Heterogeneity of Concanavalin A as Detected by Its Binding to p-Nitrophenyl2-O-a-D-Mannopyranosyl-cw-D-mannopyranoside*

The binding of p-nitrophenyl2-O-a-D-mannopyranosyl-a-D-mannopyranoside (MS) to concanavalin A was studied by equilibrium dialysis and uv difference spectroscopy. Concanavalin A, purified by conventional methods, was shown to behave heterogeneously in its interactions with Mz and Sephadex G-75. Elution of concanavalin A bound to Sephadex G-75 by 19 mM Dglucose gives a protein composed predominantly of “nicked” or fragmented polypeptide chains. A second elution with 100 mM D-glucose gives concanavalin A composed entirely of intact polypeptide chains. Both fractions, which are homogeneous with respect to their interactions with Mz, bind Mz with an association constant of 1.9 (kO.2) X lo5 M-’ and a change in the molar absorptivity of -3.2 x lo3 cm-’ M-l, at 317 nm. About 50% of the concanavalin A is contained in a third fraction which is retained on the Sephadex G-75 column. This third fraction could not be eluted from the Sephadex G-75 column by 1.0 M D-glucose, 1.0 M methyl (Y-Dmannopyranoside, or 1.0 M sodium thiocyanate. The material remaining on the Sephadex G-75 column appears to be a metal-deficient form of concanavalin A which denatures and precipitates at low ionic strength and has a lower affinity for Mz than “intact” con A. Although addition of 1 mM Ca” and 1 mM Mn2+ to conventionally prepared concanavalin A causes it to behave like pure intact concanavalin A in its interactions with Mz, the metal-deficient form of concanavalin A, after being reconstituted with metal ions, appears to lose metal ions more rapidly than intact concanavalin A. Comparison of the association constants for binding of monoand disaccharides demonstrates that intact concanavalin A has a 19-fold greater affinity for M2 than the monosaccharide p-nitrophenyl a-D-mannopyranoside (Ml). This increased affinity for the chromogenic disaccharide over its monosaccharide counterpart strongly suggests that concanavalin A interacts simultaneously with groups on both mannopyranosyl residues of M2.