Competitive Protein Adsorption Research Articles

Impact of lipid droplet characteristics on the rheology of plant protein emulsion gels: Droplet size, concentration, and interfacial properties

Plant-based meat analogs are being developed to address environmental, sustainability, health, and animal welfare concerns associated with real meat products. However, it is challenging to mimic the desirable physicochemical, functional, and sensory properties of real meat products using plant-based ingredients. Emulsion gels consisting of lipid droplets embedded in biopolymer matrices are commonly used to create products with appearances, textures, and sensory attributes like meat products. In this study, the impact of soybean oil droplet characteristics (concentration, size, and charge) on the physicochemical properties of potato protein gels was studied. The oil droplets were either coated by a non-ionic surfactant (Tween 20) or a plant protein (patatin) to obtain different surface properties. The introduction of the oil droplets caused the protein gels to change from mauve to off-white, which was attributed to increased light scattering. Increasing the oil droplet concentration in the emulsion gels decreased their shear modulus and Young’s modulus, which was mainly attributed to the fact that the oil droplets were less rigid than the surrounding protein network. Moreover, increasing the oil droplet size made this effect more pronounced, which was attributed to their greater deformability. Competitive adsorption of proteins and surfactants at the oi-water interface in the Tween emulsion promoted emulsion instability. This research highlights the complexity of the interactions between oil droplets and protein networks in emulsion gels. These insights are important for the utilization of emulsion gels in the formulation of plant-based foods with improved quality attributes.

Competitive protein adsorption in isocratic anion exchange chromatography

Protein chromatography is the dominant method of purification of biopharmaceuticals. Although all practical chromatography involves competitive absorption and separation of M. species, competitive protein absorption has remained inadequately understood. We previously introduced the measurement of equilibrium protein adsorption isotherms with all intensive variables held constant, including competitor concentration. In this work, we introduce isocratic chromatographic retention measurements of dynamic protein adsorption in the presence of a constant concentration of a competitor protein. These measurements are achieved by establishing a dynamic equilibrium with a constant concentration of competitor (insulin) in the mobile phase flowing through an ion exchange adsorbent column and following the behavior of a test protein (α-lactalbumin) injected into this environment. We observed decreased retention times for α-lactalbumin in presence of the competitor. The presence of competitor also reduces the heterogeneity of the sites available for adsorption of the test protein. This investigation provides an approach to fundamental understanding of competitive dynamics of multicomponent protein chromatography.

Separation of protein corona from nanoparticles under intracellular acidic conditions: effect of protonation on nanoparticle-protein and protein-protein interactions.

Protein coronas separate from nanoparticles under intracellular acidic conditions however, competitive adsorption of multiple proteins and their protein network formation under different pH conditions have not yet been systematically studied at the atomic scale. Herein, we report all-atom molecular dynamics simulations of plasma proteins (human serum albumin and immunoglobulin gamma-1 chain C) adsorbed to 10 nm-sized carboxyl-terminated polystyrene (PS) nanoparticles at different protonation states that mimic extracellular and intracellular pH conditions of 7, 6-5, and 4.5. Binding free energies are calculated from umbrella sampling simulations, showing the significantly weakened binding between PS particles and proteins at the protonation state at pH 4.5, in agreement with experiments showing the separation of protein corona from nanoparticles at pH 4.5. Mixtures of multiple proteins and PS particles are also simulated, showing much less protein adsorption and protein cluster formation at the protonation state at pH 4.5 than that at higher pH values, which are further confirmed by calculating the diffusivities and hydrodynamic radii of individual proteins. In particular, electrostatic particle-protein and protein-protein interactions are significantly weakened by a combination of particle and protein protonation rather than by particle protonation alone, to an extent dependent on different proteins. These findings help explain the experimental observations regarding separation of protein corona from nanoparticles under intracellular acidic conditions at pH 4.5 but not at higher pH, supporting that acidification cannot be the only reason for this separation during the process of endosome maturation.

