Thiophilic adsorption: a comparison of model protein behavior

A newly recognized type of protein-ligand interaction phenomenon has resulted in the preparation of simple, nonionic, and highly specific gel derivatives for selective adsorption chromatography. The essential structure of the immobilized ligand can be represented as agarose-CH2CH2SO2CH2CH2SCH2CH2OH, which was prepared by using mercaptoethanol to derivatize [0.9-1.0 mmol (g of dry gel)-1] divinyl sulfone activated agarose (thiophilic or T-gel). Proteins interacting with this ligand are provisionally termed "thiophilic" to recognize their affinity for the definitive sulfone-thioether constituents. To better understand the experimental variables affecting adsorption efficiency and selectivity, several well-characterized proteins with diverse physicochemical features have been evaluated for thiophilic properties. Thiophilic interaction chromatography was investigated as a function of pH as well as the type and concentration of water-structure-forming salts required to promote adsorption. The model proteins characterized varied distinctly in their individual thiophilic affinities. At acidic pH values, a salt-independent adsorption process was observed. Furthermore, a minimum in the salt-promoted thiophilic adsorption tendency at pH 5-6 was found, with varying magnitude, for each of the model proteins evaluated. Recovery of adsorbed proteins routinely varied from 90% to 100%. There does not appear as yet to be any easily recognized physicochemical property associated with either thiophilic or nonthiophilic behavior. These results suggest that thiophilic interaction chromatography is a process that utilizes a previously unrecognized protein-ligand interaction mechanism. We suggest that salt allows the protein into close proximity with the sulfone-thioether group where short-range forces are effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Thiophilic adsorption of immunoglobulins--analysis of conditions optimal for selective immobilization and purification

Immunoglobulins have been selected by their general affinity for adjacent sulfone-thioether sulfur groups as a useful model system for the characterization of thiophilic interaction chromatography. Mercaptoethanol coupled to divinylsulfone-activated agarose (thiophilic or T-gel) provided an affinity matrix for the efficient and reversible immobilization of the immunoglobulins. The adsorption/desorption process was investigated as a function of protein concentration, temperature, flow rate, and pH in different concentrations of ammonium sulfate. Immobilization of these proteins was (as a function of pH) found to be both dependent and independent of the adsorption-promoting effects of water-structure-forming salts. Buffer conditions are recommended for the selective adsorption of immunoglobulins from unfractionated human serum. These results indicate that thiophilic interaction chromatography provides a new and effective alternative for the immobilization and purification of immunoglobulins and other proteins under conditions known to preserve structure and biological activity.     

Behaviour of human immunoglobulin G subclasses on thiophilic gels: comparison with hydrophobic interaction chromatography

We have used thiophilic and hydrophobic interaction chromatography in an attempt to obtain enriched human immunoglobulin G (IgG) subclasses from a therapeutic immunoglobulin preparation. Proteins were adsorbed on a thiophilic gel and on Phenyl-, Butyl-, or Octyl-Sepharose in 1 M ammonium sulphate. Elution with a decreasing salt gradient produced no marked subclass selectivity, except with Octyl-Sepharose, which yielded a poorly adsorbed fraction somewhat enriched in IgG2, representing ca. 20% of the total initial protein. Neither thiophilic nor hydrophobic interaction chromatography appear suitable for an efficient enrichment in subclasses, which all show a broad heterogeneity in their affinity for these columns. The influence of the starting salt concentration was also studied. With thiophilic gels, in the absence of ammonium sulphate, ca. 30% of the initial load was not adsorbed, and was found to be enriched in IgG2. At 2.5 and 5% ammonium sulphate, practically no adsorption occurred. At 7.5% ammonium sulphate, the non-adsorbed fraction was enriched in IgG3. With Phenyl-Sepharose, adsorption increased smoothly with the salt concentration. It is concluded that different forces come into play for adsorption on thiophilic gels at low and high salt concentration.     

Thiophilic interaction chromatography for supercoiled plasmid DNA purification

Supercoiled plasmid DNA was selectively purified from its open circular form by thiophilic interaction chromatography, performed in the presence of high concentrations of water-structuring salts. To identify optimal conditions for purification, various aromatic thioether ligands were coupled to a chromatographic support and screened for their ability to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Selectivity of the chromatographic medium depended on the structure of the ligands, with characteristics of the substituents on the aromatic ring determining the resolution between the different plasmid DNA isoforms. Optimal resolution was obtained with ligands consisting of an thioaromate, substituted with highly electronegative groups. When 2-mercaptopyridine was used as a ligand, the difference in conductivity for eluting open circular and supercoiled plasmid DNA is only 6 mS/cm. However, with 4-nitrothiophol the resolution for plasmid DNA separation on the media increased, resulting in a 20 mS/cm difference. When used in combination with a prior group separation step, these aromatic thioether ligands facilitated the isolation of highly purified supercoiled plasmid DNA, suitable for use in gene therapy and DNA vaccine applications.     

