Steric Exclusion Effect Research Articles

Steric Exclusion Chromatography for Purification of Biomolecules—A Review

Steric exclusion chromatography (SXC) is a purification method that is based on steric exclusion effects from the surface of the target and a hydrophilic stationary phase after the addition of polyethylene glycol (PEG), which leads to an association of the target with the stationary phase without direct binding, such as covalent, electrostatic, and hydrophilic/hydrophobic interactions. The gentle nature of the method has led to an increased focus on sensitive targets such as enveloped viruses with potential for other sensitive entities, e.g., extracellular vesicles and virus-like particles. SXC is related to PEG-mediated protein precipitation, but investigation of further process parameters was crucial to gain a better understanding of the SXC method. After explaining mechanistic fundamentals and their discovery, this review summarizes the findings on SXC from its first reference 11 years ago until today. Different applications of SXC are presented, demonstrating that the method can be used for a wide variety of targets and achieves high recovery rates and impurity removal. Further, critical process parameters for successful process implementation are discussed, including technical requirements, buffer composition, and scalability.

Ultrafast loose nanofiltration membrane intercalated by in-situ grown nanoparticles for dye purification and reuse

Loose nanofiltration membrane is regarded as an effective separation technology for dye purification and reuse, but is still restrained by the trade-off between the permeance and the dye/salts selectivity. Herein, we report the fabrication of an ultrafast loose polyamide nanofiltration (NF) membrane on the nanofibrous support layer with uniform in-situ grown ZnO nanoparticles. It was found that embedding ZnO nanoparticles provides more water channels and increased effective filtration area, thus enhancing the water permeation without trading off the dye/salt selectivity. The fabricated NF membrane with an in-situ formed ZnO-nanoparticle interlayer exhibits an ultrahigh water permeance up to 375 L m−2 h−1 bar−1, which increased by approximate seven-fold than that of virgin NF membrane (54 L m−2 h−1 bar−1) without nanoparticles intercalated, and attains high rejection of organic dyes (>91 % for Congo red, >92 % for Methyl blue, and >98 % for Victoria blue B) but low rejection of inorganic salts (<5 %). The synergy of steric exclusion and charge effect dominate the high selectivity of dye and salts. This study unlocks a new avenue to synthesize ultrahighly permeable-selective loose NF membranes.

The pH-dependent phase separation kinetics of type-A gelatin and dextran with different initial microstructures

The phase separation kinetics of type A gelatin and dextran (GE/DE) with different microstructures were investigated to get information on the relation between technofunctional properties and structures using static multiple light scattering (S-MLS), fluorescence microscopy, and macroscopic observations. GE/DE mixtures of 5.0 wt%/2.0 wt% and 5.0 wt%/3.0 wt% at pH = 5.50–9.00 were selected as the typical samples with the DE-in-GE and bicontinuous microstructures, respectively. Macrophase separation was found to occur in both the two mixtures at pH = 5.50–9.00 with the upper phase rich in GE and the lower phase rich in DE. However, the effect of pH on the phase separation rates of the two mixtures was different: pH-independent in DE-in-GE mixtures, while pH-dependent in bicontinuous mixtures. Specifically, the order of phase separation rates for the bicontinuous mixtures at pH = 5.50–9.00 was pH5.50 > pH6.00 > pH9.00 > pH7.00 ≈ pH8.00. Changes in the electrostatic interaction or steric exclusion effect among GE (DE) droplets at pH = 5.50–9.00 were used to explain the different phase separation kinetics of GE/DE mixtures with GE-in-DE, DE-in-GE, and bicontinuous microstructures. This work characterized the phase separation processes of GE/DE mixtures with three kinds of microstructure and provided the parameters for the phase separation kinetics at the macro-level.

Engineering a Bacillus subtilis esterase for selective hydrolysis of d, l-menthyl acetate in an organic solvent-free system.

