Further studies on the contribution of electrostatic and hydrophobic interactions to protein adsorption on dye-ligand adsorbents.

The adsorption equilibria of bovine serum albumin (BSA), gamma-globulin, and lysozyme to three kinds of Cibacron blue 3GA (CB)-modified agarose gels, 6% agarose gel-coated steel heads (6AS), Sepharose CL-6B, and a home-made 4% agarose gel (4AB), were studied. We show that ionic strength has irregular effects on BSA adsorption to the CB-modified affinity gels by affecting the interactions between the negatively charged protein and CB as well as CB and the support matrix. At low salt concentrations, the increase in ionic strength decreases the electrostatic repulsion between negatively charged BSA and the negatively charged gel surfaces, thus resulting in the increase of BSA adsorption. This tendency depends on the pore size of the solid matrix, CB coupling density, and the net negative charges of proteins (or aqueous - phase pH value). Sepharose gel has larger average pore size, so the electrostatic repulsion-effected protein exclusion from the small gel pores is observed only for the affinity adsorbent with high CB coupling density (15.4 micromol/mL) at very low ionic strength (NaCl concentration below 0.05 M in 10 mM Tris-HCl buffer, pH 7.5). However, because CB-6AS and CB-4AB have a smaller pore size, the electrostatic exclusion effect can be found at NaCl concentrations of up to 0.2 M. The electrostatic exclusion effect is even found for CB-6AS with a CB density as low as 2.38 micromol/mL. Moreover, the electrostatic exclusion effect decreases with decreasing aqueous-phase pH due to the decrease of the net negative charges of the protein. For gamma-globulin and lysozyme with higher isoelectric points than BSA, the electrostatic exclusion effect is not observed. At higher ionic strength, protein adsorption to the CB-modified adsorbents decreases with increasing ionic strength. It is concluded that the hydrophobic interaction between CB molecules and the support matrix increases with increasing ionic strength, leading to the decrease of ligand density accessible to proteins, and then the decrease of protein adsorption. Thus, due to the hybrid effect of electrostatic and hydrophobic interactions, in most cases studied there exists a salt concentration to maximize BSA adsorption.     

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.     

Optimizing dye-ligand density with molecular analysis for affinity chromatography of rabbit muscle L-lactate dehydrogenase

Ligand density is an important factor in determining the binding capacity and separation efficiency for affinity chromatography. A molecular analysis method based on the three-dimensional structure of protein and protein-ligand interactions was introduced to optimize the dye-ligand density for target protein separation. Expanded-bed adsorption (EBA) of L-lactate dehydrogenase (LDH) from rabbit muscle crude extract with Procion Red HE-3B as the dye-ligand was used as the model. After the analysis of LDH three-dimensional molecular structure and dye-protein interaction modes, the rational dye-ligand distance was predicted at about 20 A for efficiently binding LDH. A series of dye-ligand adsorbents with different ligand densities were prepared, and the isotherm adsorption equilibria of LDH were measured. High adsorption capacity of LDH was achieved at about 1600 U/mL adsorbent. Packed-bed chromatography was performed, and the elution effects were investigated. Finally, an EBA process was achieved to capture the LDH directly from rabbit muscle crude extract. The method established in the present work could be expanded to guide the screening of ligand density for other affinity chromatographic processes.     

Affinity chromatographic separation of secreted alkaline phosphatase and glucoamylase using reactive dyes

A single-step dye affinity chromatographic separation method was developed to separate secreted alkaline phosphatase (SEAP) and glucoamylase produced in CHO cell culture and Aspergillus niger fermentation, respectively. The reactive dye, Procion^® Green H-E4BD, was found to have a good binding capacity for SEAP, whereas Procion^® Blue H-ERD was the best dye ligand for glucoamylase. However, these dyes have a relatively low selectivity for the target protein. Consequently, elution of the adsorbed proteins by KCl solution resulted in a product with many impurity proteins as evident by the multiple protein bands on SDS-PAGE. However, elution of SEAP by its substrate, phosphate, produced a relatively pure protein with a high specific enzyme activity because of the competition for active site between the substrate and the dye ligand. Also, a high-purity glucoamylase product was obtained by elution with a borate solution. The relatively inexpensive dye affinity chromatography thus can be used for purifying enzymes from cell culture and fermentation broths. The adsorption of SEAP on the dye-ligand affinity resin followed the Langmuir isotherm. An axial dispersion model with external mass transfer limitation was developed to simulate the breakthrough curve in the chromatographic column. This mathematical model can be used to scale up the protein adsorption process.     

