumping, e.g. transfer, filling, or tangential flow filtration.

Catching Speedy Gonzales: Driving forces for Protein Film Formation on Silicone Rubber Tubing During Pumping

Interfacial adsorption is a major concern in the processing of biopharmaceutics as it not only leads to a loss of protein, but also to particle formation. Protein particle formation during peristaltic pumping is linked to interfacial adsorption to the tubing and subsequent tearing of the formed protein film. In the current study, driving forces and rate of the adsorption of a monoclonal antibody to the silicone rubber surface during pumping, as well as particle formation, were studied in different formulations. Particle concentration and size distribution were influenced by the formulation parameters; specifically high ionic strength led to more particles and the build-up of particles larger than 25 µm. Formulation pH and ionic strength had an effect on the total amount of adsorbed protein. Adsorbed protein amounts increased when the Debye length of the protein was decreased, leading to a higher packing density. Atomic force microscopy and streaming potential determination revealed that the irreversible protein film formation on the hydrophobic tubing surface occurs in less than a second. Electrostatic interactions are the dominating factor for the initial adsorption speed. In intimate contact to the silicone rubber surface, hydrophobic interactions govern the protein adsorption. PS20 quickly coats the tubing surface which leads to an increase in hydrophilicity and shielding of electrostatic interactions, thereby efficiently inhibiting protein adsorption. Overall, atomic force microscopy and streaming potential determination possess great potential for the characterization of adsorbed protein films and the adsorption kinetic evaluation in high-speed mode. Protein adsorption to silicone tubing is driven by a combination of electrostatic and hydrophobic interactions which is effectively shielded by PS20.

Proteins on the Rack: Mechanistic Studies on Protein Particle Formation During Peristaltic Pumping

Peristaltic pumping can cause protein particle formation. The expected causes were unfolding by heat in the pump head, oxidative stress by cavitation generated during roller movement, interfacial adsorption to the tubing wall and mechanical stress by stretching of the tubing itself. The pump head reached 28°C during experiments stayed well below the onset of the melting point of the proteins. Thus, heat may only be a relevant root cause for proteins containing domains with very low unfolding temperature. Analysis by terephthalic acid dosimetry and protein oxidation via RP-HPLC ruled out major induction of reactive hydroxyl radicals by pumping, indicating that cavitation does not play a significant role in particle generation. Addition of surfactants suppresses protein adsorption to the tubing wall and drastically reduced protein particle formation. This indicates that interfacial protein adsorption is a key element. Repeated stretching of tubing filled with protein solution led to the formation of protein particles, demonstrating that expansion and compression of the protein film on the tubing surface is the second key component for particle formation. Thus, protein particle generation during peristaltic pumping originates from the formation of a protein film on the tubing surface which gets stretched and compressed, leading to film fragments entering the bulk solution. This interplay of protein film formation and its rupture has been also observed at liquid/liquid or liquid/air interfaces.

Off-label use of plastic syringes with silicone oil for intravenous infusion bags of antibodies

The formation of particulates in post-manufacture biopharmaceuticals continues to be a major concern in medical treatment. This study was designed to evaluate the content of micro-sized particles using flow imaging of antibodies in intravenous infusion bags. Intravenous immunoglobulin (IVIG) and Avastin® were selected as model drugs and plastic syringes with and without silicone oil (SO) were used to transfer the drugs into the bags (0.9% saline or 5% dextrose). Antibodies exposed to SO had significantly increased levels of microparticles in both diluents, suggesting SO accelerates particle formation, especially at a higher antibody concentration. Even before the drop stress, their count exceeded the USP guideline. Dropping the bags in the presence of SO produced larger microparticles. Meanwhile, air bubbles were retained longer in saline suggesting more protein film formation on its air–water interface. Overall, both drugs were conformationally stable and produced less particles in dextrose than in saline.

Interfacial film formation and film stability of high hydrostatic pressure-treated β-lactoglobulin

The mechanical stability of interfacial protein films is limited through the intermolecular interaction of the adsorbed proteins. Since the intermolecular interactions vary in dependence of the protein structure, the investigation of intentionally induced structural modifications (i.e., through high hydrostatic pressure) of interfacial active proteins is required to mechanistically understand their structure-function-relationship. The aim of this study was to link the structural analysis of pressure-treated whey protein β-lactoglobulin at the oil/water-interface with the formation of the interfacial protein film and the resulting film stability against mechanical stress. For this purpose, we treated β-lactoglobulin in water at pH 7 with hydrostatic pressures of 600 MPa for 10 min at 20 °C and analyzed the protein structure in oil/water-emulsion with Fourier-transform-infrared-spectroscopy, extrinsic fluorescence, and ζ-potential. We characterized the molecular density and interfacial film properties via Langmuir trough analysis, electron-paramagnetic-resonance-spectroscopy, interfacial shear and dilatational rheology. The structural flexibility of pressure-treated β-lactoglobulin favored its unfolding and a fast interfacial protein film formation that resulted in pronounced intermolecular interactions, including van der Waals interactions, hydrophobic associations and disulfide bonds. The high interfacial molecule density combined with many and strong intermolecular interactions stabilized the pressure-treated protein film against shear deformation and small dilatational deformation. However, against high dilatational deformation, the pressure-treated β-lactoglobulin film showed a lower stability due to a slow migration towards unoccupied interfacial area. Hence, our research contributes to the control of the mechanical protein film stability, and thus the colloidal stability of emulsions in dependence of the protein structure at the oil/water-interface.