Esterase/lipase-catalyzed selective hydrolysis of d, l-menthyl esters has become one of the promising approaches for producing l-menthol, one of the most important flavoring chemicals with extensive uses. However, the activity and l-enantioselectivity of the biocatalyst are not sufficient for meeting the industrial requirements. Herein, a highly active para-nitrobenzyl esterase from Bacillus subtilis 168 (pnbA-BS) was cloned and then engineered to enhance its l-enantioselectivity. On the basis of the strategy tailoring the steric exclusion effect and structural flexibility of the region adjacent to the substrate, the substitution of Ala400 to Pro caused a remarkable improvement in the E value from 1.0 to 466.6. The variant A400P was purified and further confirmed with strict l-enantioselectivity in the selective hydrolysis of d, l-menthyl acetate, whereas the improved l-enantioselectivity caused decreased activity. To develop an efficient, easy-to-use, and green methodology, organic solvent was omitted and substrate constant feeding was integrated into the whole-cell catalyzed system. During the catalytic process, the selective hydrolysis of 1.0 M d, l-menthyl acetate in 14 h offered a conversion of 48.9%, e.e.p value of >99%, and space-time yield of 160.52 g (l d)-1.

Influence of Electrolyte Concentration on Single-Molecule Sensing of Perfluorocarboxylic Acids.

Perfluorocarboxylic acids (PFCAs) are an emerging class of persistent organic pollutants. During the fabrication process, it is unavoidable to form PFCA homologs or isomers which exhibit distinct occurrence, bioaccumulation, and toxicity. The precision measurement of PFCAs is therefore of significant importance. However, the existing characterization techniques, such as LC-MS/MS, cannot fully meet the requirement of isomer-specific analysis, largely due to the lack of authentic standards. Single-molecule sensors (SMSs) based on nanopore electrochemistry may be a feasible solution for PFCAs determination, thanks to their ultra-high spatiotemporal resolutions. Hence, as a first step, this work was to elucidate the influence of electrolyte concentration on the four most critical indicators of nanopore measurements, and furthermore, performance of nanopore SMSs. More specifically, three of the most representative short-chain PFCAs, perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA) and perfluoroheptanoic acid (PFHpA), were adopted as the target analytes, aerolysin nanopore was employed as the sensing interface, and 2, 3 and 4 M KCl solutions were used as electrolytes. It was found that, when the concentration of KCl solution increased from 2 to 4 M, the conductance of aerolysin nanopore increased almost linearly at a rate of 0.5 nS per molar KCl within the whole voltage range, the current blockade of PFPeA at −50 mV increased from 61.74 to 66.57% owing to the enhanced steric exclusion effect, the maximum dwell time was more than doubled from 14.5 to 31.5 ms, and the barrier limited capture rate increased by 8.3 times from 0.46 to 3.85 Hz. As a result, when using 4 M KCl as the electrolyte, over 90% of the PFPeA, PFHxA and PFHpA were accurately identified from a mixed sample, and the calculated limit of detection of PFPeA reached 320 nM, more than 24 times lower than in 2 M KCl. It was thus clear that tuning the electrolyte concentration was a simple but very effective approach to improve the performance of nanopore SMSs for PFCAs determination.

Influence of multivalent-electrolyte metal solutions on the superficial properties and performance of a polyamide nanofiltration membrane

Heavy metal-ion pollution in water is a serious problem for the environment, and membrane filtration technologies such as nanofiltration (NF) have become viable treatment alternatives. However, it is necessary to understand the performance of NF as a function of effluent composition and membrane surface charge properties. This work evaluated first the impact of metal ions on the zeta potential using streaming potential measurements for a commercial NF membrane (NF270). Then, NF rejection of Ca2+, Cu2+, and Al3+ in sulphate and chloride solutions was analyzed based on zeta potential results. Reductions, neutralizations, and inversions of the surface charge of the membrane were observed through streaming zeta potential analysis. Our results also revealed significant differences of the zeta potential between divalent ions and aluminum under all experimental conditions studied. Based on the zeta potential measurements, it was possible to explain the rejects observed for the different metallic ions, through an analysis of steric and electric exclusion effects. Adsorption of ions caused also changes of membrane properties that were analyzed by ATR-FTIR, AFM and contact angles.