Steric and hydrophobic effects in alkyl isocyanide binding to Rhodospirillum molischianum cytochrome c'

Equilibrium constants for the binding of a series of alkyl isocyanides to ferrous cytochrome c' from Rhodospirillum molischianum have been measured spectrophotometrically. The equilibrium constants range from 3.3 M-1 to 2.6 x 10(2) M-1 and follow the order methyl greater than ethyl less than n-propyl less than tert-butyl less than n-butyl less than amyl less than cyclohexyl less than n-hexyl. The decrease in equilibrium constant from methyl to ethyl isocyanide provides evidence for a steric interaction between the ligand and the protein. The increase in equilibrium constant from ethyl to n-hexyl isocyanide is accounted for by a favorable partitioning of the ligand into a hydrophobic heme coordination site. The effect of steric interactions on the differences in the binding constants has been further evaluated by comparing the alkyl isocyanide and CO binding constants for the ferrous cytochrome c' to those of a sterically unconstrained model heme complex in a detergent micelle. The results indicate that the heme coordination site of the ferrous cytochrome c' is severely sterically hindered, similar to that of the reported crystal structure of Rs. molischianum ferric cytochrome c'.     

The thermodynamic principles of ligand binding in chromatography and biology

In chromatography, macromolecules do not adsorb in the traditional sense of the word but bind to ligands that are covalently bonded to the surface of the porous bead. Therefore, the adsorption must be modelled as a process where protein molecules bind to the immobilised ligands. The paper discusses the general thermodynamic principles of ligand binding. Models of the multi-component adsorption in ion-exchange and hydrophobic chromatography, HIC and RPLC, are developed. The parameters in the models have a well-defined physical significance. The models are compared to the Langmuir model. In the traditional adsorption models, the standard state Gibbs energy change of adsorption does not depend level of occupancy, but when it depends on the level of occupancy it gives rise to an adsorptive behaviour known as cooperativity. The binding of oxygen to haemoglobin is a well-known example from biology but it is also observed in chromatography due to protein-protein interactions. Retention measurements on beta-lactoglobulin A demonstrate this. A discussion of salt effects on hydrophobic interactions in precipitation and chromatography of proteins concludes the paper.     

Influence of pH and ionic strength on the steric mass-action model parameters around the isoelectric point of protein

The ion-exchange equilibrium and the dependence of the parameters in the steric mass-action (SMA) model on salt concentration and buffer pH around the isoelectric point of protein were studied. Bovine serum albumin (BSA, isoelectric point = 5.4) was used as a model protein and DEAE Sepharose FF as an ion exchanger. Finite batch adsorption experiments and isocratic elution chromatography were performed for the determination of the model parameters (i.e., characteristic charge, equilibrium constant, and steric factor). The results showed that pH had significant effects on the parameters. With an increase of pH from 4.5 to 6.5, the characteristic charge increased from 0.9 to 3.0 and leveled off as a plateau at pH above 5.5. The charge groups in the contact region of protein surface were considered to play a crucial role on the characteristic charge. The decrease of pH and increase of salt concentration lowered the absolute value of the zeta potential of the protein surface and led to a decrease of the equilibrium constant. The steric factor remained unchanged at about 31 at pH 5.5 and 6.0 and increased to 44.5 at pH 5.0 and 96.8 at pH 4.5, mainly as a result of the lower adsorption capacity of BSA at pH <5.5. Furthermore, the increase of the molecular volume of BSA at pH 4.5 would be an additional reason for the increase of the steric factor. Taking into account the effect of the pH and salt concentration on these parameters, the SMA model described the ion exchange equilibrium of protein more accurately.     