Effect of egg yolk lipoproteins on foaming properties and interfacial behavior of egg white: Insights into implications for aerated food production

Chicken eggs are often used as an ingredient in oil-containing aerated foods. Different egg yolk lipids have different influence on the foaming properties of egg white, but the exact mechanism behind this influence is not well understood. In this study, we aimed to investigate how high and low-density lipoproteins present in egg yolks impact the macroscopic foam characteristics, rheological properties, and air/water (a/w) interfacial behavior in egg white foams. We found that the addition of low-density lipoprotein (LDL) at a concentration as low as 0.1 % led to a significant decrease in foaming ability (FA) by 284.67–66.67 %. Conversely, high-density lipoproteins (HDL) improved the FA from 284.67 % to 340.67 % within the concentration range of 0.024–0.16 %. Rheological properties showed that the addition of LDL caused a decrease in the viscoelasticity of the interfacial membrane, while HDL had no significant effect on viscoelasticity. Interfacial behavior results suggested that LDL and HDL were detrimental to the diffusion of protein molecules at the a/w interface. This implied that the effects of LDL and HDL on the foaming properties of EWP were mainly due to the competitive adsorption of lipids and proteins at the A/W interface, as well as the interactions between them. This work provides new insights into the effect mechanism of different forms of egg yolk lipids on the foaming properties of egg white, which can be beneficial for regulating oil-containing aerated foods to meet production demands.

Competitive protein adsorption on charge regulating silica-like surfaces: the role of protonation equilibrium

We develop a molecular thermodynamic theory to study the interaction of some proteins with a charge regulating silica-like surface under a wide range of conditions, including pH, salt concentration and protein concentration. Proteins are modeled using their three dimensional structure from crystallographic data and the average experimental pKa of amino acid residues. As model systems, we study single-protein and binary solutions of cytochrome c, green fluorescent protein, lysozyme and myoglobin. Our results show that protonation equilibrium plays a critical role in the interactions of proteins with these type of surfaces. The terminal hydroxyl groups on the surface display considerable extent of charge regulation; protein residues with titratable side chains increase protonation according to changes in the local environment and the drop in pH near the surface. This behavior defines protein–surface interactions and leads to the emergence of several phenomena: (i) a complex non-ideal surface charge behavior; (ii) a non-monotonic adsorption of proteins as a function of pH; and (iii) the presence of two spatial regions, a protein-rich and a protein-depleted layer, that occur simultaneously at different distances from the surface when pH is slightly above the isoelectric point of the protein. In binary mixtures, protein adsorption and surface–protein interactions cannot be predicted from single-protein solution considerations.

Foaming and air-water interfacial properties of camel milk proteins compared to bovine milk proteins

The objective of this research was to explore the foaming properties of camel and bovine milk and their derived proteins fractions including sodium caseinates, sweet whey, β-casein, α-lactalbumin and β-lactoglobulin. First, camel and bovine milk proteins were identified by the reversed-phase high-performance liquid chromatography (RP-HPLC) and foaming properties (Foam capacity (FC) and stability (FS)) were analyzed. Afterwards, competitive adsorption of proteins to the air-water interface for both milk protein fractions was characterized using pendant-drop tensiometry parameters and was compared to intrinsic fluorescence results of pure proteins. Experimental results indicated that the maximum FC values were found for camel skim milk, sodium caseinates and β-casein with higher FS values for bovine β-casein. Differences in the stability and the highest tensioactive properties of camel β-casein were explained with the different molecular structure and its higher hydrophobicity when compared to its bovine counterpart. Thus, milk proteins adsorbed layers are mainly affected by the presence of β-casein which is the first adsorbed and the most abundant protein at the air-water contrary to whey proteins (α-lactalbumin and β-lactoglobulin). These globular proteins are involved in the composition of protein layers at air-water interface, giving higher viscoelastic modulus values, but could not compact well at the interface because of their rigid molecular structure. For camel milk, foaming properties and interfacial behavior are mainly maintained by camel β-casein due to its higher hydrophobicity compared to bovine β-casein and the greater exposure of tyrosine residues despite the absence of tryptophan in consistence with the intrinsic fluorescence results. Furthermore, the absence of the β-lactoglobulin leads to the dominance of the α-lactalbumin at the air-water interface which is characterized by lower hydrophobicity than its bovine counterpart leading to lower viscoelastic modulus values than those of bovine whey, and hence to weaker rheological properties of camel milk protein layer at the air-water interface.

