A model for the salt effect on adsorption equilibrium of basic protein to dye-ligand affinity adsorbent

A model describing the salt effect on adsorption equilibrium of a basic protein, lysozyme, to Cibacron Blue 3GA-modified Sepharose CL-6B (CB-Sepharose) has been developed. In this model, it is assumed that the presence of salt causes a fraction of dye-ligand molecules to lodge to the surface of the agarose gel, resulting from the induced strong hydrophobic interaction between dye ligand and agarose matrix. The salt effect on the lodging of dye-ligand is expressed by the equilibrium between salt and dye-ligand. For the interactions between protein and vacant binding sites, stoichiometric equations based either on cation exchanges or on hydrophobic interactions are proposed since the CB dye can be regarded as a cation exchanger contributed by the sulfonate groups on it. Combining with the basic concept of steric mass-action theory for ion exchange, which considers both the multipoint nature and the macromolecular steric shielding of protein adsorption, an explicit isotherm for protein adsorption equilibrium on the dye-ligand adsorbent is formulated, involving salt concentration as a variable. Analysis of the model parameters has yielded better understanding of the mechanism of salt effects on adsorption of the basic protein. Moreover, the model predictions are in good agreement with the experimental data over a wide range of salt and ligand concentrations, indicating the predictive nature of the model.     

Effect of polymer shielding on elution of G3PDH bound to dye-ligand adsorbent

Batch binding experiments were performed to assess the recovery performance of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) bound to the unshielded and polymer (polyvinyl pyrrolidone, PVP)-shielded dye-ligand (Cibacron Blue 3GA) adsorbent. The adoption of a polymer-shielded, dye-ligand technique facilitated the elution efficiency of bound G3PDH. It was demonstrated that the recovery of G3PDH using polymer-shielded dye-ligand adsorption yielded higher elution efficiency, at 60.5% and a specific activity of 42.3 IU/mg, after a low ionic strength elution (0.15 M NaCl). The unshielded dye-ligand yielded lower elution efficiency, at 6.5% and a specific activity of 10.2 IU/mg.     

Adsorption of proteins on a lipid bilayer

In our analysis of protein adsorption on a lipid bilayer, the protein surface is considered to contain one or a few charged spots, and the bilayer contains a significant amount of lipids with oppositely charged head groups. After adsorption, a folded protein is assumed to change its shape slightly due to the electrostatic attraction, so that one of the spots forms a flat contact with the oppositely charged lipid heads of the lipid bilayer. With realistic parameters, this model predicts that the contribution of electrostatic interactions to the protein adsorption energy per charged amino acid–lipid pair is 16–25 kJ/mol. Thus, a few (four or five) pairs is sufficient for irreversible adsorption.     

Fractionation of plasma proteins using dye-ligand chromatography: elution profiles on immobilized green TSK-AF

The fractionation of human plasma by chromatography on immobilized Green TSK-AF was assessed by immunological analysis of the elution profiles of 27 different plasma proteins. A three-step procedure was used to elute proteins from the column. First a low-molarity buffer (30 mM sodium phosphate, pH 7.0, I = 0.053) was applied; then a linear salt gradient (0-1.0 M NaCl in the above buffer) was followed by an additional wash with four bed volumes of 1.0 M NaCl. Tightly bound proteins were finally stripped with 0.5 M NH4SCN. The elution profile of the proteins using this procedure appears to be very reproducible. Comparison with the profile obtained upon chromatography on Cibacron Blue 3GA [Gianazza, E. and Arnaud, P. (1982) Biochem. J. 201, 129-136] indicates significant differences between the binding properties of the two gels. These differences can be used to design a "tandem-chromatography" system which provides an efficient means for the separation of several plasma proteins.     

