Agarose bound* Concanavalin A (Con A)

 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