Protein adsorption on novel acrylamido-based polymeric ion-exchangers. IV. Effects of protein size on adsorption capacity and rate

The effects of protein size on the adsorption capacity and rate is determined for an acrylamido-based polymeric anion-exchanger. The proteins lactalbumin, myoglobin, ovalbumin, BSA, conalbumin, IgG, and ferritin with molecular masses ranging from 15,000 to 450,000 were investigated. At high salt concentration (50 mM Tris-HCl containing 500 mM NaCl), only the smaller proteins lactalbumin and myoglobin gained access to a significant portion of the particle volume. The larger proteins were nearly completely excluded, in agreement with the results obtained for neutral macromolecules. By contrast, at low salt concentration (50 mM Tris-HCl), the adsorption capacity was very large (280-400 mg/ml of particle volume) for all the proteins studied except for ferritin, for which the capacity was much lower. This suggests that, provided the solute is not too large, the favorable electrostatic interaction overcomes the size exclusion effect. Adsorption rate measurements showed that mass transfer rates are also quite fast at low salt concentration. Effective diffusivities were determined by matching model and experimental results and were found to decrease substantially as the protein size increased. As previously observed, the homogeneous diffusion model was found to predict the experimentally observed trends with respect to protein concentration and boundary layer mass transfer effects.     

Surface science of DNA adsorption onto citrate-capped gold nanoparticles

Single-stranded DNA can be adsorbed by citrate capped gold nanoparticles (AuNPs), resulting in increased AuNP stability, which forms the basis of a number of biochemical and analytical applications, but the fundamental interaction of this adsorption reaction remains unclear. In this study, we measured DNA adsorption kinetics, capacity, and isotherms, demonstrating that the adsorption process is governed by electrostatic forces. The charge repulsion among DNA strands and between DNA and AuNPs can be reduced by adding salt, reducing pH or by using noncharged peptide nucleic acid (PNA). Langmuir adsorption isotherms are obtained, indicating the presence of both adsorption and desorption of DNA from AuNPs. While increasing salt concentration facilitates DNA adsorption, the desorption rate is also enhanced in higher salt due to DNA compaction. DNA adsorption capacity is determined by DNA oligomer length, DNA concentration, and salt. Previous studies indicated faster adsorption of short DNA oligomers by AuNPs, we find that once adsorbed, longer DNAs are much more effective in protecting AuNPs from aggregation. DNA adsorption is also facilitated by using low pH buffers and high alcohol concentrations. A model based on electrostatic repulsion on AuNPs is proposed to rationalize the DNA adsorption/desorption behavior.     

Analysis and modeling of fixed bed column operations on As(V) removal by adsorption onto iron oxide-coated cement (IOCC)

Fixed-bed column studies were undertaken to evaluate the performance of iron oxide-coated cement (IOCC) in removing As(V), from aqueous environment, under varying operating conditions. Investigations revealed IOCC to be a very efficient media for the removal of As(V) from water. The Bohart and Adams sorption model was employed to determine characteristic parameters such as depth of exchange zone, time required for exchange zone to move its own height, adsorption rate, and adsorption capacity, useful for process design. The IOCC media effectively removed As(V) from a 2 mg/L solution with a maximum adsorption capacity of 505.3 mg/L. Predicted data points obtained using a Freundlich isotherm model were compared against experimental data points determined in the dynamic process. The effects of important design parameters such as flow rate and influent concentration on the As(V) breakthrough curves were also investigated. The bed depth service time (BDST) model was extended to the prediction of service times of columns operated at different flow rates and influent concentrations and these predicted values were compared with the experimental values. The columns were regenerated by eluting the bound As(V) with 10% NaOH after the adsorption studies.     

Interfacial dilatational elasticity and viscosity of beta-lactoglobulin at air-water interface using pulsating bubble tensiometry