The effect of sucrose esters S1570 on partial coalescence and whipping properties

This study investigated the effect of different hydrophilic sucrose ester S1570 concentrations on interfacial properties, emulsion characteristics and whipping properties of whipped cream. S1570 at low concentration range (≤0.10 wt%) can coexist with proteins at the oil/water interface. At high concentration range (>0.10 wt%), S1570 would induce crystals piercing interfacial layers and displace the proteins from the oil/water interface. And higher concentration serum protein can prevent fat droplets from entering the air/water interface. An increase of S1570 level at low concentration range can retard partial coalescence during the whole whipping process, thus causing longer optimum whipping time, higher overrun, lower firmness and stabilities of whipped cream. The increase of S1570 at high concentration range increased shear-induced partial coalescence but retard surface-mediated partial coalescence, which caused higher overrun and shorter optimum whipped time. Furthermore, samples with 0.50 wt% S1570 exhibited higher firmness and stabilities. Correlation analysis evidenced that synergistic and competitive adsorption of proteins and sucrose ester S1570 at oil/water interface account for different influence mechanics of shear-induced partial coalescence and surface-mediated partial coalescence during whipping process.

Quantitative Measurement of Multiprotein Nanoparticle Interactions Using NMR Spectroscopy.

An effective intensity-based reference is a cornerstone for quantitative nuclear magnetic resonance (NMR) studies, as the molecular concentration is encoded in its signal. In theory, NMR is well suited for the measurement of competitive protein adsorption onto nanoparticle (NP) surfaces, but current referencing systems are not optimized for multidimensional experiments. Presented herein is a simple and novel referencing system using 15N tryptophan (Trp) as an external reference for 1H-15N 2D NMR experiments. The referencing system is validated by the determination of the binding capacity of a single protein onto gold NPs. Then, the Trp reference is applied to protein mixtures, and signals from each protein are accurately quantified. All results are consistent with previous studies, but with substantially higher precision, indicating that the Trp reference can accurately calibrate the residue peak intensities and reduce systematic errors. Finally, the proposed Trp reference is used to kinetically monitor in situ and in real time the competitive adsorption of different proteins. As a challenging test case, we successfully apply our approach to a mixture of protein variants differing by only a single residue. Our results show that the binding of one protein will affect the binding of the other, leading to an altered NP corona composition. This work therefore highlights the importance of studying protein-NP interactions in protein mixtures in situ, and the referencing system developed here enables the quantification of binding kinetics and thermodynamics of multiple proteins using various 1H-15N 2D NMR techniques.

Molecular Modeling of Protein Corona Formation and Its Interactions with Nanoparticles and Cell Membranes for Nanomedicine Applications.

The conformations and surface properties of nanoparticles have been modified to improve the efficiency of drug delivery. However, when nanoparticles flow through the bloodstream, they interact with various plasma proteins, leading to the formation of protein layers on the nanoparticle surface, called protein corona. Experiments have shown that protein corona modulates nanoparticle size, shape, and surface properties and, thus, influence the aggregation of nanoparticles and their interactions with cell membranes, which can increases or decreases the delivery efficiency. To complement these experimental findings and understand atomic-level phenomena that cannot be captured by experiments, molecular dynamics (MD) simulations have been performed for the past decade. Here, we aim to review the critical role of MD simulations to understand (1) the conformation, binding site, and strength of plasma proteins that are adsorbed onto nanoparticle surfaces, (2) the competitive adsorption and desorption of plasma proteins on nanoparticle surfaces, and (3) the interactions between protein-coated nanoparticles and cell membranes. MD simulations have successfully predicted the competitive binding and conformation of protein corona and its effect on the nanoparticle–nanoparticle and nanoparticle–membrane interactions. In particular, simulations have uncovered the mechanism regarding the competitive adsorption and desorption of plasma proteins, which helps to explain the Vroman effect. Overall, these findings indicate that simulations can now provide predications in excellent agreement with experimental observations as well as atomic-scale insights into protein corona formation and interactions.