Grafting with chondroitin sulfate on poly(vinyl alcohol) to improve antifouling property

Poly(vinyl alcohol) (PVA) hydrogels become muddy while used in artificial corneas. To enhance the antifouling property of PVA hydrogels, a PVA hydrogel was grafted with chondroitin sulfate (CdS) through a two-step reaction in this work. The surface chemical compositions, surface morphology and thermal property of the hydrogel were characterized by attenuated total reflectance FTIR, X-ray photoelectron spectroscopy, atomic force microscopy, scanning electron microscopy and thermogravimetric analysis. It was confirmed that CdS was successfully grafted onto the surface of the PVA hydrogel through a two-step method. After grafting with p(GMA-CdS) (GMA: glycidyl methacrylate), both the thermal and mechanical properties of the PVA hydrogel became weaker and the PVA hydrogel became hydrophilic. The biocompatibility of the PVA-g-p(GMA-CdS) hydrogel could be considered as non-cytotoxic according to ISO 10993-5:2009. The antifouling property of the PVA-g-p(GMA-CdS) hydrogel, namely its anti-protein adsorption and anti-cell adhesion, was significantly improved due to surface hydration, steric exclusion effect and charge surface. The anti-protein adsorption of the PVA-g-p(GMA-CdS) hydrogel increased by about 33·48% in comparison with that of the PVA hydrogel and the anti-cell adhesion increased by about 67·92%. Overall, the PVA-g-p(GMA-CdS) hydrogel is an ideal biomaterial candidate for artificial corneas.

Electrolytes under Inhomogeneous Nanoconfinement: Water Structuring-Mediated Local Ion Accumulation.

The behaviors of aqueous electrolytes confined in nanoscale spaces impact a broad range of biological processes and industrial applications. Our current microscopic understanding of confined electrolytes relies primarily on ideal model systems featuring homogeneous nanoconfinement. Here, we investigate the structure and dynamics of various electrolytes subject to inhomogeneous nanoconfinement, i.e., confined in two-dimensional nanochannels with gradually varying local channel heights, by means of molecular dynamics simulations. Our results reveal unexpected local ion accumulation in the inhomogeneous space occurring at boundaries between coexisting structured water phases, including trilayer, four-layer, and bulk-like waters. This contrasts markedly with the intuition that hydrated ions are more favorable to weakly confined regimes due to a steric exclusion effect. We further show that the location and intensity of the water structuring-mediated ion accumulation are sensitive to the nanochannel's geometry and surface wettability. The revealed anomalous ion behaviors in inhomogeneous nanoconfinement should help to improve our understanding of the microscopic mechanism underlying the operation of biological ion channels and to develop functional nanofluidic devices.

Hydrophobicity Character of A-lactalbumin Nanoparticles: An Ultrasonic Study

Effect of pH and cosolvent on the stabilization of protein structure is a well established study in protein or food science. Among the various applications of proteins, the use of protein nanoparticle as drug or bioactive compound carriers is the one which is of most interest to many diverse researchers. The synthesis of such protein nanoparticles and their characterization is of prior requirement for the realization of these drug or bioactive carriers. On this basis, the present work deals with the ultrasonic analysis of hydrophobic interactions exhibited by the a-lactalbumin nanoparticle synthesized by heat treatment using acetone as desolvating agent. In order to enrich the variations in hydrophobicity, heat or temperature and cosolvent (glucose) are included in the study. The results are interpreted in terms of the interactions existing among the components and the evolved discussions reveal the bulk nature of the medium is controlled by the existing hydrophobicity interactions. The obtained results indicate that the dependency of protein denaturation on heat and the strengthening of non- covalent interactions by the cosolvent and/or the steric exclusion effect can be attributed to the structural modifications of protein.