The ability of proteins to provide stability in foams is greatly influenced by their interfacial dilatational rheological properties. Surface tension response of a pulsatingbubble with an adsorbed layer of beta-lactoglobulin was measured for different frequencies and protein concentrations using a pulsating bubble tensiometer. A methodology, accounting for adsorption/desorption as well as variation of surface concentration due to expansion/contraction, was developed for the evaluation of surface dilatational elasticity and viscosity at different frequencies from these measurements. The adsorption rate constants were inferred from the surface pressure dynamics of protein adsorption using a Langmuir minitrough. The desorption rates were shown to be negligible for beta-lactoglobulin from the surface pressure response of a spread monolayer when subjected to compression in a Langmuir minitrough. The proposed model was employed to infer the interfacial dilatational viscosity and elasticity of an adsorbed beta-lactoglobulin layer at the air-water interface from experimental pulsating bubble data for protein concentrations in the range of 0.01-0.5 wt % at pH 7. As expected, the interfacial dilatational rheological properties were found to be higher at higher protein concentrations, this effect being less pronounced for dilatational elasticity. Heating at 80 degrees C for 30 min was found to result in higher interfacial dilatational viscosity and lower interfacial dilatational elasticity though this difference was within experimental error. The traditional approach for the inference of interfacial dilatational rheological properties is found to overpredict the interfacial dilatational elasticity whereas the viscosity values do not differ significantly from those obtained using the current analysis.     

Supermacroporous hydrophobic affinity sorbents for penicillin acylase purification

Penicillin acylase (PA, EC 3.5.1.11) is used as a raw material in the production of semi-synthetic penicillins. Although there are many methods for PA purification, affinity chromatography is advantageous as it provides efficient one step purification. In this study, poly(2-hydroxyethyl methacrylate) based cryogel column containing hydrophobic N-methacryloyl-L-tryptophan (MATrp) functional monomer as a ligand was prepared. Interaction of MATrp with amino acids in PA structure is the basis of hydrophobic interaction chromatography in this study. PHEMA and PHEMATrp cryogel columns were characterized by surface area measurements, infrared spectroscopy, swelling tests, elemental analysis and scanning electron microscopy (SEM). Initial PA concentration, pH, effect of temperature, amount of ligand, flow rate, ionic strength and time on PA adsorption on PHEMATrp cryogel were investigated. Optimum pH was determined as 5.0 for PA adsorption and maximum adsorption capacity was obtained as 6.40 mg/g. It was observed that adsorption capacity increased with the increasing of temperature. Also, PA adsorption increased up to 0.25 M salt concentration and decreased in higher salt concentrations. Data obtained in this affinity system suggests that hydrophobic interactions are dominant. In the last stage of the study, PA was purified from Penicillium chrysogenum with 76.3% yield and 332.3 purification factor.     

新型Al、Ti改性MCM-41材料对污水中Cu~（2＋）的吸附研究

研究了改性介孔硅基材料（以下简称MCM-41）对污水中重金属铜离子的吸附行为。在MCM-41材料中加入Al、Ti两种诱因金属离子来合成新的Al-Ti-MCM-41及Ti-Al-MCM-41改性材料,通过氮气吸附―脱附等温线对Al-Ti-MCM-41（1∶1）样品进行了表征,考察了吸附剂的投加量、Cu2＋初始浓度和吸附温...     

Frontal analysis for characterizing the adsorption-desorption behavior of beta-lactoglobulin on immunoadsorbents

High-performance frontal affinity chromatography was employed to study the adsorption-desorption kinetics characterizing the retention of beta-lactoglobulin (beta-LG) onto polyclonal anti-beta-lactoglobulin (anti-beta-LG) chromatographic supports. The adsorption and desorption processes were studied by analyzing two different elution fronts separated by a relatively long rinsing step. The method consists in performing two successive frontal injections of the protein. In between, the column was rinsed with a given volume of mobile phase (buffer alone). During this rinsing stage, a partial desorption may occur and a novel amount of protein could be adsorbed in the second frontal injection step. The whole process (first adsorption, possible desorption, and second adsorption) was simulated by a numerical procedure, in which the column was divided into a large number of slices. A model based on bi-Langmuir type kinetics was used to describe the adsorption of the protein on the support. The model assumes a non-uniform adsorbent with two types of binding sites. At equilibrium the adsorption isotherm is of the bi-Langmuir type. A global adsorption effect was considered which includes the effective binding process and the mass transfer resistances due to the transport to the binding site. Therefore, the column capacity and the kinetic parameters of the model (apparent adsorption and desorption rate constants) were determined from the best fit of the first and second adsorption fronts to the experimental ones. The other parameters of the model are the saturation capacities for the adsorption on each type of sites. The equilibrium affinity constants were estimated in a single experiment from the ratio of the apparent adsorption and desorption rate constants. The high values found (around 10(8) M(-1)) reveal a strong interaction of beta-LG with the immunoadsorbent. Kinetic measurements were carried out at different flow rates. Both the apparent adsorption and desorption kinetics were faster at larger flow rates, indicating an important contribution of the mass transfer resistance in the stagnant fluid at the particle boundary. However, as expected, close values were found for the resulting equilibrium constants calculated from the ratio of the apparent adsorption and desorption rate constant determined at various flow rates.     

