High-performance concanavalin a affinity chromatography of liver and hepatoma membrane proteins

Josić, Djuro; Hofmann, Werner; Habermann, R.; Reutter, Werner

doi:10.1016/s0021-9673(01)94006-3

Cited by 14 publications

(6 citation statements)

References 7 publications

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“…The siliceous materials to be used for the attachment of lectins must fulfill several important criteria: they must be (1) chemically stable; (2) resistant to high pressure; (3) macroporous; (4) hydrophilic, with a nonionic surface; and (5) possessing functional groups suitable for the ligand immobilization . Just as with the synthetically modified methacrylate-based polymers, , porous silica generally meets these criteria, except that it suffers from a partial solubility at alkaline pH and the presence of silanol groups acting as potential ion-exchange sites. Therefore, prior to coupling of the ligand, the surfaces must be additionally derivatized.…”

Section: Resultsmentioning

confidence: 99%

Combining Lectin Microcolumns with High-Resolution Separation Techniques for Enrichment of Glycoproteins and Glycopeptides

2005

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Silica-based lectin microcolumns are described in this study together with the chemical procedures necessary for their preparation. The analytical merits of Canavalia ensiformis and Sambucus nigra lectins, [immobilized on activated macroporous silica], such as binding capacity, trapping reproducibility, and substrate selectivity, have been evaluated using model glycoproteins. The described microcolumns are applicable to high-pressure analytical schemes utilizing microvalving procedures, washing steps, and quantitative desorption for LC/MS analysis. The described analytical systems are amenable to the applications aiming at fractionation of complex glycopeptide mixtures and determination of the sites of glycosylation.High-performance affinity chromatography (HPAC) offers a number of advantages 1-4 over the classical and commonly used chromatographic technique which utilizes various immobilized ligands to retain selectively an analyte or a group of analytes. These advantages primarily include speed, ease of automation, and the application versatility. During the past decade, HPAC has been employed in biomolecular purification and enrichment and chiral separations, as well as in the study of biological interactions. 5-7 The success of any affinity chromatography, including HPAC, is largely dependent on chemical procedures employed in the attachment of a small-molecule ligand or an interacting biopolymer to a respective chromatographic support. 8-10 Their immobilization should not interfere with the native state of a ligand, facilitating access of analyte molecules and proper interactions.Lectins comprise a group of proteins with unique affinities toward carbohydrate structures. They have long been used in biomolecular isolation, carbohydrate chemistry, and histochemistry, and more recently as the mimicking agents in intercellular interaction studies. For a number of years, lectins have been utilized in various glycoprotein isolation efforts, 11-14 albeit few addressed the quantitative aspects of this approach. 15,16 Lectins with different specificities toward oligosaccharides have been immobilized to agarose and other conventional biochemical matrixes. 11-14 Some of these column materials have been commercialized. In addition, lectins have been immobilized to magnetic beads, 17 gold foils, 18 silica materials, 19-21 and to affinity membranes. 22The uses of lectins in contemporary glycoproteomics and glycomics are likely to grow due to the frequent needs for fractionation and preconcentration for high-sensitivity mass spectrometry (MS). 23 Although glycoproteins and glycopeptides could be fractionated through lectins with different specificity, in most reported cases, lectin affinity procedures were employed to isolate the entire pools of glycoconjugates rather than specific structures. 24,25 Consequently, wheat germ agglutinin and concanavalin A have been widely used because of their broad specificity. Although it is assumed that different glycoproteins produced by a certain cell type possess a similar type...

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Section: Resultsmentioning

confidence: 99%

Combining Lectin Microcolumns with High-Resolution Separation Techniques for Enrichment of Glycoproteins and Glycopeptides

2005

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“…By considering both the activity and stability, we found that the optimum pH of invertase (i.e., 4.7) was not suitable for the structural integrity of Con A. In some of the previous studies, it was reported that Con A lost its binding ability against the target glycoprotein in repeated adsorption–desorption cycles 44, 61. This behavior was explained by the removal of noncrosslinked Con A subunits during the desorption of adsorbed glycoprotein.…”

Section: Discussionmentioning

confidence: 91%

“…They concluded that there was noticeable deterioration in the binding properties of the carrier in the iterative use of the support. Josic et al61 also reported that the glycoprotein binding capacity of a carrier/Con A couple may decrease because of the unwanted loss of Con A (the dissociation of the Con A tetramer). The loss of Con A during elution, especially with MMP, accounted for serious complications.…”

Section: Resultsmentioning

confidence: 99%

Concanavalin A attached poly(p‐chloromethylstyrene) beads for glycoenzyme separation

Bahar¹,

Tuncel²

2004

J of Applied Polymer Sci

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ABSTRACT:Crosslinked poly(p-chloromethylstyrene) (PCMS) beads were produced by suspension polymerization. The beads had a nonporous but reasonably rough surface structure. Because of this property, a relatively high external surface area (i.e., 14.1 m 2 /g) was obtained with the proposed carrier. A biospecific ligand commonly used in the affinity chromatography of various glycoenzymes, concanavalin A (Con A), was covalently attached onto the bead surface by a direct chemical reaction. PCMS/Con A beads were then used for the separation of a model glycoenzyme, invertase, from its crude solution. The appropriate invertase adsorption and desorption conditions of the affinity system were investigated. The desorption of invertase from the carrier was achieved by the use of methyl-␣-d-mannopyranoside (MMP) as the counterligand. The effects of the MMP and salt concentrations and the temperature on the desorption behavior of invertase were investigated. The MMP concentration and temperature were found to be the dominant parameters controlling the desorption. Iterative experiments involving five reversible adsorption-desorption cycles were performed with the same particles to monitor the changes that occurred in the invertase adsorption-desorption capacities of Con A immobilized beads. The reversible adsorption-desorption stability was significantly improved by the crosslinking of Con A immobilized on the beads.

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“…4 M MgC12 or 3 M NaCl [123,128,134]; (d) denaturants, e.g. 6 M guanidineaHC1 or 8 M urea [144]; (e) 1 M a-methylpyranoside [129,138,147]; (f) free ligand, e.g. a peptide [139], or (g) a compound which reduces the ligand resulting in a decreased aflinity of ligand and protein [124,125]; and (h) 150-…”

Section: Bioaffinity Chromatography (Bac)mentioning

confidence: 99%

Integral Membrane Proteins

Welling

Welling‐Wester

2000

Journal of Chromatography Library

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High-performance concanavalin a affinity chromatography of liver and hepatoma membrane proteins

Cited by 14 publications

References 7 publications

Combining Lectin Microcolumns with High-Resolution Separation Techniques for Enrichment of Glycoproteins and Glycopeptides

Combining Lectin Microcolumns with High-Resolution Separation Techniques for Enrichment of Glycoproteins and Glycopeptides

Concanavalin A attached poly(p‐chloromethylstyrene) beads for glycoenzyme separation

Integral Membrane Proteins

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