Partitioning Behavior and Interfacial Activity of Phenolic Acid Derivatives and their Impact on \u03b2-Lactoglobulin at the Oil-Water Interface

Proteins are able to stabilize dispersed food systems due to their amphiphilic nature, acting as emulsifiers. Their interfacial properties can be influenced by different methods, including the formation of protein-phenol nanocomplexes. In this study, the interfacial behavior of phenolic compounds and protein-phenol nanocomplexes was first characterized according to the oil-water partitioning behavior of phenolic acid derivatives according to their molecular structure and its impact on interfacial tension. The influence of the phenolic compounds on protein film formation and its properties by dilatational rheology was then evaluated. The most phenolic acid derivatives are predominantly present in the aqueous phase. Despite their hydrophobic benzene body, weak interfacial activity was observed depending on their chemical structure. This result supports possible protein-phenol nanocomplex formation in the aqueous phase and possible interactions at the oil-water interface. Protein-phenol nanocomplexes showed decreased interfacial adsorption properties and decreased viscoelastic interfacial behavior, depending on the expansion of the delocalized π-electrons in the phenol.

Effects of fatty acid saturation degree on salt-soluble pork protein conformation and interfacial adsorption characteristics at the oil/water interface

The aim of this study was to explore the effect of fatty acid saturation degree on the adsorption behavior of salt-soluble pork proteins at the oil/water interface and to reveal the mechanism between the conformational change of adsorbed proteins and the interfacial adsorption characteristics. Protein conformation, interfacial protein concentration (Γs), interfacial pressure, interfacial rheology and emulsion stability were measured in the oleic acid/linoleic acid/linolenic acid emulsions (OAE/LAE/LNAE). The results showed that the fluorescence intensity of the emulsions all decreased, which implied that tryptophan residues were exposed to the external polar environment and fatty acids influenced the polarity of micro-environment. α-Helix content in the emulsifying layer of OAE was significantly higher (P < 0.05) while β-sheets and β-turns in the emulsifying layer of OAE were significantly lower (P < 0.05) than those in the emulsifying layers of LAE and LNAE. As the saturation degree decreased, α-helices decreased, β-sheets and β-turns increased, and surface hydrophobicity decreased, implying that spatial conformational re-arrangement occurred and the number of exposed hydrophobic groups influenced the formation of interfacial protein film. In addition, the interfacial pressure of OAE/LAE/LNAE all increased as a function of adsorption time. Moreover, OAE and LAE had higher increasing adsorption rates and interfacial pressures during the initial step (0–3000 s) before interfacial pressures gradually reached the equilibrium, which indicated that low unsaturation degree facilitated the diffusion and adsorption of salt-soluble proteins. The elastic modulus (Ed) of the OAE and LAE increased rapidly within 0–1200 s, and gradually decreased. The viscous modulus (Ev) of the OAE and LAE increased. Fatty acids with low unsaturation degree could facilitate faster improvement of viscoelastic properties and the denser gel-like network structures, thus enhancing the emulsification stability.

HPP improves the emulsion properties of reduced fat and salt meat batters by promoting the adsorption of proteins at fat droplets/water interface

The aim of this study was to explore whether the state of the formation of interfacial protein film (IPF) could be utilized to increase the emulsion ability modified by high pressure processing (HPP). The meat emulsion was obtained from the meat batters with reduced fat and reduced salt (RFRS). The results revealed the highest stability and activity of meat emulsion were obtained as 68.12% and 76.89% at 200 MPa, respectively, and the minimized diameter of emulsion particles was D3,2 2.29 μm. Besides, after obtaining the interfacial proteins (IPs) and the fat droplets individually, the diameter of fat droplets and proportion of ionic bonds first decreased then rose along with increasing pressure, and the lowest values were exhibited at 200 MPa. In contrast, the adsorbed IPs content (0.657 mg/m2), zeta potential (−0.27), hydrophobic interactions (63.57%), hydrogen bonds (83.29%), and disulfide bonds (75.69%) presented the opposite trends, with the highest value also exhibited at 200 MPa. In conclusion, 200 MPa helped proteins adsorbed at the interface, corresponding with the unfolding conformational rearrangement and reorientation of IPs at the interface with the dispersed fat droplets.