Generalizing a two-conformation model for describing salt and temperature effects on protein retention and stability in hydrophobic interaction chromatography

A two-conformation adsorption model that includes the effects of salt concentration and temperature on both stability and adsorption has been developed to describe the effects of secondary protein unfolding on hydrophobic interaction chromatography (HIC). The model has been applied to a biotech protein and to beta-lactoglobulin on Phenyl Sepharose 6FF low sub HIC media. Thermodynamic property models for adsorption and protein stability with parameters obtained from experimental chromatographic data successfully describe observed chromatographic behavior over ranges of temperature and salt concentration, provide predictions of distribution among different conformers, and give a basis for calculating trends in retention strength and stability with changing conditions, that might prove useful in HIC process development.     

Possible origin of the inverse and direct Hofmeister series for lysozyme at low and high salt concentrations

Protein solubility studies below the isoelectric point exhibit a direct Hofmeister series at high salt concentrations and an inverse Hofmeister series at low salt concentrations. The efficiencies of different anions measured by salt concentrations needed to effect precipitation at fixed cations are the usual Hofmeister series (Cl(-) > NO(3)(-) > Br(-) > ClO(4)(-) > I(-) > SCN(-)). The sequence is reversed at low concentrations. This has been known for over a century. Reversal of the Hofmeister series is not peculiar to proteins. Its origin poses a key test for any theoretical model. Such specific ion effects in the cloud points of lysozyme suspensions have recently been revisited. Here, a model for lysozymes is considered that takes into account forces acting on ions that are missing from classical theory. It is shown that both direct and reverse Hofmeister effects can be predicted quantitatively. The attractive/repulsive force between two protein molecules was calculated. To do this, a modification of Poisson-Boltzmann theory is used that accounts for the effects of ion polarizabilities and ion sizes obtained from ab initio calculations. At low salt concentrations, the adsorption of the more polarizable anions is enhanced by ion-surface dispersion interactions. The increased adsorption screens the protein surface charge, thus reducing the surface forces to give an inverse Hofmeister series. At high concentrations, enhanced adsorption of the more polarizable counterions (anions) leads to an effective reversal in surface charge. Consequently, an increase in co-ion (cations) adsorption occurs, resulting in an increase in surface forces. It will be demonstrated that among the different contributions determining the predicted specific ion effect the entropic term due to anions is the main responsible for the Hofmeister sequence at low salt concentrations. Conversely, the entropic term due to cations determines the Hofmeister sequence at high salt concentrations. This behavior is a remarkable example of the charge-reversal phenomenon.     

Protein adsorption on novel acrylamido-based polymeric ion exchangers. II. Adsorption rates and column behavior

Uptake kinetics and breakthrough behavior were determined for bovine serum albumin (BSA) and alpha-chymotrypsinogen (alphaCHY) in new polymeric ion-exchange media based on acrylamido monomers. Two anion exchangers and a cation exchanger were investigated. As shown in Part I of this work, the two anion exchangers have different morphologies. The first one, BRX-Q, comprises a low-density gel with a matrix of denser polymeric aggregates. While this material has a very low size-exclusion limit for neutral probes, it exhibits an extremely high binding capacity for BSA. The second anion exchanger, BRX-QP, comprises large open pores but has a very low binding capacity. The cation exchanger, BRX-S, also comprises large open pores but exhibits an intermediate capacity; likely as a result of the presence of smaller pores. Dynamic protein uptake experiments showed that the highest mass transfer rates are obtained with BRX-Q. The apparent diffusivity is also highest for this material and increases substantially as the protein concentration is reduced. For these particles, the external film resistance is dominant at very low protein concentrations. Much lower rates and apparent diffusivities are obtained for BRX-QP. Finally intermediate rates and apparent diffusivities are found with BRX-S. The concentration dependence of the apparent pore diffusivity is much less pronounced in this case. The apparently paradoxical result that mass transfer rates are highest for the material with the smallest neutral-probe size-exclusion limit can be explained in terms of a general conceptual model where parallel pore and adsorbed-phase diffusion paths exist in these particles. In the first case, adsorbed phase diffusion in gel pores is dominant, while in the second transport is dominated by diffusion in a macroporous network. In the third case, both contributions are important. The conceptual model provides an accurate prediction of the breakthrough behavior of columns packed with these media using independently determined rate parameters. Dynamic binding capacities of 80-140 mg/ml were observed for BSA on BRX-Q in ca. 1.5 cm columns operated at 300-900 cm/h in agreement with theoretical predictions.     