A comparative investigation of random and oriented immobilization of protein A ligands on the binding of immunoglobulin G

In this work, a range of investigation were conducted to illustrate the mechanism of hIgG –protein A from Staphylococcal aureus (SpA) binding for the development of high-capacity SpA gels. Results of surface plasmon resonance demonstrated that oriented immobilization at carboxyl terminus of Z domain was more preferred than that at amino terminus and bound hIgG with the oriented domain had a flat-on orientation over chip surface. Furthermore, much negative enthalpy changes as well as lower binding stoichiometry were observed in the binding of tetrameric Z domains (denoted as Z4cys) to hIgG, indicating that each of both ligands could bind 2.1 hIgG molecules. Adsorption equilibria of hIgG adsorption showed that by far the maximum adsorption capacity for hIgG in Sepharose-based protein A gels was determined to be about 120.0 mg/g gel. Its excellent performance was further manifested by the result of dynamic binding capacity. In protein A gel, moreover, there was a critical density around 20 mg/g gel, above which the availability of the ligand decreased rapidly due to serious steric exclusion effect. The research provided insight into the IgG – SpA binding and strategic guidance for the development of high-capacity protein A gels.

Effect of pH and Glucose on the Stability of α-lactalbumin

The objective of this study is to understand the influence of pH and effect of cosolvent (glucose) on the stabilization of bovine α-lactalbumin by using ultrasonic techniques. Values of density, ultrasonic velocity and viscosity were measured for bovine α-lactalbumin (5 mg/ml) dissolved in phosphate buffer (pH 2, 5, 7, 9 and 12) solutions mixed with and without the cosolvent at 30 °C. These measurements were used to calculate few thermo-acoustical parameters such as adiabatic compressibility, intermolecular free length, acoustic impedance, relaxation time, relative association constant, the partial apparent specific volume and the partial apparent specific adiabatic compressibility for the said systems. The obtained results revealed a strong comparison between the effects of acidic and alkaline pH values on protein denaturation, i.e., the acidic pH are instantaneous and are of less magnitude whereas alkaline pH are slower but sharper. Further the present study supports the fact that the presence of glucose stabilizes α-lactalbumin against denaturation due to pH variation, which may be due to the strengthening of non-covalent interactions and the steric exclusion effect.

Self Crowding of Globular Proteins Studied by Small-Angle X-Ray Scattering

Small-angle x-ray scattering (SAXS) was used to study the behavior of equine metmyoglobin (Mb) and bovine pancreatic trypsin inhibitor (BPTI) at concentrations up to 0.4 and 0.15 g/mL, respectively, in solutions also containing 50% D2O and 1 M urea. For both proteins, significant effects because of interference between x-rays scattered by different molecules (interparticle interference) were observed, indicating nonideal behavior at high concentrations. The experimental data were analyzed by comparison of the observed scattering profiles with those predicted by crystal structures of the proteins and a hard-sphere fluid model used to represent steric exclusion effects. The Mb scattering data were well fit by the hard-sphere model using a sphere radius of 18 Å, only slightly smaller than that estimated from the three-dimensional structure (20 Å). In contrast, the scattering profiles for BPTI in phosphate buffer displayed substantially less pronounced interparticle interference than predicted by the hard-sphere model and the radius estimated from the known structure of the protein (15 Å). Replacing the phosphate buffer with 3-(N-morpolino)propane sulfonic acid (MOPS) led to increased interparticle interference, consistent with a larger effective radius and suggesting that phosphate ions may mediate attractive intermolecular interactions, as observed in some BPTI crystal structures, without the formation of stable oligomers. The scattering data were also used to estimate second virial coefficients for the two proteins: 2.0 ×10-4 cm3mol/g2 for Mb in phosphate buffer, 1.6 ×10-4 cm3mol/g2 for BPTI in phosphate buffer and 9.2 ×10-4 cm3mol/g2 for BPTI in MOPS. The results indicate that the behavior of Mb, which is nearly isoelectric under the conditions used, is well described by the hard-sphere model, but that of BPTI is considerably more complex and is likely influenced by both repulsive and attractive electrostatic interactions. The hard-sphere model may be a generally useful tool for the analysis of small-angle scattering data from concentrated macromolecular solutions.