Competitive adsorption and dilatational rheology of pork myofibrillar and sarcoplasmic proteins at the O/W emulsion interface

The competitive adsorption behavior of pork sarcoplasmic (SP) and myofibrillar (MP) proteins, alone or mixed (ratios 6:4, 5:5 and 4:6, w/w) in 0.6 M NaCl, at the oil-water interface was investigated. SP adsorbed more rapidly than MP at the oil surface establishing a ~10 mN/m dynamic interfacial pressure (π). However, the dilatational elasticity (Ed) of the SP film was low when compared with MP. For corn oil emulsions prepared with mixed proteins, the poor interfacial adsorption (29%) and low emulsifying activity (3.42 m2/g) of SP were overcome by substitutions with MP that strongly bound to oil. Unabsorbed proteins were mostly SP and secondarily tropomyosin and C-protein from MP. Smaller droplets were observed in MP emulsions than in SP emulsions. Therefore, while both SP and MP contributed to the formation of O/W emulsions, MP played a far more competitive role due to its stronger interfacial adsorption and adhesion properties.

Controlling the kinetics of visible-light-induced photocatalytic performance of gold decorated graphitic carbon nitride nanocomposite using different proteins

In this work, the interaction of different proteins with two dimensional (2D) graphitic carbon nitride (GCN) nanosheets decorated with gold nanoparticles (Au NP) is reported along with their influence in controlling the visible-light-induced photocatalytic activity. The decoration of Au NP on 2D GCN nanosheets improves the photocatalytic activity as Au NP plays an important role in trapping and storing the conduction band electrons of GCN nanosheets. The structural, morphological and optical properties of Au-GCN (AGCN) nanocomposite have been investigated using scattering and spectroscopic techniques. Methylene blue (MB) dye was chosen as a representative system for the studying of visible-light-induced photocatalytic activity of AGCN nanocomposite in the presence of different proteins. Bovine serum albumin (BSA), trypsin and cytochrome C having different chain lengths and physicochemical properties were used to control the kinetics of the photocatalytic activity of GCN and AGCN nanocomposites. Due to the simultaneous competitive adsorption of MB and proteins, the photocatalytic activity of the nanocomposites got enhanced in the presence of BSA, while it got inhibited or slowed down in the presence of trypsin and cytochrome C, respectively. The observed photocatalytic activity could be correlated with the surface charges present on the proteins and the mechanistic understanding developed in this study can be beneficial in controlling the kinetics of the photocatalytic activity of semiconductor-based photocatalysts using proteins as activators or inhibitors.

Effect of competitive adsorption of serum proteins on microfluidic surfaces

Effect of competitive adsorption of serum proteins on microfluidic surfaces

Protein adsorption dynamics to polymer surfaces revisited-A multisystems approach.

Performance and safety of materials in contact with living matter are determined by sequential and competitive protein adsorption. However, cause and consequences of these processes remain hard to be generalized and predicted. In a new attempt to address that challenge, the authors compared and analyzed the protein adsorption and displacement on various thoroughly characterized polymer substrates using a combination of surface-sensitive techniques. A multiple linear regression approach was applied to model the dependence of protein adsorption, desorption, and exchange dynamics on protein and surface characteristics. While the analysis confirmed that protein properties primarily govern the observed adsorption and retention phenomena and hydrophobicity as well as surface charge are the most relevant polymer surface properties, the authors have identified several protein-surface combinations that deviate from these patterns and deserve further investigation.

Adsorption-selectivity customization and competitive adsorption of tryptamine-based mixed-mode chromatography

