Derivatization of epoxy-activated agarose with various carbohydrates for the preparation of stable and high-capacity affinity adsorbents: Their use for affinity chromatography of carbohydrate-binding proteins

Two types of affinity adsorbents for lectins were prepared by new simple procedures. Both types of adsorbents had high ligand concentration and chemically stable linkage between ligand and Sepharose 4B. Oligosaccharide ligands were coupled by reductive amination with sodium cyanoborohydride to amino-Sepharose 4B prepared by amination of epoxy-activated Sepharose 4B. The glycamyl-Sepharose 4B thus obtained had particularly high adsorption capacities for lectins; lactamyl-Sepharose 4B, 58 mg/l ml of gel for peanut lectin; maltamyl-Sepharose 4B, 146 mg/ml for concanavalin A; and tetra-N-acetylchitotetraamyl-Sepharose 4B, 36 mg/ml for wheat germ agglutinin. Hexosamine was coupled by the aid of carbodiimide to carboxyl-Sepharose 4B prepared by succinylation of amino-Sepharose 4B. Galactosamine-Sepharose 4B adsorbed 145 mg soybean agglutinin/l ml gel. The columns turned from a semitransparent white to a milky white as they were saturated with lectins.     

Preparation of high capacity affinity adsorbents using new hydrazino-carriers and their use for low and high performance affinity chromatography of lectins

Two kinds of carriers with high concentrations of hydrazino groups were prepared by simple and convenient procedures. Hydrazino-carriers (I) and (II) were obtained on incubation of epoxy-activated carriers with hydrazine hydrate and adipic acid dihydrazide, respectively. Disaccharides were coupled to the hydrazino carriers through reductive amination in the presence of sodium cyanoborohydride. The reaction time was much shorter (24 h) than that in the case of the method involving amino-Sepharose 6B (800 h) [Matsumoto, I., Kitagaki, H., Akai, Y., Ito, Y., & Seno, N. (1981) Anal. Biochem. 116, 103-110]. The glycamyl-Sepharose thus obtained showed high adsorption capacities for lectins. Glycamyl-TSKgel G3000 PW obtained by the same method with TSKgel G3000 PW, which is a hydrophobic vinyl polymer matrix for high performance gel permeation liquid chromatography, could be successfully used for the high performance liquid affinity chromatography of lectins. N-Acetylglutamic acid was coupled to hydrazino-Sepharose 4B (I) in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The adsorbent obtained was used for the affinity chromatography of Japanese horseshoe crab lectin.     

High-performance affinity chromatography of carbohydrate-binding proteins by two-stage separation on a resin carrying a number of oligosaccharides

Oligosaccharides of ovalbumin were released by hydrazinolysis and converted to the glycamine derivatives by reductive amination. The resultant derivatives were immobilized on an epoxy-activated methacrylate polymer. Application of lectins on the column containing the resultant resin, followed by injection of the competing sugars and detection of the eluate using natural fluorescence, allowed differentiation of micro amounts of the lectins, owing to their high specificity. Stepwise elution with various competing sugars also permitted separation of lectins. Application of this method to serum samples enabled detection of various carbohydrate-binding proteins with specific affinity to the injected sugars. This method, based on two-stage separation at the adsorption and elution stages, was highly specific. It was also rapid, reproducible, and sensitive.     

125I-glycoconjugate labels for identifying sites of protein catabolism in vivo: effect of structure and chemistry of coupling to protein on label entrapment in cells after protein degradation