Adsorption-desorption of BSA to highly substituted dye-ligand adsorbent: quantitative study of the effect of ionic strength

Cibacron Blue 3GA was immobilized on Sepharose CL-6B to obtain a highly substituted dye-ligand adsorbent which dye concentration was 17.4?μmol dye per gram wet gel. This adsorbent had a highly binding capacity for bovine serum albumin (BSA). The effects of ionic strength on the adsorption and desorption of BSA to the adsorbent were studied. Adsorption isotherms were expressed by the Langmuir model. The quantitative relationships between the model parameters and the ionic strength were obtained. The desorptions were performed by adding salt to the BSA solutions in which adsorption equilibria had been reached. Adding salt to the solution resulted in the desorption of the bound protein. It was found that the isotherm obtained from the desorption experiments agreed well to the isotherm obtained from the adsorption experiments at the same ionic strength. The result demonstrated that the adsorption of BSA to the highly substituted adsorbent was reversible.     

Isotachophoretic electrodesorption of proteins from an affinity adsorbent on a microscale

Cationic isotachophoresis was used for the desorption of mouse monoclonal antibody to transferrin strongly affinity-bonded to transferrin immobilized on polyethyleneglycol terephthalate powder. The electrodesorption under nondestructive conditions was effected in the capillary isotachophoresis apparatus of our own construction which was equipped with an adsorption element. The electrodesorption is on-line connected with quantitative isotachophoretic analysis of the antibody desorbed. Only a few tens of microliters of the affinity adsorbent and several nanomoles of the antibody are needed for the characterization of the capacity of the affinity adsorbent and the conditions of adsorption and desorption.     

Adsorption of dimethylsulfoxide on proteins

A method for measuring the adsorption of dimethyl sulfoxide on native and denatured trypsin and albumin was developed. On native proteins, no positive adsorption was registered, and a slight negative adsorption within the limits of experimental error was observed. It was shown that the properties of denatured proteins depend on the mode and conditions of denaturation. On one of denatured trypsin specimens, positive adsorption of dimethyl sulfoxide was registered, on other specimens no adsorption was observed. The reason for this behavior lies in the hydrophobic nature of adsorption of dimethyl sulfoxide at the interface, while the surface of native protein globules and, probably, most denatured protein specimens is hydrophilic.     

The use of dye-ligand affinity chromatography for the purification of non-enzymatic human plasma proteins

Literature data are analysed in this review on the use of immobilized triazine dyes for the characterization, isolation and purification of non-enzymatic human plasma proteins in both conventional and high-pressure liquid chromatography systems. Attention is focused on the mode of interaction between the dyes and these proteins, as well as on the advantages over previously reported techniques. Future developments are discussed.     

Adsorption behaviors of L-arginine from aqueous solutions on a spherical cellulose adsorbent containing the sulfonic group

An investigation was conducted regarding the adsorption and desorption of L-arginine from aqueous solutions with a new spherical cellulose adsorbent containing the sulfonic group. The adsorption of L-arginine on the adsorbent was time, pH, initial concentration of L-arginine and temperature dependent. The adsorption process followed the Langmuir adsorption isotherm, and was endothermic (DeltaH =24.66 KJ/mol). Almost 100% L-arginine adsorbed on the adsorbent could be recovered with a 2.0 mol/L NH4OH or 2.0 mol/L NH4Cl aqueous solution. After 25 and 40 cycles of adsorption and desorption, the decrease in adsorption capacity reached to 4.9% and 20.3%, respectively.     

Process intensification of fluidized bed dye-ligand adsorption of G3PDH from unclarified disrupted yeast: a case study of the performance of a high-density steel-agarose pellicular adsorbent

The development of a process intensified primary capture step for the direct selective recovery of intracellular proteins from very dense particulate-containing yeast extract has been explored. The purification of glyceraldehyde 3-phosphate dehydrogenase from bakers' yeast was chosen as a potential demonstration of this approach. A high throughput (50%, w/v, yeast extracts at a superficial linear velocity of 450 cm h(-1)) was achieved by adoption of a high-density adsorbent (UpFront steel-agarose; rho = 2.65 g ml(-1)) derivatized with selective ligand chemistries (Cibacron Blue 3GA). This should ultimately minimize adsorption time and maximize process efficiency of fluidized bed adsorption.     