Inhibition of lipase adsorption at interfaces. Role of bile salt micelles and colipase

The effects of bile salts and colipase on the adsorption of lipase at an interface were studied by hydrophobic affinity chromatography on phenyl- and octyl-Sepharose. In the absence of bile salts, lipase or colipase binds separately to the gel. This is unchanged in the presence of adsorbed bile salts, when one bile salt molecule is associated per hydrophobic ligand. The same data are obtained in the presence of monomeric bile salt solutions. In contrast, lipase adsorption is totally prevented in a micellar bile salt solution. These results favor the idea that the formation of a lipase-bile salt complex in solution is responsible for the lack of interfacial lipase adsorption.     

Salt-independent binding of antibodies from human serum to thiophilic heterocyclic ligands

Several thiophilic adsorbents with mercaptoheterocyclic ligands have been analyzed for their ability to bind human serum proteins in a salt-independent way. In contrast to 2-mercaptopyrimidine, 2-mercaptopyridine derived ligands show a group-selective binding of immunoglobulins and α₂-macroglobulin, not only in the presence of high concentrations of sodium sulphate but in buffers with low ionic strength. The binding is restricted to thiophilic gels obtained by coupling 2-mercaptopyridine to a vinylsulphone-activated matrix and is not achieved on epichlorohydrin-activated gels. A novel thiophilic ligand based on mercaptonicotinic acid, containing a carboxylic group together with the thiophilic pattern of thioaromatic adsorbents, is demonstrated to be useful as an alternative purification scheme for antibodies.     

Chromatographic separations of serum proteins on immobilized metal ion stationary phases

The separation of proteins on stationary phases consisting of a bound organic chelator and a chelated divalent transition metal has been studied as a function of (A) metal ion species; (B) mobile phase composition and pH; and (C) anion and cation concentration. Optimum separation was observed at alkaline pH on chelated nickel stationary phases. Ammonium and Tris salts reduced the affinity of the metal chelate packing for serum proteins. Halide ions caused the proteins to be more strongly bound to the stationary phase. High salt concentrations had only a small effect on the binding of serum proteins in the absence of amine containing buffers or salts. It was also observed that the ease of elution and the recovery of protein were dependent on pH and upon the presence of halides. The general order of elution of serum proteins, based on isoelectric focusing, was independent of metal ion species and elution conditions, suggesting that a single mechanism or a unique sequence of mechanisms was operative. The results suggest that ligand exchange is the major mechanism of separation under basic conditions and that hydrophobic effects are the result of the competition of nonnitrogen ions with ammonium ions or amines for ligand binding sites modifying or participating in protein binding. Protein binding studies under weak acidic conditions are also presented although the mechanism responsible for protein binding is unclear.     

Novel heterocyclic ligands for the thiophilic purification of antibodies

Novel thiophilic ligands based on mercaptoheterocycles were synthesized for use in one-step purification of antibodies. In order to better characterize these new structures, affinity constants were measured, as well as the influence of pH and salt on adsorption and elution. The ligand concentration was optimized for efficient and fast adsorption and elution of antibodies from ascites and serum. The purification of antibodies from cell culture supernatant proved difficult due the indicator phenol red of the growth media.     

Sulfone-aromatic ligands for thiophilic adsorption chromatography: purification of human and mouse immunoglobulins.

New thiophilic matrices and new procedures were used for the purification of immunoglobulins both from human serum and from hybridoma cell cultures containing fetal calf serum. A range of aromatic and heteroaromatic ligands containing hydroxyl or amino groups have been coupled to divinyl sulfone-activated agarose. The resulting affinity matrices have the general formula M-O-CH2-CH2-SO2-CH2-CH2-X-Y, where M is the agarose matrix, X is oxygen or nitrogen, and Y is an aromatic or heteroaromatic compound. Contrary to earlier expectations these matrices showed pronounced thiophilic binding patterns when tested for the selective binding of immunoglobulins from human serum. The binding is influenced by the structure of the aromatic part of the ligand, the ligand concentration, and the concentration and type of lyotropic salt. 2-Hydroxypyridine coupled to divinyl sulfone-activated agarose was used to purify murine monoclonal antibodies (IgG1 and IgM) from hybridoma cell cultures containing fetal calf serum. Compared to previous methods, significantly increased binding capacity (300-1500%) was obtained by using 1.0-1.2 M ammonium sulfate. Purity of the monoclonal antibody may be optimized for each individual clone by washing the column with either a low concentration of ammonium sulfate or polyethylene glycol before elution.     

