Influence of the nature of coupling agents on insulin adsorption on supports grafted with sialic acid for high-performance affinity chromatography

Porous silica exhibits excellent mechanical properties for use as a stationary phase for high-performance liquid chromatography. However, negative surface charges make it unusable in its native state. For this reason, silica beads are coated with dextran polymers carrying a calculated amount of diethylaminoethyl groups. Both the minimization of non-specific interactions and the hydrophilic character of such supports allow their functionalization with biospecific ligands and finally their use in high-performance affinity chromatography of biological products. The use of these modified supports in high-performance affinity chromatography requires a better understanding of various characteristics of stationary phases. For this purpose, several techniques were utilized, in particular, size-exclusion chromatography and adsorption of radiolabelled albumin. These methods provided complementary information on the structure of these supports. Coated silica-based supports were functionalized with sialic acid by means of different coupling agents. The affinity of these supports for insulin was determined by the establishment of adsorption isotherms and by high-performance affinity chromatography, to evidence the relationships between structural characteristics of the supports and their separation properties. The study of interactions between these supports and insulin allowed us to show the importance of the coupling method on the performances of supports in affinity chromatography.     

High-performance affinity chromatography for the purification of heparin-binding proteins from detergent-solubilized smooth muscle cell membranes

Heparin and heparan sulfates are regulators of cellular events including adhesion, proliferation and migration. In particular, the antiproliferative effect of heparin on smooth muscle cell (SMC) growth is well described. However, its mechanism of action remains unclear. Numerous results suggest an endocytosis mediated by a still unknown heparin receptor on vascular SMCs. In order to identify a putative heparin receptor on SMCs that could be involved in heparin signalling, affinity chromatography supports were developed. In this paper, we describe high-performance liquid affinity chromatography (HPLAC) supports obtained from silica beads coated with dextran polymer substituted by a calculated amount of diethylaminoethyl functions. With a polysaccharide dextran layer, this type of support can be grafted with specific ligands, such as heparin, using conventional coupling methods. In a previous work, we demonstrated, using butanedioldiglycidyl ether, that silica stationary phases coupled to heparin could be used for the fast elution and good peak resolution of heparin-binding proteins. In the present work, an affinity chromatographic fraction of SMC membrane extracts was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and six heparin-binding proteins from dodecyloctaethyleneglycol monoether-solubilized SMCs were observed. Their M_r values were between 40 and 70 kDa, with three major protein bands at 66, 45 and 41 kDa. These results indicate the usefulness of the chromatographic method for purifying heparin binding proteins from SMC membrane.     

Affinity of mouse immunoglobulin G subclasses for sialic acid derivatives immobilized on dextran-coated supports

High-performance liquid affinity chromatography is a powerful method for the purification of biological compounds owing to its specificity, rapidity and high resolution. In our laboratory, we develop chromatographic supports based on porous silica beads. However, in order to minimize non-specific interactions between the inorganic surface and proteins in aqueous solution, the silica beads are coated with modified dextran. As previously reported, many affinity ligands can be covalently grafted onto dextran-coated silica. In this study, N-acetylneuramic acid, which belongs to the sialic acid family and is present in immunoglobulin G (IgG) epitopes, is used as an active ligand. The interactions of this affinity support and IgG subclasses are analyzed. This immobilized ligand enables purification of IgG3 antibodies.     

"Glutaraldehyde-P", a stable, reactive aldehyde matrix for affinity chromatography

A high-performance affinity chromatography support based on silica has been developed for the immobilization of proteins containing primary amino groups. A hydrophilic polymer covalently bound to the silica surface minimizes nonspecific protein binding to the support while preserving high binding capacity. The Schiff base reaction involved in the coupling of a ligand to the affinity medium is rapid, allows the use of mild conditions during the coupling process, and results in a very stable linkage. Reaction parameters were studied for protein coupling to the affinity support to determine optimum binding conditions and dynamic capacity as a function of protein size. The stability of the ligand-matrix bond was determined. The performance and reproducibility of the affinity support are demonstrated by its use in the analysis of nitrophenyl sugar derivatives, purification of glycoproteins, and isolation of anti-bovine immunoglobulin G developed in rabbit.     

