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PubMed1989

Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography.

Immobilized metal ion affinity chromatography (IMAC) has been explored as a probe into the topography of histidyl residues of a protein molecule. An evaluation of the chromatographic behavior of selected model proteins--thioredoxin, ubiquitin, calmodulin, lysozyme, cytochrome c, and myoglobin on immobilized transition metal ions (Co2+, Ni2+, Cu2+, and Zn2+)--allows establishment of the following facets of the histidyl side chain distribution: (i) either interior or surface; (ii) when localized on the surface, accessible or unaccessible for coordination; (iii) single or multiple; (iv) when multiple, either distant or vicinal. Moreover, proteins displaying single histidyl side chains on their surfaces may, in some instances, be resolved by IMAC; apparently, the microenvironments of histidyl residues are sufficiently diverse to result in different affinities for the immobilized metal ions. IMAC, previously introduced as an approach to the fractionation of proteins, has become also, upon closer examination, a facile probe into the topography of histidyl residues. This is possible because of the inherent versatility of IMAC; an appropriate metal ion (M2+) can be selected to suit the analytical purpose and a particular chromatographic protocol can be applied (isocratic pH, falling pH, and imidazole elution).

Calmodulin↗

PubMed1996

Polystyrene as an affinity chromatography matrix for the purification of antibodies.

Affinity chromatography is used for the purification of diagnostic polyclonal antibodies in order to ensure specificity. Most commonly, activated bead-formed agarose or its derivatives are used as gel matrices. Alternative matrix materials have been described, but as yet they do not appear to offer important advantages. In this study, pulverized polystyrene (PS 158K, BASF, Mannheim, Germany) was used as a solid phase for the immobilisation of bovine immunoglobulins (Ig). Affinity chromatography was performed using these coated polystyrene beads as the column matrix material in the purification of anti-bovine Ig. The polystyrene binding capacity for the different bovine Ig classes was compared using the Mancini single radial immunodiffusion technique, and ELISA procedures were used to monitor the antibody reactivity of purified and unpurified antibodies. The degree of purification was comparable to the most commonly used procedure using gel matrices from activated bead-formed agarose (e.g. CNBr-activated Sepharose 4B, Pharmacia/LKB Biotechnology, Uppsala, Sweden), but the antibody yield per ml column volume was distinctly lower. In order to raise the yield, such polystyrene bead columns with immobilized antigen can be re-used without loss of activity or larger column volumes can be used to raise the binding capacity. The polystyrene material is quite durable, chemically and immunologically inert and has a long shelf life. We conclude that polystyrene based affinity chromatography is efficient, simple and cheap.

Adsorption↗

PubMed1980

Affinity chromatography: a tool for enzyme immunoassay.

Affinity chromatography has been widely used for enzyme purification and for the preparation of antigen-enzyme conjugates. Most enzyme immunoassay use immunoadsorbent (specific or double insolubilized antibodies) which makes automation difficult as centrifugation and washing steps are necessary. We propose affinity chromatography for rapid separation of antigen-enzyme bound to antibodies from free conjugate and its application in enzyme immunoassay. We describe enzyme immunoassays for human placental lactogen and progesterone which use delta 5,3-ketosteroid isomerase as marker and depend upon retention by specific absorbent of enzyme conjugate not bound to antibodies.

Adsorption↗

PubMed1993

Hydrophobic affinity chromatography of human thrombin.

Hydrophobic affinity chromatography on p-chlorobenzylamido-agarose (p-CBA-agarose) was used to characterize various modified forms of human thrombin. Native alpha-thrombin bound tightly to the column and was eluted with either acetonitrile or 1,4-dioxane, while the catalytically inactive prethrombin 2 did not bind to the matrix. Site-specific chemical modification with pyridoxal 5'-phosphate resulted in the loss of at least 80% of fibrinogen clotting activity but did not influence the binding of thrombin to p-CBA agarose. Modification of thrombin with pyridoxal 5'-phosphate is thought to occur at the fibrinogen-binding site and the heparin-binding site. In contrast, binding of thrombin to p-CBA agarose was eliminated by modification of the active site histidine using either H-D-phenylalanyl-L-prolyl-L-arginine chloromethylketone or dansyl-L-glutamyl-glycyl-L-arginine chloromethylketone but not with tosyl-L-lysine chloromethylketone. The presence of either hirudin or heparin blocked the binding of thrombin to p-CBA-agarose but dansyl-arginine-N-(3-ethyl-1,5-pentanediyl)amide had no effect. These results indicate that p-CBA agarose binds to thrombin outside of the enzyme active site and its use should be valuable in characterizing site-specific modified thrombins obtained by either protein engineering or chemical modification.