Mixed-mode chromatography (MMC) shows great promise for applications in protein purification. Therefore, novel MMC ligands are always desired. However, achieving high adsorption selectivity of ligands is a major challenge due to competitive adsorption. Herein, MMC with tryptamine (4 F F-TA) exhibited equal adsorption capacity for bovine serum albumin (BSA) and bovine immunoglobulin (bIgG), affording a unique competitive adsorption process, but non ideal purity. Molecular imprinting technology (MIT) was adopted for the customized adsorption selectivity of MMC, and BSA and bIgG were used as imprinting templates. Static and dynamic binding performances under competitive adsorption before and after imprinting were investigated at different pH values, salt concentrations, and BSA/bIgG mass ratios. The results showed that the adsorption capacity of the target protein remained similar level but competitive protein adsorption dropped significantly (below 40 mg/g resin). This indicated that the adsorption selectivity of 4 F F-TA could be customized by changing the template protein used during imprinting. In addition, the pH-dependence and salt tolerance of MMC were unchanged after imprinting. The binding behaviors of BSA and bIgG in column chromatographic separation further confirmed the improved in adsorption selectivity. The purity of BSA increased from 55.4% to 78.2% for 4 F F-TA@BSA and that of bIgG from 40.7% to 71.2% for 4 F F-TA@bIgG. This study indicated that MIT is an effective strategy to change and improve the adsorption selectivity of MMC.

Characterization of Sample Loss Caused by Competitive Adsorption of Proteins in Vials Using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.

Sample loss caused by competitive protein adsorption on solid surfaces from complex samples remains to be a major hurdle in sensitive analyses of proteins. No label-free techniques can easily quantify individual proteins adsorbed on irregular surfaces of Eppendorf vials or Falcon tubes, which are commonly used to contain complex biological samples. Multiplexed characterization of such adsorption by different proteins is technically challenging. Herein, we developed a direct protein analysis based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the characterization of sample loss occurred on the curved surface with limited area. Using this simple and easily accessible method, we discovered the effect of ethylenediaminetetraacetic acid on surface adsorption of different milk proteins, specifically an augmented loss of milk proteins in low-binding sample vials. In this study, we also identified severe biases of silver staining and established proteomics-based mapping of protein distribution in biological samples for absolute quantification of competitive protein adsorption on irregular surfaces.

Development and performance of a biomimetic artificial perilymph for in vitro testing of medical devices

Objective. Cochlear implants interface with the fluid in the cochlea called perilymph. The volume of this fluid present in human and animal model cochlea is prohibitively low for isolation for in vitro studies. Thus, there is a need for an artificial perilymph that reflects the complexity of this fluid in terms of competitive protein adsorption. Approach. This study established a biomimetic artificial perilymph (BAP) comprising serum albumin, immunoglobulin G, transferrin, inter-alpha-trypsin inhibitor, apolipoprotein A1 and complement C3 to represent the major components of human perilymph. Adsorption of the BAP components to platinum was analysed. Main results. It was established that this six component BAP provided competitive and complex adsorption behaviours consistent with biologically derived complex fluids. Additionally, adsorption of the BAP components to platinum cochlear electrodes resulted in a change in polarisation impedance consistent with that observed for the cochlear device in vivo. Significance. This study established a BAP fluid suitable for furthering the understanding of the implant environment for electroactive devices that interface with the biological environment.

The Competitive Dynamic Binding of Some Blood Proteins Adsorbed on Gold Nanoparticles

AbstractNanoparticle (NP) surfaces are modified immediately by the adsorption of proteins when injected into human blood, leading to the formation of a protein corona. The protein‐coated NPs may be recognized by living cells. Furthermore, the adsorption of serum proteins is a continuous competitive dynamic process that is the key to exploring the bioapplication and biosafety of NPs. In this study, the competitive dynamic adsorption of some serum proteins on gold nanoparticles (AuNPs) is investigated by fluorescence emission, dynamic light scattering, and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Serum proteins with different AuNPs binding affinities are used to address the competitive dynamic process of protein‐AuNP interactions in vitro. The results show that more abundant serum proteins, such as human serum albumin, adsorb on AuNPs first, and then the higher binding affinity and lower concentration serum proteins, such as fibrinogen (FIB), replace the abundant and lower binding affinity serum proteins. However, the lower binding affinity serum proteins, such as hemoglobin, do not replace the higher binding affinity proteins from the protein‐AuNP conjugates. During the dynamic exchange process, the larger the binding affinities difference between two proteins, the faster the exchange rate. This dynamic exchange process usually takes longer in inner protein‐AuNP conjugates (hard corona) than the external surface of protein‐AuNP conjugates (soft corona).