Immobilized artificial membrane chromatography: rapid purification of functional membrane proteins

A solid-phase membrane mimetic system, denoted as immobilized artificial membranes (IAM), has been developed and utilized as a novel high-performance liquid chromatography (HPLC) matrix for the first step in the rapid purification of functional membrane proteins. IAM phases consist of monolayers of amphiphilic membrane lipid molecules covalently bonded to a rigid silica particle. These monolayers of lipids have proved remarkably effective for the chromatography of biomolecules. Several cytochrome P450 isozymes, an extremely important family of hydrophobic membrane proteins with a labile heme catalytic center, have been partially purified in functional conformations from rat liver, kidney, and adrenal microsomes on IAM supports. Functionality of purified P450 and P450 reductase has been demonstrated by optical difference spectroscopy, by carbon monoxide binding, and by reconstitution of enzymatic activity in vitro. Other membrane proteins, including rat liver plasma membrane NADH oxidase and ferricyanide oxidoreductase have also been partially purified by IAM HPLC. The methods for purification of these proteins are described.     

Recombinant human insulin V Optimization of the reversed-phase high-performance liquid chromatographic separation

The applicability of reversed-phase high-performance liquid chromatography (HPLC) to the analysis of the products of recombinant insulin was studied. The influence of several mobile phases in reversed-phase and ion-pair HPLC on selectivity, resolution and sensitivity was investigated. Optimum conditions for the separation of insulin-related proteins on commercial and laboratory-made supports were established by means of three-dimensional optimizations of selectivity and resolution as a function of pH and ionic strength (μ). A mechanism for the separation of proteins with a mobile phase containing a high salt concentration and a pH near the isoelectric point of proteins is proposed. The questions of scaling up are considered. The proposed techniques allow the analysis of the main impurities and ensures a high quality of active insulin production.     

Peptide binding domains determined through chemical modification of the side-chain functional groups.

A clear understanding of the specific secondary structure and binding domain resulting from the interactions of proteins and peptides with lipid surfaces will provide insight into the specific functions of biologically active molecules. We have shown in earlier studies that the stationary phases used in reverse-phase high-performance liquid chromatography represent a model artificial lipid surface for the study of induced conformational states of peptides on lipid interaction. We have now used reverse-phase high-performance liquid chromatography to determine the binding domains of peptides and, by extension, of proteins to a lipid surface. This approach consists of performing chemical modifications of specific amino acid side-chain functionalities after the interaction of the peptides with the reverse-phase high-performance liquid chromatography C18 groups. The susceptibility to oxidation was also studied after binding of the same peptides to liposomes. Oxidation of a single methionine residue "walked" through an amphipathic alpha-helical 18-mer peptide was selected to illustrate this approach. The extent of oxidation was found to be clearly dictated by the accessibility of the methionine residue to the aqueous mobile phase. The binding domain found for the peptide in its lipid-induced conformational state was unequivocally the entire hydrophobic face of the amphipathic alpha-helix.     

Affinity surfactants as reversibly bound ligands for high-performance affinity chromatography

Pyridine was coupled covalently to a nonionic ethoxylated alcohol: octaethylene glycol n-hexadecyl ether. This modified surfactant was found to be a reversible, competitive inhibitor of horse serum cholinesterase. The surfactant bound irreversibly, in aqueous media, to octadecyl-bounded reverse phase silica particles commonly used for high-performance liquid chromatography. The amount of ligand bound was found to be 550 mumol/ml of packing, a concentration that is over 100 times higher than what can be normally bound to agarose affinity chromatography supports. With this packing, a 280-fold purification of cholinesterase from horse serum and a 79-fold purification of human serum cholinesterase were accomplished, with yields greater than 80%, using a 2-cm-long column and a 7-min elution time. The affinity surfactant could be eluted from the column using a 6:4 (v/v) mixture of methanol and isopropanol. This technique should be generally applicable in the development of biospecific supports for high-performance affinity chromatography.     

Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection

Normal phase, isocratic high-performance liquid chromatography methods are described for the separation of neutral lipid and fatty acid classes using low wavelength detection. Prior to high-performance liquid chromatography, methods were developed and are described for the separation of phospholipids from neutral lipids and fatty acids using small (600 mg) silica Sep-PaksTM. Recoveries of cholesteryl esters, triglycerides, fatty acids, and phospholipids from the silica columns were greater than 95%. Two mobile phases are described for lipid class separation by high-performance liquid chromatography. The first mobile phase, hexane-2-propanol-acetic acid 100:0.5:01, resulted in incomplete separation of cholesteryl ester and triglyceride but excellent separations of fatty acids and cholesterol. The second mobile phase, hexane-n-butyl chloride-acetonitrile-acetic acid 90:10:1.5:0.01, resulted in complete separation of the four lipid classes. This mobile phase also separated individual triglycerides and fatty acids based on the number of double bonds. Recoveries of radiolabeled lipids for the four lipid classes from high-performance liquid chromatography was greater than 95% with both mobile phases.     

Binding of substituted phenol and aniline derivatives to the corn protein zein studied by high-performance liquid chromatography

The interaction of 12 substituted phenol, three aminophenol and four substituted aniline derivatives with the corn protein zein was studied on zein-coated silica and alumina stationary phases by high-performance liquid chromatography using bidistilled water as mobile phase. Solutes were eluted from the zein-coated supports with different retention times indicating that they bind to the protein with different forces. They were more strongly retained on silica-based than on alumina-based support proving that the original adsorptive character of the support remains even after impregnation. The retention of solutes on both zein-coated stationary phases significantly depended on the steric and electronic parameters of solutes and was independent of the calculated and measured lipophilicity parameters, indicating that hydrophobic forces are not included in the interaction of zein with these class of solutes. It has been concluded that the interaction is governed by steric and electrostatic forces.     

Use of weak monoclonal antibodies for affinity chromatography

When using weakly interacting ligands in affinity chromatography, it is possible to take advantage of a true chromatographic process in the separation, as compared with traditional affinity chromatography which is rather an on/off process. In this work, weak monoclonal antibodies were immobilized on a silica and a perfusion-type support (POROS AL) and used for high-performance liquid affinity chromatography (HPLAC). Similar carbohydrate antigens were separated under isocratic conditions according to their weak interaction with the immobilized monoclonal antibody. The affinity of the antibodies was adjusted with temperature and pH to modify the separation. The productivity of the chromatographic system was increased with the immobilized perfusion support but at the expense of loss of plate numbers. This study clearly demonstrates the potential of weak affinity biological interactions as a basis for chromatographic separation.     

Conformation,orientation, and adsorption kinetics of dermaseptin B2 onto synthetic supports at aqueous/solid interface

The antimicrobial activity of cationic amphipathic peptides is due mainly to the adsorption of peptides onto target membranes, which can be modulated by such physicochemical parameters as charge and hydrophobicity. We investigated the structure of dermaseptin B2 (Drs B2) at the aqueous/synthetic solid support interface and its adsorption kinetics using attenuated total reflection Fourier transform infrared spectroscopy and surface plasmon resonance. We determined the conformation and affinity of Drs B2 adsorbed onto negatively charged (silica or dextran) and hydrophobic supports. Synthetic supports of differing hydrophobicity were obtained by modifying silica or gold with omega-functionalized alkylsilanes (bromo, vinyl, phenyl, methyl) or alkylthiols. The peptide molecules adsorbed onto negatively charged supports mostly had a beta-type conformation. In contrast, a monolayer of Drs B2, mainly in the alpha-helical conformation, was adsorbed irreversibly onto the hydrophobic synthetic supports. The conformational changes during formation of the adsorbed monolayer were monitored by two-dimensional Fourier transform infrared spectroscopy correlation; they showed the influence of peptide-peptide interactions on alpha-helix folding on the most hydrophobic support. The orientation of the alpha-helical Drs B2 with respect to the hydrophobic support was determined by polarized attenuated total reflection; it was around 15 +/- 5 degrees. This orientation was confirmed and illustrated by a molecular dynamics study. These combined data demonstrate that specific chemical environments influence the structure of Drs B2, which could explain the many functions of antimicrobial peptides.     