Adsorption Characteristics of Fungal Family 1 Cellulose-Binding Domain from Trichoderma reesei Cellobiohydrolase I on Crystalline Cellulose: Negative Cooperative Adsorption via a Steric Exclusion Effect

Cellobiohydrolases (CBHs) hydrolyzing crystalline cellulose share a two-domain structure of catalytic domain (CD) and cellulose-binding domain (CBD). To focus on the binding characteristics of CBD, we analyzed the adsorption of fusion protein of fungal family 1 CBD from Trichoderma reesei CBH I and red-fluorescent protein on crystalline and amorphous celluloses. Binding data were better fitted by Hill's model with negative cooperativity than by other adsorption models, suggesting the occurrence of a steric exclusion effect among the fusion molecules on the cellulose surfaces. The degree of negative cooperativity depended on the nature of the cellulose. The significance of this phenomenon for catalysis by intact CBHI is discussed.

Cation⊗3π: Cooperative Interaction of a Cation and Three Benzenes with an Anomalous Order in Binding Energy

Cation-π or cation-π-π interaction between one cation and one or two structures bearing rich π-electrons (such as benzene, aromatic rings, graphene, and carbon nanotubes) plays a ubiquitous role in various areas. Here, we analyzed a new type interaction, cation⊗3π, whereby one cation simultaneously binds with three separate π-electron-rich structures. Surprisingly, we found an anomalous increase in the order of the one-benzene binding strength of the cation⊗3π interaction, with K(+) > Na(+) > Li(+). This was at odds with the conventional ranking of the binding strength which usually increases as the radii of the cations decrease. The key to the present unexpected observations was the cooperative interaction of the cation with the three benzenes and also between the three benzenes, in which a steric-exclusion effect between the three benzenes played an important role. Moreover, the binding energy of cation⊗3π was comparable to cation⊗2π for K(+) and Na(+), showing the particular importance of cation⊗3π interaction in biological systems.

Impact of cosolvent (glucose) on the stabilization of ovalbumin

Proteins are stabilized by glucose against denaturation due to extremes of pH. This was studied by means of density, ultrasonic velocity, viscosity and surface tension measurements in the ovalbumin (5 mg/ml) dissolved in phosphate buffer (pH 2, 5, 7, 9 and 12). Few thermo-acoustical parameters such as adiabatic compressibility, intermolecular free length, acoustic impedance, the partial apparent specific volume and the partial apparent specific adiabatic compressibility were calculated for the said systems. Obtained results suggest that the stabilization of ovalbumin occurs in the presence of glucose through strengthening of hydrophobic interactions supported by other non-covalent interactions and the steric exclusion effect of the cosolvent molecules.

Mathematical study of non-ideal electrostatic correlations in equilibrium electrolytes

We undertake the mathematical analysis of a model describing equilibrium binary electrolytes surrounded by charged solid walls. The problem is formulated in terms of the electrostatic potential and the ionic concentrations which have prescribed spatial mean values. The free energy of the system is decomposed as the difference of the internal energy and entropy functionals. The entropy functional is the sum of an ideal entropy and an excess entropy, the latter taking into account non-ideality due to electrostatic correlations at low ionic concentrations and steric exclusion effects at high ionic concentrations. We derive sufficient conditions to achieve convexity of the entropy functional, yielding a convex–concave free energy functional. Our main result is the existence and uniqueness of the saddle point of the free energy functional and its characterization as a solution of the original model problem. The proof hinges on positive uniform lower bounds for the ionic concentrations and uniform upper bounds for the ionic concentrations and the electrostatic potential. Some numerical experiments are presented in the case where the excess entropy is evaluated using the mean spherical approximation.