Residualizing radioactive labels are designed to remain entrapped within cells following degradation of a carrier protein, and have been used for identification of the tissue and cellular sites of plasma protein catabolism. In this study we describe a convenient synthesis and purification of a series of 125I-labeled glycoconjugates, and an evaluation of their efficiency of retention in liver following degradation of a model carrier protein, asialofetuin. Glycoconjugates were prepared in 65-90% yield by reductive amination of reducing sugars with aromatic amines using NaBH3CN. The products were purified in a single ion-exchange chromatographic step, and then labeled with 125I. The derivatives prepared were mono-and disubstituted lactitol-,cellobiitol-and glucitol-[125I]tyramine and lactitol-[125I]tyrosine. 125I-Glycoconjugates were coupled to asialofetuin using either cyanuric chloride or, for lactose-containing labels, by treatment with galactose oxidase followed by reductive amination with NaBH3CN. Attachment of labels by either procedure did not affect the normal rapid clearance of asialofetuin from the rat circulation nor its uptake and degradation in liver lysosomes. Leakage of 125I-labeled degradation products from cells was measured by following the kinetics of loss of whole-body radioactivity. We observed that degradation products from larger, disubstituted glycoconjugates were retained more efficiently than those from smaller and monosubstituted derivatives, and that glycoconjugates coupled to protein via reductive amination were retained in the body more efficiently than those coupled by cyanuric chloride. Overall, dilactitol-[125I]tyramine coupled to protein by reductive amination was entrapped most efficiently in liver.     

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 of proteins on oxidized crosslinked Sepharose preparations by reductive amination

Mild periodate oxidation of certain commercially available crosslinked agarose beads (Sepharose CL-4B and CL-6B) results in the generation of aldehydo groups which were useful for immobilization of amino compounds by reductive amination using pyridine borane. Consumption of periodate ion and production of formaldehyde were only observed with crosslinked Sepharose preparations and were correlated with a binding capacity much greater than that of uncross-linked gels when subjected to the reductive amination reaction. Up to 50 mg (approximately 0.73 mumol) of bovine serum albumin and 30 mumol of glycylglycine were coupled per gram of moist oxidized Sepharose CL-6B. The immobilization reaction was shown to proceed at neutral pH requiring about 12 h for completion and to be relatively insensitive to temperature and pyridine borane concentration. The oxidized gel was shown to be stable for at least 2 months upon storage in 0.1 M acetic acid. This method has proven to be useful for the preparation of a variety of affinity matrices and immobilized enzymes.     

Effect of conjugation methodology on the immunogenicity and protective efficacy of meningococcal group C polysaccharide-P64k protein conjugates

Neisseria meningitidis serogroup C polysaccharide (CCPS) was conjugated to the carrier protein P64k using two different conjugation procedures, condensation mediated by carbodiimide with adipic acid dihydrazide as spacer and the reductive amination method. BALB/c mice were immunized with the resultant polysaccharide-protein conjugates and the immune response was evaluated. All conjugates assayed generated at least 10-fold higher antibody titers than the free polysaccharide. The reductive amination method rendered the best conjugate (CCPS-P64kR) that was able to elicit antibody titers statistically higher than the titer elicited by the plain CCPS (P<0.001). The sera of the group immunized with CCPS-P64kR showed a three-fold higher bactericidal response than the sera of the group immunized with the plain CCPS and they were able to protect against challenge with meningococci in the infant rat protection model. In addition, three different conjugates were obtained from polysaccharides with molecular relative sizes of 2000-4000 Da, 4000-10,000 Da or 10,000-50,000 Da, but no differences were detected in the immune response obtained against the three conjugates. Our experiments demonstrate that it is possible to generate a protective, T-cell-dependent response against CCPS using the P64k protein as carrier.     