Supports for reverse-phase high-performance liquid chromatography of large proteins

Reverse-phase high-performance liquid chromatography supports have been developed for use in separating proteins up to 300,000 M_r. They are based on silica supports to which octyl, cyanopropyl, or diphenyl groups are covalently bonded. Their effectiveness in rapidly separating several standard proteins is demonstrated. Applications presented include the separation of the α₁ and α₂ chains of chick Type I collagen within 1 h and the separation of the α and β components of human Type I collagen.     

High-performance liquid affinity chromatography with phenylboronic acid, benzamidine, tri-L-alanine, and concanavalin A immobilized on 3-isothiocyanatopropyltriethoxysilane-activated nonporous monodisperse silicas

Nonporous, microparticulate, monodisperse silicas with particle diameters between 0.7 and 2.1 microns are introduced as stationary phases in high-performance affinity chromatography. The immobilization of m-aminophenylboronic acid, p-aminobenzamidine, tri-L-alanine, and concanavalin A onto these silicas was successfully achieved using 3-isothiocyanatopropyl-triethoxysilane as an activation reagent. Immobilized phenylboronic acid was applied to the isolation of nucleosides, nucleotides, and glycoprotein hormones such as bovine follicotropin and human chorionic gonadotropin, while immobilized benzamidine was employed for the isolation of the serine proteases thrombin and trypsin, immobilized tri-L-alanine for the separation of pig pancreatic elastase and human leukocyte elastase, and immobilized concanavalin A for the isolation of horseradish peroxidase. In all affinity chromatographic systems studied, the nonporous monodisperse silicas showed improved chromatographic performance compared to results obtained with porous silica supports using identical activation and immobilization procedures. Furthermore, frontal analysis was used as a method to evaluate the influence of experimental parameters on biological activity and accessible ligand densities. Only minor changes in bioactivity were found with the nonporous affinity supports, where accessibilities were typically higher than ca. 60%. The immobilization of affinity ligands onto porous supports as used in this and associated papers thus represents a successful general procedure for the preparation of stable matrices with fast kinetics for use in high-performance affinity chromatography.     

Separation of drug enantiomers by liquid chromatography and capillary electrophoresis, using immobilized proteins as chiral selectors

Proteins display interesting chiral discrimination properties owing to multiple possibilities of intermolecular interactions with chiral compounds. This review deals with proteins which have been used as immobilized chiral selectors for the enantioseparation of drugs in liquid chromatography and capillary electrophoresis. The main procedures allowing the immobilization of proteins onto matrices, such as silica and zirconia particles, membranes and capillaries are first presented. Then the factors affecting the enantioseparation of drugs in liquid chromatography, using various protein-based chiral stationary phases (CSPs), are reviewed and discussed. Last, chiral separations already achieved using immobilized protein selectors in affinity capillary electrochromatography (ACEC) are presented and compared in terms of efficiency, stability and reproducibility.     

Caenorhabditis elegans N-glycans containing a Gal-Fuc disaccharide unit linked to the innermost GlcNAc residue are recognized by C. elegans galectin LEC-6

We report a detailed structural analysis of the N-glycans of Caenorhabditis elegans recognized by C. elegans galectin LEC-6. Glycoproteins of C. elegans captured by an immobilized LEC-6 affinity adsorbent were isolated. The N-glycans of these glycoproteins were then liberated by hydrazinolysis and labeled with the fluorophore 2-aminopyridine (PA). The derived pyridylaminated (PA)-sugars were further fractionated by rechromatography on immobilized LEC-6 adsorbent and by reversed-phase high-performance liquid chromatography (HPLC). The structures of the PA-sugars thus obtained were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS) in conjunction with glycosidase digestion. We confirmed that all PA-sugars having affinity for LEC-6 contain a Gal-Fuc disaccharide unit, and that this unit is bound to the innermost GlcNAc residue of the N-glycan chain. The dissociation constants of LEC-6 for these glycans were measured by frontal affinity chromatography. LEC-6 exhibited higher affinity for oligosaccharides having a Gal-Fuc unit linked to position 6 of the innermost GlcNAc residue than for those having Galbeta1-4GlcNAc units. Affinity for the former disappeared, however, following treatment with beta-galactosidase. If the glycan contained a Hex-Fuc disaccharide linked to the penultimate GlcNAc residue, the affinity would be diminished. We propose, therefore, that the galectins of C. elegans utilize the Gal-Fuc disaccharide unit for recognition instead of the Gal-GlcNAc unit that is common in vertebrates.     

Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose

Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(11-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid beta-glucosidase (beta-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal beta-Glc/ml of settled gel, respectively. The purified normal enzyme (14-18% yield) had a specific activity of 1.6 X 10(6) nmol/h/mg protein and was homogeneous as evidenced by a single protein species of Mr = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the beta-Glc cDNA. Amino acid composition analyses of beta-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11%. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.     

Carbohydrate structures of acetylcholine receptor from Torpedo californica and distribution of oligosaccharides among the subunits

The structure of carbohydrates in acetylcholine receptor (AChR) from Torpedo californica is reported. Oligosaccharides released quantitatively from the whole molecule by N-oligosaccharide glycopeptidase digestion were fractionated by thin-layer chromatography and further purified by high-performance liquid chromatography. We show that more than 70% of the total oligosaccharide chains in Torpedo AChR are of the high-mannose type with the structures (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2. The structure of these oligosaccharides were determined by proton nuclear magnetic resonance spectroscopy. These two types of oligosaccharides were shown to be distributed different proportions in all subunits of Torpedo AChR. We also show that several kinds of complex-type oligosaccharides comprising the rest of the carbohydrate in the protein exist mainly in the gamma and delta subunits. The structure of the carbohydrate moiety that is distributed on the four subunits of AChR was also examined by susceptibility to endo-beta-N-acetylglucosaminidase and sialidase and by binding affinity to lectins, e.g. concanavalin A, leucoagglutinating phytohemagglutinin, and wheat germ agglutinin.     

The examination and quantitation of tissue cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using hydroxylapatite

Ion-exchange chromatography of proteins and peptides has been most successfully achieved historically on hydrophilic gel matrices. The poor mechanical strength of these organic gels has necessitated the development of new supports for high-performance separations. High-performance supports are of three types: totally inorganic, totally organic, and composite inorganic-organic materials. Several ionic species such as diethylaminoethyl ethanol and poly-ethylene imine have been used as stationary phases with similar results. Pore-diameter selection has been shown to be important in both resolution and loading capacity. Capacity is maximum for proteins of 50 to 100 kilodaltons on 300-Å-pore-diameter supports. Maximum resolution of high-molecular-weight species also requires macroporous supports. Interestingly, column length is of minor importance in the resolution of proteins. Columns of 5-cm length have approximately the same resolution as those of 30-cm length. Application of high-performance ion-exchange chromatography to a variety of protein mixtures has now been reported. These supports generally give recoveries of enzyme activity equivalent to the classical supports.     

Structures of sugar chains of the third component of human complement

Human C3, the third component of human complement, contained mannose and N-acetylglucosamine as sugar components. The sugar chains were liberated from the polypeptide chains by hydrazinolysis, and the free amino groups were N-acetylated. The reducing end residues of the sugar chains thus obtained were tagged with 2-aminopyridine, and the pyridylamino (PA-) derivatives of sugar chains were separated by high-performance liquid chromatography. The structures of purified PA-sugar chains were analyzed by a combination of stepwise exoglycosidase digestions, size determination by paper electrophoresis, methylation analysis, Smith degradation, and partial acetolysis. These results showed that C3 contained two high-mannose type sugar chains ranging from Man5GlcNAc2 to Man9GlcNAc2. Analyses of the sugar chains of alpha- and beta-chains of C3 indicated that the alpha-chain contained mainly Man8GlcNAc2 and Man9GlcNAc2, while the beta-chain contained mainly Man5GlcNAc2 and Man6GlcNAc2.