Energetics of lysozyme adsorption on mesostructured cellular foam silica: effect of salt concentration

The heat of lysozyme adsorption on mesostructured cellular foam (MCF) silica was measured using flow microcalorimetry (FMC) to investigate the influence of a neutral salt, sodium sulfate. At concentrations up to 0.5 M sodium sulfate, a complex initial exotherm was followed by an endotherm. Protein surface coverage, the magnitudes of the exothermic heat signals and the magnitudes of the net heat of adsorption increased with sodium sulfate concentration. These observations suggest that electrostatic interactions are the principal driving force at low ionic strengths; van der Waals interactions become dominant at higher salt concentrations. Each exotherm could be deconvoluted into two exotherms, indicating multiple modes of lysozyme attachment to the silica surface. The endothermic peak, associated with protein desorption, disappeared at the highest sodium sulfate concentration (1.0 M), indicating irreversible adsorption of the protein on the MCF silica surface. The data are consistent with an adsorption mechanism in which the initial attachment of lysozyme to the surface is followed by a reorientation and formation of a secondary or stronger attachment to the surface.     

Kinetic Adsorption of Cd onto Nanometer Al203/Carbon Fibre

A new nanometer material, nano-Al2O3 with carbon fibre as the carrier, was employed for the removal of Cd with low concentrations from polluted water. The characterization of the material was carried out by means of SEM and TEM. Batch adsorption and elution experiments were carried out to determine the adsorption properties of Cd on the new adsorbent. The classical Thomas model was applied to estimating the equilibrium coefficients of Cd adsorption and the saturated adsorption ability. The results show that the Thomas model is fit for describing the kinetic adsorption process, and the maximum adsorption capacity of the nanometer Al2O3/carbon is 69.29 mg/g. The resulting information also indicates that the desorption of Cd eluted with de-ionized water at a rate of 9.8 mL/min can be neglected. With the advantage of a high adsorption capacity for removing low concentration Cd, the Al2O3/carbon fibre possesses the potentiality to be an effective adsorbent for the removal of Cd from polluted water.     

Kinetic Adsorption of Cd onto Nanometer Al2O3/Carbon Fibre

Introduction There are many vast water areas where water con-tains higher contents of metals than the recommendedconcentrations with the rapid development of machinemanufacturing, metallurgical industry, organic chemi-cal industry, petrochemical industry, and mining in-dustry in recent years. Ingesting the water with metalconcentrations exceeding the limit exerts a harmfuleffect on human health.Removal of metals from aqueous media can beachieved by electrodialysis, chemical precipitation, ad-s…     

Protein adsorption and transport in agarose and dextran-grafted agarose media for ion exchange chromatography: Effect of ionic strength and protein characteristics

This work investigates the effects of ionic strength and protein characteristics on adsorption and transport of lysozyme, BSA, and IgG in agarose-based cation exchangers with short ligand chemistry and with charged dextran grafts. In all cases, the adsorption equilibrium capacity decreased with increasing salt. However, the adsorption kinetics was strongly influenced by the adsorbent structure and protein characteristics. For the smaller and positively charged lysozyme, the effective pore diffusivity was only weakly dependent on salt for the dextran-free media, but declined sharply with salt for the dextran-grafted materials. For this protein, the dextran grafts enhanced the adsorption kinetics at low salt, but the enhancement vanished at higher salt concentrations. For BSA, which was near its isoelectric point for the experimental conditions studied, the effective diffusivity was low for all materials and almost independent of salt. Finally, for the larger and positively charged IgG, the effective diffusivity varied with salt, reaching an apparent maximum at intermediate concentrations for both dextran-free and dextran-grafted media with the kinetics substantially enhanced by the dextran grafts for these conditions. Microscopic observations of the particles during protein adsorption at low ionic strengths showed transient patterns characterized by sharp adsorption fronts for all materials. A theory taking into account surface or adsorbed phase diffusion with electrostatic coupling of diffusion fluxes is introduced to explain the mechanism for the enhanced adsorption kinetics observed for the positively charged proteins.     

