Concanavalin A (Con A), Agarose bound

Con A recognizes α-linked mannose present as part of a core oligosaccharide in many serum and membrane glycoproteins. At neutral and alkaline pH, Con A exists as a tetramer of four identical subunits; below pH 5.6, Con A dissociates into active dimers of 52 kDa. Acetylation, succinylation, or other derivatizations can also produce stable forms with dimeric structures. (See succinylated Con A). Nicks in the sequence are often present in the purest preparations due to hydrolytic damage within the seeds. Con A requires calcium or manganese ions at each of its four saccharide binding sites. Although these divalent metal ions are bound tightly to the polypeptide structure, buffers which can bind calcium (such as phosphate) generally should be avoided in diluting Con A, since a gradual loss in activity may occur. Agarose bound* Con A is prepared using our affinity-purified lectins. Heat stable, cross-linked 4% agarose beads with a molecular weight exclusion limit of about 2x107 daltons are used as the solid-phase matrix to which the lectins are covalently coupled. The attachment of the lectins to the beads is carefully controlled to preserve lectin activity and minimize conformational changes of the bound lectins that might result in nonspecific ionic or hydrophobic interactions. The technique we have developed to couple lectins to agarose beads inserts a hydrophilic spacer arm between the lectin and the matrix. This coupling method provides several advantages over the traditional cyanogen bromide procedure: Maximum carbohydrate binding activity of the coupled lectins is retained. Linkage is stable over a range of pH values. Conjugated proteins are not leached off the beads by Tris or other routinely used buffers. No residual charges are present after conjugation. This minimizes non-specific binding to the matrix. Our agarose bound lectins are supplied at a constant concentration of lectin per ml of settled beads. The concentration for each lectin is selected to achieve the highest glycoconjugate binding capacity per mg of lectin present in the beads. Each lot is tested for its binding capacity using glycoproteins known to bind the lectin. This provides a guideline for the user and assures the quality of our agarose bound lectins. Inhibiting/Eluting Sugar: mixture of 200 mM α-methylmannoside/200 mM α-methylglucoside. *6 mg lectin/ml gel.

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