Investigations on the specificity of thiophilic interaction for monoclonal antibodies of different subclasses

A comparative study was carried out to investigate the influence of different mouse antibody subclasses on the chromatographic behaviour on thiophilic supports. Cell-free supernatants from different mouse-mouse hybridoma cultures in a standard medium were purified on thiophilic agarose and Fractogel EMD TA. The adsorption capacities and purification factors were monitored under optimised adsorption conditions. The different isotypes did not differ significantly regarding capacity of the thiophilic matrix, but the purity of the eluted antibody fractions was significantly lower for the IgG_2a subclass compared to all other murine antibodies. A significant copurification of proteins from cell culture supernatant with antibodies of the IgG_2a subclass indicated a restriction in the universal nature of thiophilic interaction.     

Adsorption of biopolymers at hydrophilic cellulose-water interface

The extent of adsorption (Gamma2(1)) of bovine serum albumin (BSA), beta-lactoglobulin, lysozyme, gelatin, and DNA from aqueous solution onto the hydrophilic surface of cellulose has been measured as function of biopolymer concentration at different temperatures, pHs, and ionic strengths, and in the presence of a high concentration of inorganic salts and denaturants. In all cases, the value of Gamma2(1) increases with the increase of biopolymer concentration (X2) in bulk and it attains a maximum value at a critical mole fraction concentration X2m. The value of Gamma2m depends upon the nature of protein, temperature, pH, and ionic strength, as well as the nature of neutral salts present in excess. Gamma2m for proteins at a fixed physicochemical condition stands in the following order: Gelatin>betalactoglobulin>lysozyme>BSA. The isotherms for adsorption of DNA nucleotides on cellulose surface at pH 4.0 have been compared at different temperatures and ionic strengths, and in the presence of high concentration of inorganic salts LiCl, NaCl, KCl, and Na2SO4. Values of Gamma2m for different systems have been evaluated and critically compared. At pH 6.0 and 8.0, Gamma2(1) values of DNA nucleotides on cellulose are all negative due to the excess positive hydration of cellulose. At pH 4.0, adsorption of nucleotides of acid, alkali, and heat-denatured DNA widely differ from each other and in the presence of excess concentration of urea becomes negative. The probable mechanisms of biopolymer-cellulose adsorption in terms of polymer hydration, steric interaction, London-van der Waals, hydrophobic, and other types of interactions have been discussed qualitatively. The standard free energy change for the adsorption of protein and DNA nucleotides on the cellulose surface at the state of adsorption saturation has been calculated in kJ per kg of cellulose using an integrated form of the Gibbs adsorption equation. The relation between DeltaG degrees and maximum affinities between biopolymers and the polysaccharide interface have been discussed for various systems.     

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.     

Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system

The correlation between protein solubility and the preferential interactions of proteins with solvent components was critically examined with aqueous MgCl2 as the solvent system. Preferential interaction and solubility measurements with three proteins, beta-lactoglobulin, bovine serum albumin, and lysozyme, resulted in similar patterns of interaction. At acid pH (pH 2-3) and lower salt concentrations (less than 2 M), the proteins were preferentially hydrated, while at higher salt concentrations, the interaction was either that of preferential salt binding or low salt exclusion. At pH 4.5-5, all three proteins exhibited either very low preferential hydration or preferential binding of MgCl2. These results were analyzed in terms of the balance between salt binding and salt exclusion attributed to the increase in the surface tension of water by salts, which is invariant with conditions. It was shown that the increase in salt binding at high salt concentration is a reflection of mass action, while its decrease at acid pH is due to the electrostatic repulsion between Mg2+ ions and the high net positive charge on the protein. The preferential interaction pattern was paralleled by the variation of protein solubility with solvent conditions. Calculation of the transfer free energies from water to the salt solutions for proteins in solution and in the precipitate showed dependencies on salt concentration. This indicates that the nature of interactions between proteins and solvent components is the same in solution and in the solid state, which implies no change in protein structure during precipitation. Analysis of the transfer free energies and preferential interaction parameter in terms of the salting-in, salting-out, and weak ion binding contributions has led to the conclusions that, when the weak ion binding contribution is small, the predominant protein-salt interaction must be that of preferential salt exclusion most probably caused by the increase of the surface tension of water by addition of the salt. A necessary consequence of this is salting-out of the protein, if the protein structure is to remain unaltered.     