Hydrophobic interaction chromatography of proteins. III. Unfolding of proteins upon adsorption

Hydrophobic interaction chromatography (HIC) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. In contrast to reversed-phase chromatography, this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. Hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. When proteins are injected on HIC columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. The higher the salt concentration, the lower is the amount of eluted protein. The rest can be desorbed with a buffer of low salt concentration or water. It has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. This has been also corroborated by physicochemical measurements. The retention data of 5 different model proteins and 10 different stationary phases were evaluated. Partial unfolding of proteins upon adsorption on surfaces of HIC media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. Furthermore, we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain.     

Albumin separation with Cibacron Blue carrying macroporous chitosan and chitin affinity membranes

Cibacron Blue F3GA, Procion Red HE-3B and Procion Blue MX-R were immobilized on macroporous chitosan and chitin membranes with concentrations as high as 10–200 μmol/ml membrane. These dyed membranes were chemically and mechanically stable, could be reproducibly prepared, and operated at high flow rates. Human serum albumin (HSA) and bovine serum albumin (BSA) were selected as model proteins, and their adsorption on and desorption from the dyed chitosan membranes investigated. The Cibacron Blue F3GA membranes had a higher protein adsorption capacity, much greater for HSA than BSA, than the other dyed membranes. About 8.4 mg HSA/ml membrane were adsorbed at saturation by Cibacron Blue F3GA–chitosan membranes from a 0.05 M Tris–HCl/0.05 M NaCl, pH 8 solution. The chitin membranes had a lower dye content and hence a lower protein adsorption capacity than the chitosan membranes. The effects of important operation parameters (flow rate, protein concentration and loading) were also investigated. Cibacron Blue F3GA–chitosan membranes were employed for the separation of HSA from human plasma and high purity HSA thus obtained. This suggests that these membranes could be used for large-scale plasma fractionation.     

Understanding the Impact of Key Wine Components on the Use of a Non-Swelling Ion-Exchange Resin for Wine Protein Fining Treatment

The impact of key classes of compounds found in wine on protein removal by the ion-exchange resin, Macro-Prep^® High S, was examined by adsorption isotherm experiments. A model wine system, which contained a prototypical protein Bovine Serum Albumin (BSA), was used. We systematically changed concentrations of individual chemical components to generate and compare adsorption isotherm plots and to quantify adsorption affinity or capacity parameters of Macro-Prep^® High S ion-exchange resin. The pH (hydronium ion concentration), ethanol concentration, and prototypical phenolics and polysaccharide compounds are known to impact interactions with proteins and thus could alter the adsorption affinity and capacity of Macro-Prep^® High S ion-exchange resin. At low equilibrium protein concentrations (< ~0.3 (g BSA)/L) and at high equilibrium protein concentrations in model wines at various pH, the adsorption behavior followed the Langmuir isotherm, most likely due to the resin acting as a monolayer adsorbent. The resulting range of BSA capacity was between 0.15–0.18 (g BSA)/(g Macro-Prep^® High S resin). With the addition of ethanol, catechin, caffeic acid, and polysaccharides, the protein adsorption behavior was observed to differ at higher equilibrium protein concentrations (> ~0.3 (g BSA)/L), likely as a result of Macro-Prep^® acting as an unrestricted multilayer adsorbent at these conditions. These data can be used to inform the design and scale-up of ion-exchange columns for removing proteins from wines.     

Comparison of the adsorption equilibrium of a few low-molecular mass compounds on a monolithic and a packed column in reversed-phase liquid chromatography

Adsorption isotherm data were acquired by frontal analysis for several low-molecular mass compounds (3-phenyl 1-propanol, 4-tert.-butylphenol, butylbenzene, and butyl benzoate) on a classical packed column and a monolithic column using methanol-water RP-HPLC conditions. These columns have similar characteristics (C18-bonded silica, close specific surface areas and bonding densities). In each case, the isotherm model best accounting for the data was the same on both columns. The solute polarity determines the class of this model. For the two -OH compounds it was a Langmuirian adsorption isotherm. The hydrocarbon data were best modeled by an anti-Langmuir convex-downward isotherm model. The adsorption data for the aromatic ester exhibited a nearly linear behavior, depending on the methanol concentration of the mobile phase. A slightly convex downward isotherm was obtained at high methanol concentrations while the best fitting was obtained with a liquid-solid extended multilayer B.E.T. isotherm model at low concentrations. The validation of these models is discussed in detail. In all cases, similar values of the adsorption-desorption constants were found, underlining the closeness of the adsorption energies on both columns. By contrast, the adsorption capacity of the monolithic column was found to be approximately 1.4 greater than that of the packed column in spite of the close values of the surface areas of the silica in both columns.