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)     

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 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.     

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.     

Immobilization of proteins on liposome surface

Different methods for the immobilization of proteins (enzymes and immunoglobulins) on liposomes are reviewed. These methods include adsorption, incorporation, covalent binding and binding of preliminarily modified proteins with the liposomal surface. Literature data are compared, optimal immobilization conditions are discussed and requirements for the immobilization processes are formulated. The possibility of using liposome-protein conjugates for drug targeting is especially discussed.     

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.     

Interaction of the alpha-mannosidase from Canavalia ensiformis with the lectin from the same plant, concanavalin A

The specific interaction between the lectin concanavalin A and the alpha-mannosidase from the Leguminosa Canavalia ensiformis was studied by means of laser nephelometry and affinity chromatography. Both proteins react optimally within a certain stoichiometrical range. Interaction is restricted to a narrow pH interval (around pH 5) and to low ionic strengths (less than 10mM NaCl). Neither the sugar-binding site of the lectin nor the catalytic and the hydrophobic sites of the enzyme participate in the interaction. The conformation of the enzyme at pH 5 which favours the interaction can be arrested by immobilization. After this, the enzyme is able to bind the lectin even at pH 8 where no interaction takes place between the dissolved proteins.     

Immobilization of immunoglobulins on polystyrene latex beads: characterization by density gradient centrifugation.

Isopycnic banding by density gradient centrifugation was used to measure density changes in complexes formed by the immobilization of each of four different immunoglobulins (IgG) (bovine, dog, rabbit, and sheep) on polystyrene latex beads (0.109 +/- 0.0025 micrometer diameter). Subtractive measurements of density changes allowed calculation of the mass of immobilized IgG under varying experimental conditions. The immobilization data were correlated with adsorption isotherms which incorporated charge repulsion forces. The effects of pH and NaCl concentration on the immobilization were studied for the latex-bovine IgG system. It was found that the mass of immobilized immunoglobulins was increased from 10 to 20% by removing the IgG from its isoelectric range.     

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.     

蕲蛇蛇毒中一种新型自降解抗凝血酶AA-ACA-I的纯化和表征

利用嗜硫色谱和POROS HQ20离子交换色谱从蕲蛇蛇毒中纯化得到一种新型P-Ⅲ型蛇毒金属蛋白酶（snake venom metalloproteases SVMP) AA-ACA-I.该酶经SDS-PAGE测得分子量为 47.6 kD,含有链内二硫键,加DTT处理后,分子量为50.8 kD,中性糖含量为3.5%,具有出血毒性和抗凝血活性.在70℃和pH 9.0条件下保温60 min,该酶会自降解成分子量30 kD左右的新蛋白,降解得到的新蛋白抗凝血活性高于原蛋白     

Thiophilic adsorption revisited

Specific and efficient selection of serum immunoglobulins, but not other proteins, on T-gel remains difficult. T-gel capacity was determined for different activation conditions and serum loadings. Mass spectrometry analysis was used to identify the proteins found in the flow-through and in the eluted fractions. Alpha-2-macroglobulin and albumin were the major contaminants of the eluates. The influence of the competition between immunoglobulins and the other serum proteins on the adsorption was also studied. Using a serum depleted in immunoglobulins (flow-through of a first chromatography on T-gel), many serum proteins were retained on the T-gel, including albumin. We conclude that T-gel selectivity is less than absolute and may reflect for a large part the experimental conditions of the adsorption.     

Detection of an immunoglobulin-like serum protein possessing a high affinity for collagen

Fibronectin isolated from bovine serum by affinity chromatography on collagen-Sepharose was found to contain a great number of concomitant proteins. Polyacrylamide gel electrophoresis of experimental samples pretreated with beta-mercaptoethanol under denaturation conditions resulted in the polypeptide fractions with Mr of 25, 54 and 82 KD, while the non-treated samples contained only one protein of non-fibronectin type (Mr = = 180-190 KD). This protein was isolated from the total preparations of collagen-binding proteins by the procedures generally employed for the isolation of purified preparations of immunoglobulins G; this protein was also isolated from purified immunoglobulins G using affinity chromatography on collagen-Sepharose. In terms of its molecular weight, subunit composition and immunological and chromatographical behaviour this protein can be related to immunoglobulins. The immunoglobulin-like protein isolated together with fibronectin revealed an affinity for denatured collagen, but not for fibronectin or Sepharose. The content of immunoglobulin with an affinity for denatured collagen in the total fraction of immunoglobulins G is 0.3-0.5%.     

