Saccharide binding to transition metal ion free concanavalin A

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Abstract
Saccharide binding was observed with demetallized concanavalin A [Canavalia ensiformis] in the presence of Ca2+ only, using the fluorescent sugar 4-methylumbelliferyl .alpha.-D-mannopyranoside. At pH 7.2 both the nicked and intact forms of concanavalin A bound 4-methylumbelliferyl .alpha.-D-mannopyranoside with similar affinities. Competitive binding with methyl .alpha.-D-mannopyranoside was demonstrated. The association constants at 5.degree. C were 9.6 .+-. 0.6 .times. 104 M-1 for 4-methylumbelliferyl .alpha.-D-mannopyranoside and 1.1 .+-. 0.3 .times. 104 M-1 for methyl .alpha.-D-mannopyranoside. 4-Methylumbelliferyl .alpha.-D-mannopyranoside binding was also observed if demetallized concanavalin A was remetallized with less than stoichiometric amounts of Ca2+. The association constants with low Ca2+ concentrations were similar to those determined with saturating Ca2+. With less than stoichiometric levels of Ca2+, the number of sugar molecules bound per protein subunit was a reflection of the fraction of activated lectin subunits. These results show that saccharide binding activity of concanavalin A does not require a transition metal ion at pH 7.2; only Ca2+ is required. At pH values near 5, where most previous studies were carried out, both a transition metal ion and Ca2+ are necessary.