Critical Dipole Length for the Wetting Transition Due to Collective Water-dipoles Interactions

The wetting behavior of water on the solid surfaces is fundamental to various physical, chemical and biological processes. Conventionally, the surface with charges or charge dipoles is hydrophilic, whereas the non-polar surface is hydrophobic though some exceptions were recently reported. Using molecular dynamics simulations, we show that there is a critical length of the charge dipoles on the solid surface. The solid surface still exhibited hydrophobic behavior when the dipole length was less than the critical value, indicating that the water molecules on the solid surface seemed not “feel” attractive interactions from the charge dipoles on the solid surface. Those unexpected observations result from the collective interactions between the water molecules and charge dipoles on the solid surface, where the steric exclusion effect between water molecules greatly reduces the water-dipole interactions. Remarkably, the steric exclusion effect is also important for surfaces with charge dipole lengths greater than this critical length.

Physicochemical basis for cosolvent modulation of β-lactoglobulin functionality: Interfacial tension study

Abstract The impact of neutral cosolvents on the thermal stability of globular proteins in aqueous solutions depends on the nature of the cosolvent, e . g ., sorbitol causes a pronounced increase in the thermal denaturation temperature ( T m ) of β-lactoglobulin (β-lg), whereas glycerol does not. When a protein unfolds there is a change in the exposed surface area and in the type of molecular interactions that occur at the protein–solvent–cosolvent interface. These changes contribute to the free energy change associated with protein denaturation and depend on cosolvent type. In this study we measured the equilibrium interfacial tensions of aqueous glycerol (0–70% w/w) and sorbitol (0–55% w/w) solutions as a function of temperature to provide insights into the role of the interfacial energy on the thermal stability of β-lg. There was a slight increase in interfacial tension with increasing sorbitol concentration, indicating its preferential exclusion from the oil–water interface. On the other hand, there was an appreciable decrease in interfacial tension with increasing glycerol concentration, indicating its preferential accumulation at the oil–water interface. These changes were largely independent of the measurement temperature (30–80 °C). Our results suggest that sorbitol increases the T m of β-lg mainly through a steric exclusion effect, whereas glycerol has little effect on T m because the steric exclusion effect is counter-balanced by a differential interaction effect.

The effect of binary cosolvent systems (glycerol–sucrose mixtures) on the heat‐induced gelation mechanism of bovine serum albumin

SummaryThe influence of cosolvent composition on the thermal denaturation and gelation of bovine serum albumin (BSA) has been studied. Cosolvent composition was varied by changing the ratio of glycerol to sucrose in 40 wt% cosolvent solutions containing BSA. Differential scanning calorimetry measurements on 0.5 wt% BSA solutions showed that the thermal denaturation temperature of the protein increased with increasing sucrose content. Temperature scanning dynamic shear rheology and turbidity measurements on 4 wt% BSA solutions showed that the gelation temperature and final gel strength increased with increasing sucrose concentration. These observations were attributed to the fact that sucrose was more effective than glycerol at increasing the thermal stability and attraction between globular BSA molecules through a steric exclusion effect. The molecular origin of these effects is the tendency for the system to minimize the contact area of the protein molecules with the surrounding cosolvent solution.

Application of short monolithic columns for fast purification of plasmid DNA

Anion-exchange chromatography is one of the most important methods in downstream processing of plasmid DNA, both as a process and as an analytical technique. Separation of plasmid DNA on traditional particle-based anion-exchange supports is usually slow. Moreover, such supports have a low capacity for plasmid DNA due to the steric exclusion effects. In this work, the separation of plasmid DNA using short monolithic columns, Convective Interaction Media, will be presented. It will be demonstrated that plasmid DNA can be purified from bacterial cells using alkaline lysis followed by chromatography on a very short weak anion-exchange chromatographic columns—disks—with good purity and quality within a short time. Furthermore, the separation of plasmid DNA from cell RNA can be carried out without the need of adding RNAse. Fast and efficient method for in- process control of the purified plasmid will be described as well.