Immunostaining on thin-layer chromatograms of oligosaccharides released from gangliosides by endoglycoceramidase

A method for immobilizing oligosaccharides on a TLC plate for immunostaining has been developed. N-Glycolylneuraminic acid (NeuGc)-containing oligosaccharides derived from II3NeuGc-LacCer, IV3NeuGc-nLcOse4Cer, II3NeuGc-GgOse3Cer, and II3(NeuGc)2-LacCer by digestion with our newly isolated endoglycoceramidase (Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282) and sialyllactose were chromatographed on polyamide 11 TLC or NH2-HPTLC plates, and covalently linked to the plates by reductive amination with sodium cyanoborohydride (NaBH3-CN). The immobilized oligosaccharides were detected by enzyme-immunostaining using NeuGc-specific chicken anti-NeuGc-LacCer and horseradish peroxidase-conjugated rabbit anti-chicken IgG. II3NeuGc-nLcOse4 showed the highest reactivity with the antibody, followed by II3NeuGc-GgOse3. As little as 0.8 nmol of the NeuGc-containing oligosaccharides was detected. The polyamide 11 TLC aluminum plate was found to be more suitable for the immunostaining than the NH2-HPTLC plate under the conditions used. For binding of the oligosaccharides to the NH2-HPTLC plate, reductive amination was found to be superior to the heating method reported earlier.     

Utilization of an exopolysaccharide produced by Chryseomonas luteola TEM05 in alginate beads for adsorption of cadmium and cobalt ions

Cadmium and cobalt adsorption from aqueous solution onto calcium alginate, sodium alginate with an extracellular polysaccharide (EPS) produced by the activated sludge bacterium Chryseomonas luteola TEM05 and immobilized C. luteola TEM05 was studied. In addition, solutions containing both of these ions were prepared and partial competitive adsorption of these mixtures was investigated. Metal adsorption onto gel beads was carried out at pH 6.0 and 25 degrees C. The maximum adsorption capacities determined by fitting Langmuir isotherms to the data for calcium alginate, calcium alginate+EPS, calcium alginate + C. luteola TEM05 and calcium alginate + EPS + C. luteola TEM05 were 45.87, 55.25, 49.26, 51.81 mg g(-1) for Co(II) and 52.91, 64.10, 62.5, 61.73 mg g(-1) for Cd(II), respectively. The biosorption capacity of the carrier for both metal ions together in competition was lower than those obtained when each was present alone.     

Chromogenic labeling of milk oligosaccharides: purification by affinity chromatography and structure determination

Oligosaccharides from human milk were derivatized with 4'-N,N-dimethylamino-4-amino-azobenzene (DAAB) by reductive amination and purified by affinity chromatography on immobilized antibodies followed by resolution of the retained antigenic molecules by adsorption chromatography on HPLC. The visibility to the naked eye and the favorable handling properties of the DAAB-oligosaccharides (desalting, quantification) offered distinctive advantages over underivatized oligosaccharides. Analysis by MS and NMR identified the two major antigens as the Lewis a active pentasaccharide and the Lewis b active hexasaccharide, respectively. Further derivation of DAAB-oligosaccharides by palmitoylamidoacetaldehyde generated glycolipid-like compounds suitable for immunological detection by in situ overlay techniques after separation by thin-layer chromatography.     

Affinity purification of cytosolic epoxide hydrolase using derivatized epoxy-activated Sepharose gels

Improved affinity chromatography procedures for the purification of cytosolic epoxide hydrolase are described. An earlier affinity purification method using immobilized 7-methoxycitronellyl thiol (MCT) sporadically produced final enzyme preparations containing major impurities. To eliminate these impurities, we tested alternate ligands, spacer arms, and ligand concentrations. A series of alkyl and aryl thiols coupled to epoxy-activated Sepharose were found to exhibit markedly different binding characteristics as compared with commercially available alkyl- and aryl-Sepharose gels. Using one of these new matrices, benzylthio-Sepharose, cytosolic epoxide hydrolase from mouse liver was purified over 100-fold, appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was obtained with 60-90% recovery of enzyme activity. The impurities previously observed with the MCT-Sepharose procedure were reduced or eliminated by using an MCT ligand concentration of 5 microequivalents per gram or less. MCT-Sepharose and benzylthio-Sepharose provide rapid and convenient one-step procedures for obtaining purified cytosolic epoxide hydrolase from numerous species and tissues.     