Acetonitriles↗

PubMed1977

Improved methods for separation and purification by affinity chromatography.

Defined conditions for simplified Affinity Chromatography suitable for instructional and research purposes, were developed. The standard method of activation of polysaccharide gels using BrCN was modified and improved using a highly buffered reaction solution. A batchwise protocol for the immunoadsorbent isolation of anti-BSA was used (with commonly employed dissociation agents) and comparative data were obtained.

Acetonitriles↗

PubMed1987

Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies.

Immuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture. The procedure was monitored by testing the respective fractions in an infectivity titration system, in an ELISA, in a haemagglutination assay and by negative contrast electron microscopy to quantify CPV or CPV antigen. The degree of purification was further estimated by testing the fractions for total protein content in a colorimetric method, for bovine serum albumin content in an ELISA and by SDS-PAGE. Over 99% of the contaminating proteins proved to be removed, and 20% or 70-90% of infectious CPV or CPV antigen, respectively, was recovered.

Animals↗

PubMed2000

Biomimetic dyes as affinity chromatography tools in enzyme purification.

Affinity adsorbents based on immobilized triazine dyes offer important advantages circumventing many of the problems associated with biological ligands. The main drawback of dyes is their moderate selectivity for proteins. Rational attempts to tackle this problem are realized through the biomimetic dye concept according to which new dyes, the biomimetic dyes, are designed to mimic natural ligands. Biomimetic dyes are expected to exhibit increased affinity and purifying ability for the targeted proteins. Biocomputing offers a powerful approach to biomimetic ligand design. The successful exploitation of contemporary computational techniques in molecular design requires the knowledge of the three-dimensional structure of the target protein, or at least, the amino acid sequence of the target protein and the three-dimensional structure of a highly homologous protein. From such information one can then design, on a graphics workstation, the model of the protein and also a number of suitable synthetic ligands which mimic natural biological ligands of the protein. There are several examples of enzyme purifications (trypsin, urokinase, kallikrein, alkaline phosphatase, malate dehydrogenase, formate dehydrogenase, oxaloacetate decarboxylase and lactate dehydrogenase) where synthetic biomimetic dyes have been used successfully as affinity chromatography tools.

Chromatography, Affinity↗

PubMed2001

Exploiting lectin affinity chromatography in clinical diagnosis.

Lectin affinity chromatography (LAC) offers a tool that aids purification of cell surface glycoconjugates in sufficient quantities so that studies addressing their structural elucidation could be carried out. It has several advantages over the conventional biochemical methods, such as immunoprecipitation and/or immunoaffinity chromatography, used for the purification of various glycoconjugates. Serial LAC (SLAC) not only helps establish the identity of a glycoprotein or allows purification of a glycoprotein to homogeneity from among a mixture of glycoproteins, but it also successfully resolves the microheterogeneity in these glycoproteins, which is an otherwise impracticable problem to address. Specific cases of the altered expression and maintenance of microheterogeneity of some of the glycoproteins in pathological conditions vis a vis during normal biology are presented. The application of LAC in (i) itself, (ii) a serial fashion, and (iii) conjunction with other techniques such as two-dimensional electrophoresis, capillary electrophoresis, mass spectrometry, etc. in the diagnosis of certain pathological conditions, and the possibility of using this knowledge in designing treatments for various diseases, is discussed.

Cell Membrane↗

PubMed1977

DTNB modification of SH groups of isocitrate dehydrogenase from Bacillus stearothermophilus purified by affinity chromatography.