Protein interactions in hydrophobic charge induction chromatography (HCIC)

A quantitative understanding of how proteins interact with hydrophobic charge induction chromatographic resins is provided. Selectivity on this mode of chromatography for monoclonal antibodies as compared to other model proteins is probed by means of a linear retention vs pH plot. The pH-dependent adsorption behavior on this mode of chromatography for a hydrophobic, charged solute is described by taking into account the equilibrium between a hydrophobic, charged solute and an ionizable, heterocyclic ligand. By analogy, an equation that is seen to adequately describe macromolecular retention under linear conditions over a range of pH is developed. A preparative, nonlinear isotherm that can capture both pH and salt concentration dependency for proteins is proposed by using an exponentially modified Langmuir isotherm model. This model is seen to successfully simulate adsorption isotherms for a variety of proteins over a range of pHs and mobile phase salt concentrations. Finally, the widely differing retention characteristics of two monoclonal antibodies are used to derive two different strategies for improving separations on this mode of chromatography. A better understanding of protein binding to this class of resins is seen as an important step to future exploitation of this mode of chromatography for industrial scale purification of proteins.     

Biospecific adsorption of lysozyme onto monoclonal antibody ligand immobilized on nonporous silica particles

It is very important to understand the equilibrium and dynamic characteristics of biospecific adsorption (affinity chromatography) for both scientific and application purposes. Experimental equilibrium and dynamic column data are presented on the adsorption of lysozyme onto antibody immobilized on nonporous silica particles. The Langmuir model is found to represent the equilibrium experimental data satisfactorily, and the equilibrium association constants and heats of adsorption have been estimated for two systems with different ligand densities. The effects of nonspecific interactions are more pronounced in the system with low-density ligand. The dynamic interaction kinetic parameters are estimated by matching the predictions of a fixed-bed model with the experimental breakthrough curves. The agreement between theory and experiment is good for the initial phases of breakthrough, where the mechanism of biospecific adsorption is dominant. In the later phase (saturation neighborhood) of breakthrough, the effects of nonspecific interactions appear to be greater in the low-density ligand system. The kinetics of the nonspecific interactions were estimated from the data of the later phase of breakthrough and were found to be considerably slower than those attributed to biospecific adsorption.     

Dye-Ligand Affinity Chromatography: The Interaction of Cibacron Blue F3GA® with Proteins and Enzyme

Abstract

The dye Cibacron Blue F3GA has a high affinity for many proteins and enzymes. It has therefore been attached to various solid supports such as Sephadex, Sepharose, polyacrylamide, and the like. In the immobilized form the dye has rapidly been exploited as an affinity chromatographic medium to separate and purify a variety of proteins including dehydrogenases, kinases, serum albumin, interferons, several plasma proteins, and a host of other proteins. Such a diversity shown by the blue dye in binding several unrelated classes of proteins has generated considerable work in terms of studies of the chromophore itself and also the immobilized ligand. As a prelude to realizing the full potential of the immobilized Cibacron Blue F3GA, an understanding of the basic interactions of the dye with its surroundings must be gained. It has been recognized that the dye is capable of hydrophobic and/or electrostatic interactions at the instance of the ambient conditions. The study of interactions of the dye with salts, solvents, and other small molecules indicates the nature of the interactions of the dye with different kinds of groups at the interacting sites of proteins. The review will cover such interactions of the dye with the proteins, the interactions of the proteins with the immobilized ligand, and the media used to elute the bound protein in several cases, and thus consolidate the available information on such studies into a cogent and comprehensive explanation.     

Mechanistic analysis on the effects of salt concentration and pH on protein adsorption onto a mixed-mode adsorbent with cation ligand

Streamline Direct HST is a new kind of mixed-mode adsorbent with cation exchange ligand, especially developed for the expanded bed adsorption process, which can capture target protein directly from the moderate ionic strength feedstock without the need of dilution or other additives. In this study, the isotherm adsorption behaviors and the isocratic retention factors of bovine serum albumin (BSA) on Streamline Direct HST were measured, and the corresponding adsorption mechanisms were also described. The results indicated that Streamline Direct HST shows the typical property of salt-independent adsorption and the maximum binding capacity of BSA occurs near the isoelectric point of BSA. When there are some amounts of electrostatic repulsion protein-adsorbent interactions, the multilayer adsorption could be found, and high salt concentration does not favor the adsorption of protein. A patch-controlled adsorption process and an oriented adsorption model are proposed for describing the adsorption behaviors under electrostatic repulsion condition.     