Carp maturational-ovulatory gonadotropin but not carp vitellogenic gonadotropin or salmon maturational-ovulatory gonadotropin stimulates testosterone production by rat Leydig cells in vitro

Carp (Cyprinus carpio) maturational-ovulatory gonadotropin, prepared from the fraction of pituitary extract adsorbed on Con A-Sepharose (Con A II) and subsequently adsorbed on CM-cellulose (Whatman CM-52), stimulated testosterone production by isolated rat Leydig cells. The fraction of carp pituitary extract unadsorbed on the immobilized lectin (Con A I) with a mol. wt of 30,000, which had previously been shown to contain vitellogenic gonadotropin, was devoid of steroidogenic activity. Salmon (Oncorhynchus keta) pituitary Con A I and Con A II fractions containing vitellogenic and maturational-ovulatory gonadotropin respectively did not enhance steroidogenesis in the same assay system. The results indicated that carp maturational-ovulatory gonadotropin resembled mammalian luteinizing hormone (LH) in its chromatographic behavior on Con A-Sepharose and CM-cellulose and also in its steroidogenic activity in rat Leydig cells. However, not all teleost maturational-ovulatory gonadotropins are LH-like: the salmon hormone is a notable exception. The data further supports the distinctiveness of carp vitellogenic gonadotropin and maturational-ovulatory gonadotropin.     

Protein A immobilized polyhydroxyethylmethacrylate beads for affinity sorption of human immunoglobulin G

Protein A immobilized polyhydroxylmethyacrylate (PHEMA) microbeads were investigated for the specific removal of HIgG from aqueous solutions and from human plasma. PHEMA microbeads were prepared by a suspension polymerization technique and activated by CNBr in an alkaline medium (pH 11.5). Protein A was then immobilized by covalent binding onto these microbeads. The amount of immobilized protein A was controlled by changing pH and the initial concentrations of CNBr and protein A. The maximum protein A immobilization was observed at pH 9.5. Up to 3.5 mg protein A/g PHEMA was immobilized on the CNBr activated PHEMA microbeads. The maximum HIgG adsorption on the protein A immobilized PHEMA microbeads was observed at pH 8.0. The non-specific HIgG adsorption onto the plain PHEMA microbeads was low (about 0.167 mg of HIgG/g PHEMA). Higher adsorption values (up to 6.0 mg of HIgG/g PHEMA) were obtained in which the protein A immobilized PHEMA microbeads were used. Much higher amounts of HIgG (up to 24.0 mg of HIgG/g PHEMA) were adsorbed from human plasma.     

Aromatic amino acids and their derivatives as ligands for the isolation of aspartic proteinases

Affinity chromatography was used to study an interaction of aspartic proteinases with immobilized aromatic amino acids and their derivatives. The following ligands were used: L-tyrosine, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, L-phenylalanine, p-iodo-L-phenylalanine and N-acetyl-L-phenylalanine. With the exception of the last one, ligands were coupled directly to divinyl sulfone activated Sepharose 4B. For the preparation of immobilized N-acetyl-L-phenylalanine, divinyl sulfone activated Sepharose 4-B with linked ethylene diamine was used. Porcine pepsin was used for the evaluation of the capacity of the prepared affinity carriers. The capacity of the immobilized amino acid derivatives significantly increased in comparison with the non-derivatized amino acids. The prepared immobilized ligands were further used for the separation of human pepsinogens.     

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30-60 and 1,4-butanediol-diglycidyl ether: 16-36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a Mr of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and processcompatible alternative to other types of chromatography.     