Adsorption chromatography of proteins

A method for adsorption chromatography of proteins is proposed. A protein solution is passed through a cellulose column at a pH value corresponding to an isoelectric point of the protein. Depending on the charge of unwanted proteins, they either remain at the origin (if charges of protein and ion-exchanger are opposite) or are released from the column (if charges of protein and ion-exchanger coincide). Elution volume of the purified protein is higher than for the second group of unwanted proteins because movement of the uncharged protein of interest includes its adsorption on cellulose followed by subsequent desorption caused by the elution buffer. Problems of optimization of buffers and adsorbents are discussed. Applicability of the method of adsorption chromatography is illustrated using purification of horseradish peroxidase as an example.     

Butyl-Toyopearl 650 as a new hydrophobic adsorbent for water-soluble enzyme proteins

Butyl-Toyopearl 650, a butyl derivative of Toyopearl HW-65, was synthesized for use in hydrophobic chromatography. Water-soluble enzyme proteins were adsorbed on butyl-Toyopearl 650 in the presence of ammonium sulfate and eluted easily in the absence of the salt. Cytochrome c, myoglobin, and chymotrypsinogen A were successfully separated on a butyl-Toyopearl 650 column in order of their individual hydrophobicity by decreasing the concentration of ammonium sulfate contained in the buffer eluant. Based on these results, the use of butyl-Toyopearl 650 is demonstrated for the hydrophobic separation of water-soluble enzyme proteins.     

Effect of adsorbent porosity on performance of expanded bed chromatography of proteins

Expanded bed or fluidized bed adsorption has emerged as an important unit operation in downstream processing of proteins. A number of specifically designed commercial adsorbents are available today for expanded bed purification of proteins. Protein purification essentially requires adsorbent matrices that have large pore size. Very large pore size or macroporous adsorbents can provide high efficiency in packed beds even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the macropores. This is reflected in leveling off of HETP (height equivalent to theoretical plate) versus flow curve after a threshold velocity. Expanded bed operation, on the other hand, can also show plateauing of the HETP curve, but not necessarily on account of macroporosity of adsorbent. It is shown in this article how any adsorbent intended for protein adsorption in expanded bed mode can give plateauing HETP curve, regardless of pore size. As a result, RTD measurements on an expanded bed can give equal, and at times better, performance than a corresponding packed bed. Large pore size, on the other hand, can result in lesser retention of biomass and easy flushing of the adsorbent to obtain an entirely particulate-free adsorbent prior to the product elution step. Adsorbent with larger pores is also shown to provide faster and more efficient elution both in packed and expanded bed modes.     

Adsorption and separation of proteins by a smectitic clay mineral

The adsorption of proteins by a smectitic clay mineral was investigated. The clay used in this study is a mixture of montmorillonite and amorphous SiO₂. Due to the high porosity the montmorillonite units are accessible for protein adsorption. The amorphous silica prevents the montmorillonite from swelling and allows column packing. Protein adsorption was performed at different pH under static conditions. Furthermore, static capacities were determined. The material reveals high adsorption capacities for proteins under static conditions (270–408 mg/g), whereby proteins are mainly adsorbed via electrostatic interactions. The Freundlich isotherm is suggested as an adsorption model. For desorption a pH shift was found to be most effective. Binding and elution of human serum albumin and ovalbumin were tested under dynamic conditions. Dynamic capacities of about 40 mg/g for ovalbumin at 764 cm/h were found. The clay mineral provides suitable properties for the application as cost-efficient, alternative separation material.     