Use of pH Gradients in Responsive Polymer Hydrogels for the Separation and Localization of Proteins from Binary Mixtures

Using a molecular-level equilibrium theory where proteins are described using their crystallographic structure, we have studied protein adsorption from binary and ternary mixtures of myoglobin, lysozyme, and cytochrome c to poly(methacrylic acid) hydrogel films. The pH gradients these films induce can lead to selective protein adsorption, where the solution pH provides a sensible dial to externally control protein separation. Changing the chemical composition of the polymer network, adding either another acidic or a neutral comonomer, allows for protein localization to controlled spatial regions of the film with nanometer resolution. As pH-sensitive polymer hydrogels are promising candidates for smart, responsive biomaterials, understanding the complexity of competitive protein adsorption is essential. In this work, we highlight the decisive role of amino acid protonation in selective protein adsorption. We present conditions such that the hydrogel film will selectively incorporate the more weakly charged...

Conformational and Organizational Insights into Serum Proteins during Competitive Adsorption on Self-Assembled Monolayers.

Physicochemical interactions of proteins with surfaces mediate the interactions between the implant and the biological system. Surface chemistry of the implant is crucial as it regulates the events at the interface. The objective of this study was to explore the performance of modified surfaces for such interactions relevant to various biomedical applications. Because of a wide range of surface wettability, we aimed to study protein behavior (i.e., conformational changes and their packing) during competitive protein adsorption. Three serum proteins (bovine serum albumin, BSA; fibrinogen, FB; and immunoglobulin G, IgG) were tested for their conformational changes and orientation upon adsorption on hydrophilic (COOH and amine), moderately hydrophobic (mixed and hybrid), and hydrophobic (octyl) surfaces generated via silanization. Modified surfaces were characterized using Fourier-transform infrared spectroscopy, contact angle, and atomic force microscopy (AFM) techniques. Adsorbed masses of proteins from single and binary protein solutions on different surfaces were quantified along with their secondary structure analyses. Maximum adsorbed protein masses were found to be on negatively charged and hydrophobic (octyl) surfaces because of ionic and hydrophobic interactions between protein molecules and surfaces, respectively. Side-on and end-on orientations of adsorbed protein molecules were analyzed using theoretical and AFM analyses. We observed compact and elongated forms of BSA molecules on hydrophilic and hydrophobic surfaces, respectively. We further found a linear increase in the α-helix content of BSA and β-sheet contents of FB and IgG proteins with the increasing side-on (%)-oriented protein molecules on the surfaces. This indicates that side-on orientations of adsorbed FB and IgG lead to the formation of β-sheets. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was employed to quantify the protein types and their ratio in competitively adsorbed proteins on different surfaces. A theoretical analysis was also used to determine the % secondary structures of competitively adsorbed proteins from BSA/FB and BSA/IgG solutions, which very well agreed with experimental results. The competitive protein adsorption from both BSA/FB and BSA/IgG solutions was found to be entropy-driven, as revealed by thermodynamic studies performed using isothermal titration calorimetry.

Competitive adsorption of multiple proteins to nanoparticles: the Vroman effect revisited

ABSTRACTProteins adsorbed from the blood plasma change nanoparticles interactions with the surrounding biological environment. In general, the adsorption of multiple proteins has a non-monotonic time dependence, that is, proteins adsorbed at first may slowly be replaced by others. This ‘Vroman effect’ leads to a highly dynamic protein corona on nanoparticles that profoundly influences the immune response of the body. Thus, the temporal evolution of the corona must be taken into account when considering applications of nanocarriers in, e.g., nanomedicine or drug delivery. Up to now, the Vroman effect is explained solely in terms of diffusion: Smaller proteins which diffuse faster are adsorbed first, while larger ones, having a stronger interaction with the surface, are preferred at equilibrium. Here we use dynamic density functional theory (DDFT) including steric and electrostatic interactions to provide a full model for the temporal evolution of the protein corona. In particular, we demonstrate that proper consideration of all interactions leads to Vroman-like adsorption signatures in widely different scenarios. Moreover, consideration of energetic terms predicts both competitive as well as cooperative adsorption. In this way, DDFT provides a reacher picture of the evolution of the dynamic protein corona.