Polymerized liposome as ligand carrier for affinity precipitation of proteins

A polymerized liposome (PLS) was prepared using a synthesized phospholipid with a diacetylene moiety in the hydrophobic chain and an amino group in the hydrophilic head. The PLS was used as a novel ligand carrier for affinity precipitation of proteins because it showed a reversibly precipitable property on salt addition and removal. Soybean trypsin inhibitor (STI) was easily immobilized on the PLS by a one-step carbodiimide reaction. The PLS showed no nonspecific adsoprtion of proteins. It had a large ligand coupling capacity, and then a large adsorption capacity for trypsin after STI immobilization. The PLS with immpbilized STI was recycled three times for the purification of trypsin from a crude pancreatic extract. Although the degree of purification was compromised by the impurity of the STI employed, in each run the purification factor reached about 6 and more than 80% of trypsin activity was recovered. The results indicated that the PLS was a potential ligand carrier for affinity precipitation of proteins. (c) 1995 John Wiley & Sons, Inc.     

Preparation and characterisation of immobilised metal ion hollow-fibre polysulphone membranes. Their application in high-speed pectic enzyme fractionation

Chelating hollow-fibre membranes were prepared from epoxy-activated polysulphone microfiltration fibres by introducing iminodiacetic acid (IDA) groups in the presence of dimethyl sulphoxide. Fibres with 160, 350 and 620 μmol epoxy groups/ml provided ligand densities of 69, 134 and 203 μmol IDA/ml and pure water fluxes of 7.8, 5.8 and 0.42 cm/min, respectively. However, lysozyme capacity was close to 4 μmol/ml for all fibres. Adsorption isotherms for lysozyme and pectinesterase did not fit Langmuir-type curves and the existence of two types of ligand (A and B) with different accessibility to proteins was assumed. For pectinesterase, maximum capacities of 5100 and 2900 U/ml and dissociation constants of 25 and 316 U/ml were found, respectively, for ligands A and B. For lysozyme, maximum capacities were 2.9 and 0.9 μmol/ml and dissociation constants 5.0 and 102 μM, respectively, for said ligands. A cartridge assembled with IDA hollow fibres had a dynamic capacity for pectinesterase of 7509 U/ml. Productivity of this cartridge for pectic enzyme fractionation was 750 pectinesterase U/ml min, far higher than that obtained with a chelating soft gel (81 pectinesterase U/ml min).     

Affinity chromatography of the muscarinic acetylcholine receptor

A novel compound, 3-(2'-aminobenzhydryloxy)-tropane (ABT), and an ABT-agarose gel were synthesized and used for the purification of solubilized muscarinic receptors. ABT had a high affinity with an apparent dissociation constant (Kd) of 7 nM for the muscarinic receptors solubilized from the porcine brain by digitonin. An ABT-agarose gel was prepared by coupling ABT with epoxy-activated Sepharose 6B, and the degree of substitution to the gel was determined to be 4-5 mumol/ml of the gel by UV absorption spectrum. During affinity chromatography using 10 ml of the ABT-agarose gel and 100 ml of the digitonin-solubilized preparation, 70% of muscarinic receptors were adsorbed to the gel, in marked contrast with the adsorption of only 2% of proteins. Approximately 25% of muscarinic receptors applied to the gel were eluted biospecifically with 1 mM muscarinic ligands. The purified fraction showed a high affinity for [3H]quinuclidinyl benzylate with a Kd of 0.4 nM and similar specificity for muscarinic ligands to that of unpurified soluble receptors. The protein concentration of the purified fraction was too low to be determined accurately, but very approximately a purification of 10(3)-fold was indicated.     

One-step affinity purification of urease from jack beans

Jack bean (Canivalia ensiformis) urease (EC3.5.1.5) was purified in one-step by ligand affinity chromatography using epoxy-activated Sepharose 6B-urea. The yield of the purified enzyme was about 80% with a specific activity of about 500 U/mg of protein. The enzyme was apparently homogeneous when analyzed by SDS-PAGE and native PAGE. The protein band on native PAGE coincided with the stained band of urease activity. The affinity column could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either acetamide or semicarbazide as affinity ligands were also found to be useful for the isolation of urease.     

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30–60 and 1,4-butanediol-diglycidyl ether: 16–36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a M _r of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and process-compatible alternative to other types of chromatography.