A new method of affinity chromatography using blue dextran-Sepharose 4B resin was established to purify NADP+-dependent isocitrate dehydrogenase [EC 1.1.1.42] from Bacillus stearothermophilus in high yield. The purified preparation was found to be homogeneous on disc gel electrophoresis. The SH groups of the enzyme were modified with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to determine the number of SH groups per molecule and their contribution to the enzyme activity. One SH group was titrated with DTNB per subunit (the native enzyme consisted of two subunits) and after complete denaturation with 4 M guanidine-HCl the number of titratable SH groups remained unchanged. ORD and CD measurements showed that the alpha-helical conformation of the polypeptide backbone was unaffected by DTNB modification, though the near ultraviolet CD spectrum was evidently altered. The fluorescence derived from tryptophanyl residue(s) was quenched by the modification to 30% of the native level, which may indicate the presence of SH in the vicinity of tryptophanyl residue(s). A remarkable decrease of the enzyme activity was detected upon modification with DTNB, but there was some discrepancy between the rate of inactivation and that of modification of SH groups. The presence of substrate and Mg2+ gave partial protection against modification of the SH groups by DTNB. Complete protection of the native enzyme activity against heating at 65 degrees was observed in the presence of substrate and Mg2+, but the thermostability of the enzyme was markedly reduced by modification of the SH groups.

Chromatography, Affinity↗

PubMed1978

Identification of a breast tumor-associated orosomucoid by concanavalin A affinity chromatography.

Con A-Sepharose affinity chromatography was utilized to examine the glycoproteins in phosphosaline extracts of normal and breast tumor tissues and breast patient sera. In extracts of normal breast tissue, normal sera and patient sera, all glycoproteins were eluted from the Con A-Sepharose with a linear gradient of 0.0-0.5 M alpha-methylmannose. Using breast tumor extracts, a glycoprotein peak which could not be eluted as with normal tissue extracts was observed. This tightly-binding peak could be eluted from the Con A-Sepharose with acetate buffer containing 1.0 M KCl. Polyacrylamide electrophoresis of this tightly-binding glycoprotein peak revealed one major glycoprotein and four minor glycoproteins. The major glycoprotein obtained from electrophoresis represented about 60% of the Con A-Sepharose tightly-binding protein and reacted with antiserum to human orosomucoid (alpha 1-acid glycoprotein). All glycoproteins isolated from tumor tissue extracts appeared to represent normal serum constituents as they were retained on an immunoadsorbent containing antibodies to normal serum proteins. The possible significance of the isolated tumor-associated orosomucoid is discussed.

Adenocarcinoma↗

PubMed1990

Purification of swine haptoglobin by affinity chromatography.

A globin-agarose affinity chromatography technique was used to purify swine haptoglobin. This technique provides a highly specific, single-step purification method without the contamination of extraneous serum proteins reported by previous studies. Complex formation between the haptoglobin isolate and swine hemoglobin confirmed that biological activity was maintained during the purification process. Immunoelectrophoretic and Ouchterlony immunodiffusion methods revealed that the swine haptoglobin isolate cross-reacted with polyvalent antisera against human haptoglobin.

Animals↗

PubMed1979

Na+/K+-dependent adenosinetriphosphatase in rabbit kidney. Separation by affinity chromatography.

We attempted separations by affinity chromatography of the Na+/K+-dependent adenosinetriphosphatase, using Sepharose 6B covalently coupled with ouabain, either on microsomal fractions or on preparations purified by discontinuous sucrose density gradient ultracentrifugation. The material, specifically eluted with ouabain, was tested to evaluate the recovery of ATPase activity and ouabain binding capacity. The results have shown the efficiency of this technique for (Na+/K+)-APTase isolation.

Animals↗

PubMed1981

Study of hemoglobin synthesis by affinity chromatography on Sepharose haptoglobin.

An affinity chromatography system, using haptoglobin bound covalently to Sepharose 4B, has been developed to purify human hemoglobin from soluble non-heme proteins. Agarose-haptoglobin specifically binds hemoglobin. Globin chains were eluted from the agarose-haptoglobin after removal of the heme. This method has allowed accurate measurements of globin chain synthesis in blood and bone marrow samples from adults and in culture of early erythroid precursors present in adult blood or bone marrow.

Bone Marrow↗

PubMedDate not supplied

Measurement of glycosylated hemoglobins using boronate affinity chromatography.

A boronate affinity column method for the measurement of glycosylated hemoglobins was evaluated. In the procedure the glycosylated hemoglobins were bound by immobilized boronic acid to separate them from nonglycosylated hemoglobins. Elution of bound glycosylated hemoglobins was carried out with sorbitol buffer, and the absorbance was read at 414 nm. The method was linear to a glycosylated hemoglobin concentration of at least 20 percent. The precision of the method ranged from 1.2 to 2.8 percent (C.V.) within-run, and 3.4 to 5.3 percent day-to-day. The reference interval was 4.8 to 6.4 percent. The method correlated with a cation exchange resin mini-column method (r = 0.94) and a colorimetric method (r = 0.93) but results from the boronate affinity method were higher in diabetic patients. The measured glycosylated hemoglobin was significantly correlated with estimated one-day-mean plasma glucose in diabetic patients (r = 0.54, n = 52, p less than 0.002). The affinity method provides an attractive alternative to earlier methods for measuring glycosylated hemoglobins.