Orientation and Conformation of Proteins at the Air-Water Interface Determined from Integrative Molecular Dynamics Simulations and Sum Frequency Generation Spectroscopy.

Understanding the assembly of proteins at the air-water interface (AWI) informs the formation of protein films, emulsion properties, and protein aggregation. Determination of protein conformation and orientation at an interface is difficult to resolve with a single experimental or simulation technique alone. To date, the interfacial structure of even one of the most widely studied proteins, lysozyme, at the AWI remains unresolved. In this study, molecular dynamics (MD) simulations are used to determine if the protein adopts a side-on, head-on, or axial orientation at the AWI with two different forcefields, GROMOS-53a6 + SPC/E and a99SB-disp + TIP4P-D. Vibrational sum frequency generation (SFG) spectroscopy experiments and spectral SFG calculations validate consistency between the structure determined from MD and experiments. Overall, we show with strong agreement that lysozyme adopts an axial conformation at pH 7. Further, we provide molecular-level insight as to how pH influences the binding domains of lysozyme resulting in side-on adsorption near the isoelectric point of the lysozyme.

Synergistic effects of flow and interfaces on antibody aggregation

During the manufacturing process, solutions of protein-based drugs are exposed to hydrodynamic forces, which can potentially affect protein stability and aggregation. Despite being an area of extensive investigation, the effect of hydrodynamic flow on protein aggregation is still controversial. In this study, we designed an experimental setup that allowed us to investigate flow- and interface-induced protein aggregation of two model immunoglobulins in the presence of well-defined flow stresses and solid-liquid interfaces. Within the range of shear rates typically encountered in bioprocessing ( ), we observed that increasing the shear rate by three orders of magnitude had a negligible effect on protein aggregation. By contrast, changes in the materials of the syringe barrels had a dramatic effect on the monomer loss, demonstrating the key role of solid-liquid interfaces in flow-induced aggregation. This finding was confirmed by the observed inverse dependence of the aggregation rate on the initial protein concentration, which is inconsistent with mechanisms of protein aggregation in bulk solution. Overall, our results reveal the presence of a synergistic effect of interfaces and hydrodynamic flow in flow-induced protein aggregation, which arises from the formation of protein particles or films on interfaces followed by displacement by flow or mechanical scraping.

Protein film formation on cell culture surfaces investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy

Conventional cell culture surfaces typically consist of polystyrene, with or without surface modifications created through plasma treatment or protein/peptide coating strategies. Other polymers such as fluorinated ethylene propylene are increasingly being implemented in the design of closed cell culture vessels, for example to facilitate the production of cells for cancer immunotherapy. Cultured cells are sensitive to culture vessel material changes through different mechanisms including cell-surface interactions, which are in turn dependent on the amount, type, and conformation of proteins adsorbed on the surface. Here, we investigate the protein deposition from cell culture medium onto untreated polystyrene and fluoropolymer surfaces using quartz crystal microbalance with dissipation monitoring and atomic force microscopy. Both of these non-polar surfaces showed comparable protein deposition kinetics and resulted in similar mechanical and topographical film properties. At protein concentrations found in typical serum-free media used to culture dendritic cells, two deposition phases can be observed. The protein layers form within the first few minutes of contact with the cell culture medium and likely consist almost exclusively of albumin. It is indicated that initial protein film formation will be completed prior to cell settling and initial cell contact will be established with the secondary protein layer. The structural properties of the protein film surface will strongly depend on the albumin concentration in the medium and presumably be less affected by the chemical composition of the cell culture surface.

Alternative Assembly of α-Synuclein Leading to Protein Film Formation and Its Application for Developing Polydiacetylene-Based Sensing Materials

Understanding the self-assembly process of amyloidogenic protein is valuable not only to find its pathological implication but also to prepare protein-based biomaterials. α-Synuclein (αS), a pathological component of Parkinson's disease, producing one-dimensional (1D) amyloid fibrils, has been employed to generate two-dimensional (2D) protein films by encouraging an alternative self-assembly process. At a high temperature of 50 °C, αS molecules self-assembled into 2D films instead of 1D amyloid fibrils, whereas the fibrils were the major product at 37 °C. Based on circular dichroism and Fourier transform infrared spectroscopy analyses, the film was produced via a structural transition from the initial random to still undefined but mostly the turn or loop structure, which was distinctive from the β-sheet formation observed with the amyloid fibrils. The αS 2D film was also routinely prepared at the oil-water interface and used as a matrix to produce polydiacetylene-based sensing materials. 10,12-Pentacosadiynoic acids (PCDA) were aligned on the film and photopolymerized to form a π-conjugated molecular assembly yielding a blue color. Its colorimetric transition to red was induced by increasing the temperature. This functionalized protein film increased its height from 40 to 55 nm upon PCDA immobilization and exhibited enhanced physical and chemical stability. In addition, the modified film showed remarkably high electrical conductivity only in the red state. This film, therefore, can be considered as a robust protein-based hybrid biomaterial capable of simultaneously recognizing various external stimuli (heat, pH, and solvents) with changes in color and conductivity, and it is expected to be utilized as a basic material for the development of biocompatible sensors.