Salt-independent adsorption of human serum proteins on cyanocarbon gels

Electron donor acceptor gels based on cyanocarbons have been tested for human serum protein adsorption in the absence of salt-promotion by water-structuring salt. This phenomenon was compared with a normal adsorption process in the presence of salt. The tricyanoaminopropene–divinyl sulfone–agarose displayed unusual protein adsorption properties as binding could occur both independently or dependently of the salt-promotion. The absence of hydrophobic or ionic character of the salt-independent interaction suggests an electron donor acceptor adsorption mechanism which is shown, for the first time, to occur independently of salt-promotion in aqueous solution. Study of the protein adsorption specificity showed similar protein selectivity for the fractions adsorbed in both conditions.     

Salt-independent adsorption of human serum proteins on cyanocarbon gels

Electron donor acceptor gels based on cyanocarbons have been tested for human serum protein adsorption in the absence of salt-promotion by water-structuring salt. This phenomenon was compared with a normal adsorption process in the presence of salt. The tricyanoaminopropene–divinyl sulfone–agarose displayed unusual protein adsorption properties as binding could occur both independently or dependently of the salt-promotion. The absence of hydrophobic or ionic character of the salt-independent interaction suggests an electron donor acceptor adsorption mechanism which is shown, for the first time, to occur independently of salt-promotion in aqueous solution. Study of the protein adsorption specificity showed similar protein selectivity for the fractions adsorbed in both conditions.     

Surface hydrophobic amino acid residues in cellulase molecules as a structural factor responsible for their high denim-washing performance

The denim-washing performance of six purified fungal cellulases (four endo-1,4-beta-D-glucanases and two cellobiohydrolases) was compared using a model microassay. The performance of cellobiohydrolases per mg of protein was much lower than that of endoglucanases. For endoglucanases, it varied up to 5 times between the best and the worst enzyme. Experiments with amino acids immobilized on cross-linked agarose showed that their side chains may bind indigo owing to hydrophobic interactions and formation of hydrogen bonds. The best binding effects provided Tyr and Phe. Analysis of three-dimensional structures of cellulase molecules showed that a certain correlation exists between the washing performance of enzyme and (i) quantity (percentage) of aromatic residues exposed to solvent on the surface of protein globule or (ii) overall percentage of the surface hydrophobic residues. Data presented provide an evidence that the molecules of certain cellulases, which have hydrophobic domains (clusters of closely located non-polar residues) on their surface, may bind indigo and thus act as emulsifiers helping the dye to float out of cellulose fibers to the bulk solution.     

Protein adsorption onto monoliths: A surface energetics study

This part of work was done to explore the basic understanding of the adsorption chromatography by determining the interaction of selected model proteins (n = 5) to monolithic chromatographic materials, with varying densities of butyl and phenyl ligands. Surface energetics approach was applied to study the interaction behavior. The physicochemical properties of the proteins and monolithic chromatographic materials were explored by contact angle and zeta potential values. These values were used to study protein to monolith interaction under various operating conditions. Surface energetics approach allowed the calculation of interaction energy as a function of distance, i.e. energy minimum values. Calculations were performed at various conditions to analyze the effect of major operating parameters on the interaction strength. The interaction strength exposed the hydrophobic nature of the monoliths which increases with increasing ligand density. Further, interaction energy of proteins were higher with monolith with butyl ligand compared to monolith with phenyl ligand. For instance, lactoferrin interaction to monoliths with butyl represents more interaction, i.e. 24.38 kT as compared to monoliths with phenyl i.e. 23.28 kT, keeping lambda as 0.2 nm and salt concentration as 100 mM of ammonium sulphate. Hence, more energy and time will be consumed for elution of proteins immobilized to monoliths with butyl. Similarly, the effect of solid surface for proteins immobilization, effect of ligand density and effect of lambda showed some interesting insights on the interaction behavior. The knowledge generated from the present work will help in the basic understanding as well as development of an efficient, low cost downstream processing design and may mimic the real chromatographic experiments.     

Factorial design of experiments in the determination of adsorption equilibrium constants for basic methylene blue using biopolymer

Equilibrium conditions in the adsorption of a basic dye on Chitosan were studied. The Factorial Design methods and Analysis of Variance have been applied in the experimental determination of adsorption equilibrium constants. Factorial design with three levels of temperature (30v°C, 45v°C, 60v°C), pH (6.7, 8.1, 9.5), particle size (0.177 mm, 0.914 mm, 1.651 mm) was used in identification of significant effects and interactions in the calculation of the equilibrium constants. The dye adsorption capacity of chitosan was found to increase by decreasing the particle size and increasing temperature and pH. The methodology identifies the principal experimental variables, which have the greatest effect on the adsorption process.     