Adolescent↗

PubMed1977

Erythropoietin: isolation by affinity chromatography with lectin-agarose derivatives.

Affinity chromatography using agarose-bound lectins was used to isolate erythropoietin from crude preparations of sheep plasma and human urinary erythropoietin. On the basis of previous estimates of the sugar content of the hormone, six lectins (wheat germ agglutinin, phytohemagglutinin, Ricinus communis 120, soybean agglutinin, concanavalin A, and limulin) were chosen for study. Only wheat germ agglutinin-agarose and phytohemagglutinin-agarose derivatives had significant affinity for erythropoietin. By use of wheat germ aggutinin-agarose columns, erythropoietin could be separated from over 95% of the initial starting protein, resulting in an 8-to 100-fold purification and a recovery of at least 40% depending on the source of the hormone. Affinity chromatography with agarose-bound lectins provides a simple rapid method for isolating erythropoietin from crude preparations of the hormone.

Animals↗

PubMed1990

[Isolation of tubular proximal cells of the human kidney by affinity chromatography using Lotus tetragonolobus lectin].

An affinity chromatography technique, using a lectin issued from Lotus tetragonolobus as ligand, and coupled to Sepharose 6MB support, has been chosen to enrich a cellular population in tubular proximal cells. Cell desorption was obtained with a competitive sugar, L Fucose. Intensive enzymatic brush border activity, shown in selected cells compared with initial cellular mixture, evidenced an enrichment in proximal tubular cells. These results prove that lectin use as ligand in affinity chromatography permits the separation of renal cell populations and presents the advantage that the bound cells can be recovered by a gentle competitive elution for further cultures.

Cell Separation↗

PubMed1988

Novel approach to affinity chromatography using "weak" monoclonal antibodies.

Affinity purification generally relies on specific high-affinity recognition between two species of biological molecules: one molecular species (the ligate) dissolved in a mobile phase is selectively adsorbed to the other species (the ligand) coupled to a solid support. Desorption of the ligate often requires harsh conditions that degrade biological activity of the purified product. As an alternative to this general procedure, we have studied affinity chromatography in a weak affinity mode, where ligand-ligate interactions are in dynamic equilibrium. Ligates recognized with low affinities (dissociation constant greater than 10(-4) M) elute from affinity columns under mild, isocratic conditions as retarded peaks, separated from noninteracting solutes that elute in the void volume. To illustrate the procedure, we report chromatography of an oligosaccharide on a 2-ml column containing 86 mg of a monoclonal antibody coupled to 10-micron microparticulate silica particles. Using a temperature-sensitive antibody, we observed that when the ligand-ligate dissociation constant is greater than 10(-3) M, performance of the system exceeds 300 theoretical plates/10 cm column length and approaches the efficiencies generally associated with high-performance liquid chromatography.

Antibodies, Monoclonal↗

PubMed2001

Affinity chromatography in the industrial purification of plasma proteins for therapeutic use.

Affinity chromatography is a powerful technique for the purification of many proteins in human plasma. Applications cover the isolation of proteins for research purposes but also, to a large extent, for the production of therapeutic products. In industrial plasma fractionation, affinity chromatography has been found to be particularly advantageous for fine and rapid capture of plasma proteins from industrial plasma fractions pre-purified by ethanol fractionation or by ion-exchange chromatography. To date, affinity chromatography is being used in the production of various licensed therapeutic plasma products, such as the concentrates of Factor VIII, Factor IX, von Willebrand Factor, Protein C, Antithrombin III, and Factor XI. Most commonly used ligands are heparin, gelatin, murine antibodies, and, to a lesser extent, Cu(2+). Possible development of the use of affinity chromatography in industrial plasma fractionation should be associated to the current development of phage display and combinatorial chemistry. Both approaches may lead to the development of tailor-made synthetic ligands that would allow implementation of protein capture technology, providing improved productivity and yield for plasma products.

Anticoagulants↗