Formation of Mixed Protein Films Using Proteins with Different Heat Stabilities

Formation of Mixed Protein Films Using Proteins with Different Heat Stabilities

In-situ 2D bacterial crystal growth as a function of protein concentration: An atomic force microscopy study.

The interplay between protein concentration and (observation) time has been investigated for the adsorption and crystal growth of the bacterial SbpA proteins on hydrophobic fluoride‐functionalized SiO2 surfaces. For this purpose, atomic force microscopy (AFM) has been performed in real‐time for monitoring protein crystal growth at different protein concentrations. Results reveal that (1) crystal formation occurs at concentrations above 0.08 µM and (2) the compliance of the formed crystal decreases by increasing protein concentration. All the crystal domains observed presented similar lattice parameters (being the mean value for the unit cell: a = 14.8 ± 0.5 nm, b = 14.7 ± 0.5 nm, γ = 90 ° ± 2). Protein film formation is shown to take place from initial nucleation points which originate a gradual and fast extension of the crystalline domains. The Avrami equation describes well the experimental results. Overall, the results suggest that protein‐substrate interactions prevail over protein–protein interactions.Research Highlights AFM enables to monitor protein crystallization in real‐time.AFM high‐resolution determines lattice parameters and viscoelastic properties.S‐layer crystal growth rate increases with protein concentration.Avrami equation models protein crystal growth.

A new approach to ordered protein film formation

A new approach to ordered protein film formation

Influence of molybdate ion and pH on the fretting corrosion of a CoCrMo – Titanium alloy couple

Recent findings suggest that during wear CoCrMo alloys develop a protein-rich protective tribochemical film formed by interaction of released molybdate ion with proteins in the lubricant. The purpose of this study was to determine the effect of adding molybdate ions directly to the medium on the fretting corrosion of a high carbon CoCrMo alloy in contact with a Ti6Al4V alloy. We also examined the effect of lowered pH to simulate acidification that may occur in enclosed areas, as within a modular joint.High-carbon CoCrMo cylindrical pins underwent fretting (±50μm, 17N, 60MPa) against a Ti6Al4V rod in a flat-on-cylinder configuration in a custom-designed fretting corrosion set-up that included a standard three-electrode configuration for electrochemical measurements. Four electrolytes were used, consisting of diluted bovine serum (30g/L of proteins), with or without added sodium molybdate (32mM), and at pH4.5 or 7.6. All the tests were performed potentiostatically at −0.25V versus SCE. Total Co, Cr, and Ti release to the media was determined by ICP-MS.The addition of molybdate ion decreased the release of cobalt by 32% and increased the after-to-before ratio of the polarization resistance by 8× at pH4.5, but had not effect on these parameters at pH7.6. Film deposits in the wear scar were more pronounced in the presence of molybdate ion. The addition of molybdate ion to the medium had a significant protective effect for the CoCrMo only at pH4.5, possibly because the lower pH allowed for more molybdate-induced protein film formation.

In situ observation of lubricant film formation in THR considering real conformity: The effect of diameter, clearance and material

The aim of the present study is to provide an analysis of protein film formation in hip joint replacements considering real conformity based on in situ observation of the contact zone. The main attention is focused on the effect of implant nominal diameter, diametric clearance and material. For this purpose, a pendulum hip joint simulator equipped with electromagnetic motors enabling to apply continuous swinging flexion–extension motion was employed. The experimental configuration consists of femoral component (CoCrMo, BIOLOX®forte, BIOLOX®delta) and acetabular cup from optical glass fabricated according to the dimensions of real cups. Two nominal diameters were studied, 28 and 36mm, respectively, while different diametric clearances were considered. Initially, a static test focused on the protein adsorption onto rubbing surfaces was performed with 36mm implants. It was found that the development of adsorbed layer is much more stable in the case of metal head, indicating that the adsorption forces are stronger compared to ceramic. A consequential swinging test revealed that the fundamental parameter influencing the protein film formation is diametric clearance. Independently of implant diameter, film was much thicker when a smaller clearance was considered. An increase of implant size from 28mm to 36mm did not cause a substantial difference in film formation; however, the total film thickness was higher for smaller implant. In terms of material, metal heads formed a thicker film, while this fact can be, among others, also attributed to clearance, which is more than two times higher in the case of ceramic implant.