Adsorption equilibrium and dynamics of lactase/CM-Sephadex system

Summary Partitioning behaviour and adsorption isotherms of lactase/CM-Sephadex system at equilibrium were investigated together with the adsorption kinetics in this study. Maximum adsorption was obtained at the pH values between 5.5–6.0. Adsorption isotherm was a close fit to the Langmuir model.Nomenclature a specific mass transfer area - D_m molecular diffusion coefficient (m²/sec) - e₁, e₂ charge of the protein and the gel - k apparent mass transfer coefficient (s^-1) - ka global mass transfer coefficient - f partition coefficient - K_p dissociation constant for adsorbent-adsorbate complex, (mg/mL solvent) - p equilibrium concentration of free enzyme, (mg free enzyme/mL solution) - q equilibrium concentration of adsorbed enzyme, (mg ads./mL gel) - q_m maximum adsorption capacity, (mg ads./ml gel) - Re particle Reynolds number - Sh Sherwood number - V_g/V gel volume (mL)/bulk solvent volume (mL) - Z dimensionless extent of adsorption - K_p/P_o , model parameter - (/) +1 , model parameter - V_g q_m / V P_o , model parameter     

Cell/adsorbent interactions in expanded bed adsorption of proteins

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from unclarified feedstock. A method is presented which allows a qualitative and quantitative understanding of the main mechanisms governing the interaction of biomass with fluidised resins. A pulse response technique was used to determine the adsorption of various cell types (yeast, Gram positive and Gram negative bacteria, mammalian cells and yeast homogenate) to a range of commercially available matrices for EBA. Cells and cell debris were found to interact with the ligands of agarose based resins mainly by electrostatic forces. From the adsorbents investigated the anion exchange matrix showed the most severe interactions, while cation exchange and affinity adsorbents appeared to be less affected. Within the range of biologic systems under study E. coli cells had the lowest tendency of binding to all matrices while hybridoma cells attached to all the adsorbents except the protein A affinity matrix. The method presented may be employed for screening of suitable biomass/adsorbent combinations, which yield a robust and reliable initial capture step by expanded bed adsorption from unclarified feedstock.     

Adsorption of phenol on formaldehyde-pretreated Pinus pinaster bark: equilibrium and kinetics

This work studies phenol adsorption on Pinus pinaster bark that has been previously treated with formaldehyde in acid medium. The influence of several variables such as solid/liquid ratio, pH and initial concentration of phenol in the solution on the adsorption capacity of the bark has been analysed. A kinetic model based on phenol diffusion within the pores of the adsorbent was in agreement with the results obtained for high initial concentrations of phenol, allowing the determination of diffusion coefficients. Adsorption equilibrium data were fitted by the Freundlich and BET isotherms. From their parameters phenol adsorption capacity and intensity, as well as the specific surface (BET) of the adsorbent, were determined.     

IgG and hybridoma partitioning in aqueous two-phase systems containing a dye-ligand

The effect of the important ATPS- and bufferparameters on IgG and hybridoma partitioning in ATPSscontaining a PEG-dye-ligand was studied. Objective wasto establish selection criteria for effective ligandsfor extractive fermentations with animal cells inATPSs.In the presence of 1% PEG-dye-ligand the binding ofIgG to the PEG-ligand was affected severely by theNa-chloride concentration. The tie-line length and pHaffected IgG partitioning to a lesser extent. Thedesired partitioning of IgG into the top phase, wasonly obtained when, in addition to the 10 mmol/kgK-phosphate buffer, no Na-chloride was present. In anATPS culture medium, with ± 35 mmol/kg Na-bicarbonateand 60 mmol/kg Na-chloride, increasing thePEG-dye-ligand concentration up to 100% did increasethe partition coefficient, but was not effective inconcentrating the IgG in the top phase of ATPS culturemedium at a pH of 7.8.Furthermore, addition of the PEG-dye-ligand to ATPSculture medium changed the hybridoma cell partitioningfrom the bottom phase to the interface.     

Adsorption of Ni2+ on the surface of molecularly imprinted adsorbent from Penicillium chysogenum mycelium

The adsorption capacity for Ni²⁺ on to the surface molecular imprinting adsorbent on Penicillium chysogenum mycelium (the surface-imprinted adsorbent) was 40–45 mg g^–1 (using 200 mg Ni²⁺ l^–1), two times of the mycelium adsorbent. The surface-imprinted adsorbent had good stability at pH 28. The optimal concentration of EDTA for desorption was 0.1 to 0.5 g l^–1. The surface imprinted adsorbent could be reused 15 times without losing its uptake.