Salt stimulation of serum insulin-like growth factor binding protein activity

Insulin-like growth factors (IGF)-I and -II are bound to carrier or binding proteins in serum. There are at least two classes of binding protein: a high molecular weight complex and a low molecular weight species that is relatively unsaturated. Total binding capacity in serum generally is determined by incubating [125I]IGF with protein that has been stripped of IGF by acid gel filtration. We found that addition of NaCl to the assay increased binding to stripped guinea pig binding protein to about two to four times the level measured in the absence of salt. Stimulation by NaCl was optimal between concentrations of 0.6 and 1.4 M and also was observed when fetal calf or human sera were used as sources of stripped binding protein or when IGF-II was the ligand. Using chloride salts, the order of activity with respect to cations was Na+ greater than K+ greater than Li+. Na2HPO4 at 0.6 M was as stimulatory as 1.2 M NaCl but 0.6 M Na2SO4 was less effective. NH4HCO3 was as effective as NaCl at 0.6 M. Scatchard plots of data from competitive dilution experiments with [125I]IGF-I and unlabeled IGF-I showed that binding was heterogeneous in the absence of 0.6 M NaCl but linear in its presence. NaCl did not stimulate binding when whole serum was used, but after gel filtration of serum on Sephacryl 200 at pH 8, which does not dissociate IGFs from binding protein, binding to individual fractions was stimulated three- to fourfold by NaCl. Fractions stimulated included those containing the large complex or the unsaturated binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thiophilic adsorption chromatography: purification of Equ c2 and Equ c3, two horse allergens from horse sweat

Purification of two allergens from horse (Equus caballus) sweat, Equ c2 and Equ c3, by means of salt-promoted chromatography on a “thiophilic” (T-gel) adsorbent is described. Immobilization of these proteins was found to be dependent on the presence of water-structure-forming salts where the ammonium sulphate concentration in the equilibration buffer was 2 M. Equ c2 showed higher affinity towards the thiophilic matrix than Equ c3. Their molecular mass (M_r) values established by SDS–polyacrylamide gel electrophoresis were for Equ c2 ≈17 000 and for Equ c3 ≈16 000, and both proteins showed a low isoelectric point of ≈3.8. Their allergenic properties were also investigated using sera from horse-sensitized patients, where it was demonstrated that these proteins exhibited an IgE antibody binding capacity. In this report we show the broad potential applications of thiophilic adsorption chromatography for the efficient purification of allergens.     

Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis

It is well established that salt enhances the interaction between solutes (e.g., proteins, displacers) and the weak hydrophobic ligands in hydrophobic interaction chromatography (HIC) and that various salts (e.g., kosmotropes, chaotropes, and neutral) have different effects on protein retention. In this article, the solute affinity in kosmotropic, chaotropic, and neutral mobile phases are compared and the selectivity of solutes in the presence of these salts is examined. Since solute binding in HIC systems is driven by the release of water molecules, the total number of released water molecules in the presence of various types of salts was calculated using the preferential interaction theory. Chromatographic retention times and selectivity reversals of both proteins and displacers were found to be consistent with the total number of released water molecules. Finally, the solute surface hydrophobicity was also found to have a significant effect on its retention in HIC systems.     

New method for the selective capture of antibodies under physiolgical conditions

Hydrophobic charge induction chromatography is a recently developed method for protein separation based on the use of dual-mode ligands. They are designed in such a way so as to combine a molecular interaction supported by a mild hydrophobic association effect in the absence of salts. When environmental pH is changed, the ligand becomes ionically charged resulting into the desorption of the protein. This method is applied to the separation of antibodies from ascite fluids and culture supernatants from hybridomas cultured in the presence of fetal bovine serum or in protein free environment. Typically adsorption from cell culture supernatants is accomplished without any pH or ionic strength adjustment; the column is then washed with a typical buffer to eliminate protein impurities. Antibodies are then desorbed using acetate buffer, pH 4. Antibody binding capacity is in the range of 30 mg per ml of resin at 10% breakthrough. Antibody purity varies according to the initial feed stock and can reach values higher than 90% in a single pass. One example of antibody purification process involving hydrophobic charge induction chromatography as a capture step followed by a polishing phase with DEAE Ceramic HyperD is described. Longevity and ligand leakage are compatible with large-scale applications.     

Conformational studies of two non-histone chromosomal proteins and their interactions with DNA.

The conformational properties of two non-histone chromosomal proteins (high-mobility-group proteins 1 and 2) have been studied by spectroscopic methods. The interaction of high-mobility-group protein 1 with DNA has also been studied. 1. Circular dichroism results indicate that in the presence of salt both proteins are 40-50% helical between pH 1 and 9. Above pH 9 denaturation takes place. In the absence of salt the proteins denature below pH 4. 2. Nuclear magnetic resonance spectra show the presence of ring-current shifted peaks and perturbed aromatic resonances, demonstrating that the helix formation is accompanied by specific tertiary folding. 3. Nuclear magnetic resonance spectra of compelxes between high mobility group protein 1 and DNA demonstrate that a low ionic strength a portion of the molecule rich in lysine and containing all the aromatic residues is bound to DNA, whilst a more acidic region of the chain remains free from the DNA.