Lyotropic salt effects in hydrophobic chromatography.

Pure hydrophobic chromatography can be observed with agarose gels containing caprylyl hydrazide. These nonionic gels show increased avidity in protein adsorption with higher content of caprylyl groups. Lyotropic salt effects can be used to control chromatographic behavior of proteins. Salting-out agents enhance binding of protein to sorbent, while salting-in agents diminish this binding. Based on these findings, the hydrophobic separation of β-lactoglobulin, ovalbumin, and bovine serum albumin is demonstrated.     

Hydrophobic interaction chromatography in alkaline pH

Hydrophobic interaction chromatography is a very powerful protein purification technique which is dependent on strong salting-out salts to increase the hydrophobic interactions between the protein and the ligand. Ammonium sulfate is the salt most commonly used for this purpose, but it cannot be used at very alkaline pH. Monosodium glutamate was therefore tested as a salt for hydrophobic interaction chromatography at pH 9.5. When ribonuclease A, ovalbumin, and beta-lactoglobulin were individually applied to a phenyl superose column in 2 M monosodium glutamate, all three proteins bound to the column and could be subsequently eluted by decreasing the salt concentration. Using this salt, it was possible to separate commercially obtained beta-lactoglobulin into authentic protein and contaminants and to purify the individual proteins from a mixture of ovalbumin and beta-lactoglobulin. These results demonstrate that monosodium glutamate is a useful salt for hydrophobic interaction chromatography. Guanidine and sodium sulfate and sodium aspartate were also examined at the same pH, demonstrating that they also resulted in the binding and elution of the proteins examined.     

What Drives Amyloid Molecules To Assemble into Oligomers and Fibrils?

We develop a theory for three states of equilibrium of amyloid peptides: the monomer, oligomer, and fibril. We assume that the oligomeric state is a disordered micellelike collection of a few peptide chains held together loosely by hydrophobic interactions into a spherical hydrophobic core. We assume that fibrillar amyloid chains are aligned and further stabilized by steric zipper interactions—hydrogen bonding, steric packing, and specific hydrophobic side-chain contacts. The model makes a broad set of predictions that are consistent with experimental results: 1), Similar to surfactant micellization, amyloid oligomerization should increase with peptide concentration in solution. 2), The onset of fibrillization limits the concentration of oligomers in the solution. 3), The extent of Aβ fibrillization increases with peptide concentration. 4), The predicted average fibril length versus monomer concentration agrees with data on α-synuclein. 5), Full fibril length distributions agree with data on α-synuclein. 6), Denaturants should melt out fibrils. And finally, 7), added salt should stabilize fibrils by reducing repulsions between amyloid peptide chains. It is of interest that small changes in solvent conditions can tip the equilibrium balance between oligomer and fibril and cause large changes in rates through effects on the transition-state barrier. This model may provide useful insights into the physical processes underlying amyloid diseases.     

Covalent chromatography and salt-promoted thiophilic adsorption

Covalent chromatography on 3-(2-pyridyl disulfido)-2-hydroxypropyl agarose, abbreviated PyS2, turns out to involve more complex interactions than has been supposed heretofore. Unexpectedly, the sorption is highly salt dependent. The relative affinities for serum proteins have therefore been determined in the absence and presence of different types of salts at different salt concentrations and with different degrees of ligand substitution on the adsorbent. In the presence of water-structuring salts the PyS2-gel shows an adsorption pattern for serum proteins resembling that of the "thiophilic" T-gel (J. Porath; F. Maisano, and M. Belew (1985) FEBS Lett. 185, 306-310). Superimposed on thiophilic adsorption we have found, as expected, covalent attachment of thiol-containing proteins. Also the thiol-disulfide exchange increases from 4-5% in the absence of potassium sulfate or sodium chloride up to about 40% of the applied serum proteins when such a water-structuring salt is present. We have thus shown that the interaction of a protein with the ligand is greatly facilitated by a water-structuring salt--and in this case the product is a covalently as well as a thiophilically immobilized protein. A cautious interpretation of protein interaction phenomena is justified whenever ligands containing sulfide, disulfide, or pi-electron-rich structures such as aromatic moieties are involved.