Affinity chromatography of porcine pepsin and pepsinogen using immobilized ligands derived from the specific substrate for this enzyme

Affinity chromatography of porcine protease and its zymogen was carried out on immobilized components of specific substrate used for the pepsin determination. For the immobilization of N-acetyl-L-phenylalanine and iodinated derivative of L-tyrosine, divinyl sulfone activated Sepharose was used. Ligands with blocked amino group and free carboxyl one were linked to Sepharose via ethylene diamine spacer using carbodiimide reaction. Conditions of affinity chromatography of porcine pepsin and pepsinogen on the prepared carriers were optimized: the effect of pH, ionic strength and a nature of the buffers used on adsorption of the enzyme and zymogen to an affinity carrier, as well as their elution was studied. The following parameters were taken into consideration: capacity of the prepared affinity matrices, reproducibility of experiments and the enzyme stability. Pepsin was adsorbed to both immobilized ligands at pH 3.5-4.0; for the elution of the enzyme it was necessary to increase ionic strength (up to 0.5 M). For the adsorption of pepsinogen pH 5.2 was found to be optimum, for its desorption, an increase of ionic strength was used.     

Avidin column as a highly efficient and stable alternative for immobilization of ligands for affinity chromatography

The avidin/biotin system was applied as a general mediator in the adsorption/desorption or immobilization of biologically active macromolecules to solid supports. In this context, model biotinylated proteins (lectins and antibodies) were attached to avidin-coupled Sepharose. As examples for affinity chromatography, peanut agglutinin and anti-transferrin antibody were used to isolate asialofetuin and transferrin, respectively. The capacity and product yields were significantly better than those achieved with conventional affinity chromatography on CNBr-activated Sepharose columns containing the same lectin or antibody. Moreover, the columns were characterized by improved stability properties exhibiting remarkably low levels of leakage.     

Thiophilic paramagnetic particles as a batch separation medium for the purification of antibodies from various source materials

A preparation of thiophilic agarose-based paramagnetic particles (T-Gel) has been developed with physical characteristics (particle size and particle density) that facilitate its use as a batch separation medium suitable for the large-scale purification and isolation of immunoglobulins. The medium was used to extract immunoglobulins from a wide range of starting materials, including sera, ascites fluid, tissue culture medium, and whole blood. None of these starting materials required pretreatment such as clarification by centrifugation or filtration prior to antibody extraction. The antibody purity obtained using T-Gel compared well with that obtained using protein A agarose column chromatography. Yields were approximately 30 mg of immunoglobulins per milliliter of T-Gel, and little was required in the way of specialist equipment. The method is uncomplicated and involves a roll mix extraction overnight, followed by magnetic separation to facilitate supernatant removal and subsequent washing of the particles. Elution of bound antibodies was carried out at neutral pH to yield a concentration of immunoglobulins that was approximately 7 mg/ml. The method was found to be applicable to antibody purification from the blood serum of seven different mammalian species and for all immunoglobulin classes.     

Insulin adsorption on coated silica based supports grafted with N-acetylglucosamine by liquid affinity chromatography

Silica beads are coated with dextran carrying a calculated amount of positively charged diethylassminoethyl groups (DEAE) in order to neutralize negative charged silanol groups at the silica surface and in this way to minimize non specific interactions between silica surface and proteins in solution. Dextran-coated silica supports are potentially excellent stationary phases for high-performance liquid chromatography of proteins. These supports combine the advantages of polysaccharide phases with the excellent mechanical characteristics of silica. These supports (silica-dextran-DEAE = SID) are easily functionalized by grafting N-acetylglucosamine (GlcNAc) using conventional coupling methods. The performances of the support bearing GlcNAc are studied by high-performance liquid affinity chromatography (HPLAC) of insulin, the hypoglycemic peptide hormone of the human organism. The study shows that these supports exhibit a reversible and specific affinity towards insulin and allow separations with high purification yields. Moreover, the influence of different physico-chemical parameters (pH, NaCl and insulin concentration) on insulin retention on the support was analysed. This allowed us to optimize the conditions of adsorption and to better understand the interaction mechanisms between insulin and GlcNAc as biospecific ligand.     

Essential role of the concentration of immobilized ligands in affinity chromatography:: Purification of guanidinobenzoatase on an ionized ligand

Guanidinobenzoatase, a plasma protein with possible application as a ‘tumor marker’, has been fully purified by one-step affinity chromatography. The affinity matrix was prepared by ‘controlled’ immobilization of an enzyme inhibitor (agmatine) onto commercial agarose gels containing carboxyl moieties activated as N-hydroxysuccinimide esters. In this way, agmatine becomes immobilized through an amido bond and preserves an ionized guanidino moiety. Different matrices with different concentration of ligands were prepared in order to evaluate their properties as affinity supports. Interestingly, matrices with a very low concentration of immobilized ligands (2 μmol/ml, corresponding to the modification of only 5% of active groups in the commercial resins) exhibited a low capacity for unspecific adsorption of proteins (as anion-exchange resins) and displayed also a high capacity for specific adsorption of our target protein. On the other hand, when affinity matrices possessed a moderate concentration of agmatine (10 μmol/ml of gel or higher), two undesirable phenomena were observed: (a) the matrix behaves as a very good anionic exchange support able to non-specifically adsorb most of plasma proteins and (b) the specific adsorption of our target protein becomes much lower. The latter phenomenon could be due to steric hindrances promoted by the interaction between each individual immobilized ligand and the corresponding binding pocket in the target protein. These hindrances could also be promoted by the presence of a fairly dense layer of immobilized ligands covering the support surface, thus preventing interactions between immobilized ligands and partially buried protein-binding pockets. In this way, a successful affinity purification (23.5% yield, ×220 purification factor, a unique electrophoretic band) could be achieved by combination of three approaches: (i) the use of affinity matrices possessing a very low density of immobilized ligands, (ii) performing affinity adsorption at high ionic strength and (iii) performing specific desorption with substrates or substrate analogues.     

Immobilization and affinity purification of recombinant proteins using histidine peptide fusions

A gene fusion approach to simplify protein immobilization and purification is described. A gene encoding the protein of interest is fused to a gene fragment encoding the affinity peptide Ala-His-Gly-His-Arg-Pro. The expressed fusion proteins can be purified using immobilized metal affinity chromatography. A vector, designed to ensure obligate head-to-tail polymerization of oligonucleotide linkers was constructed by in vitro mutagenesis. A linker encoding the affinity peptide, was synthesized and polymerized to two, four and eight copies. These linkers were fused to the 3' end of a structural gene encoding a two-domain protein A molecule, ZZ, and to the 5' end of a gene encoding beta-galactosidase. Fusion proteins, of both types, with zero or two copies of the linker showed little or no binding to immobilized Zn2+, while a relatively strong interaction could be observed for the fusions based on four or eight copies of the linker. Using a pH gradient, the ZZ fusions were found to be eluted from the resin at different pHs depending on the number of the affinity peptide. These results demonstrate that genetic engineering can be used to facilitate purification and immobilization of proteins to immobilized Zn2+ and that the multiplicity of the affinity peptide is an important factor determining the binding characteristics.     

Screening of PC and PMMA-binding peptides for site-specific immobilization of proteins

In the present study, we used proteomic research technology to develop a method for the screening and evaluation of material-binding peptides for protein immobilization. Using this screening method, soluble Escherichia coli proteins that preferentially adsorbed onto polycarbonate (PC) and poly(methylmethacrylate) (PMMA) as model plastic materials were first isolated and identified by 2-dimensional electrophoresis (2DE) combined with peptide mass fingerprinting (PMF). The genes of identified protein candidates (ELN, MLT, OMP, and BIF) that exhibited a hexahistidine tag (6×His-tag) were over-expressed by E. coli BL21 (DE3), and the proteins were purified by IMAC affinity chromatography. The candidates for PC and PMMA-binding peptides were isolated from peptide fragments from affinity protein candidates, which were digested with trypsin and chymotrypsin. Consequently, 5 candidates for the PC-binding peptide and 2 candidates for the PMMA-binding peptide were successfully identified by MALDI-TOF MS. All of the peptides identified were introduced to the C-terminus of glutathione S-transferase (GST) as a model protein for immobilization. Adsorption of peptide-fused and wild-type GSTs onto the plastic surfaces was directly monitored using a quartz crystal microbalance (QCM) device. Consequently, genetic fusion of PC-MLT8 and PC-OMP6 as PC-binders and PM-OMP25 as a PMMA-binder significantly enhanced the adsorption rates of GST, achieving an adsorption density that was more than 10 times higher than that of wild-type GST. Furthermore, the residual activity levels of GST-PC-OMP6 and GST-PM-OMP25 in the adsorption state were 2 times higher than that of wild-type GST. Thus, the PC and PMMA-binding peptides identified in this study, namely PC-OMP6 and PM-OMP25, were considerably useful for site-specific immobilization of proteins, while maintaining a higher adsorption density and residual activity levels. The method demonstrated in this study will be applicable to the isolation of a variety of material-binding peptides against the surfaces of unique materials.     

Interaction of immunoglobulins with charged biopolymers: significance in the regulation of humoral immunity

When subjected to ion exchange chromatography on QAE-Sephadex A-50 or gel filtration of Sephadex G-200 under conditions that cause the dissociation of immune complexes at pH 4.05, immunoglobulins both from serum and its immunoglobulin fraction increase their interaction with charged antigens as native DNA and cardiolipin. Ion exchange chromatography also leads to the deaggregation of complexes. It was demonstrated that immunoglobulins bind DNA molecule through its F(ab)2 fragments. Based on data obtained, the suggestion was made that interaction between immunoglobulins and charged serum biopolymers is an important factor in humoral immunity regulation. Namely, high specificity of immunological reactions may be supported by elimination of non-specific binding provided electrostatic interactions from all the potential spectrum of antigen-antibody reactions.