THERMODYNAMICS OF LECTIN-CARBOHYDRATE INTERACTIONS - TITRATION MICROCALORIMETRY MEASUREMENTS OF THE BINDING OF N-LINKED CARBOHYDRATES AND OVALBUMIN TO CONCANAVALIN-A

The thermodynamics of binding of concanavalin A (Con;A) with a series of linear and branched chain oligosaccharides including certain N-link ed complex type and oligomannose type carbohydrates and a fraction of quail ovalbumin containing Man7 and Man8 oligomannose chains have been determined using titration microcalorimetry. Methyl -(alpha-D-mannopy ranosyl)-alpha-D-mannopyranoside, a branch chain trisaccharide moiety found in all N-linked carbohydrates which possesses approximately 60-f old higher affinity than methyl alpha-D-mannopyranoside, exhibited a c hange in enthalpy of binding (Delta H) of -14.4 kcal mol(-1) as compar ed to -8.2 kcal mol(-1) for the monosaccharide. This demonstrates that Con A possesses an extended binding site for the trimannoside. Howeve r, a biantennary complex type carbohydrate with terminal beta(1,2)-Glc NAc residues which binds with 3-fold higher affinity than the trimanno side possesses a Delta H of only -10.6 kcal mol(-1). A plot of -Delta H versus -T Delta S for the carbohydrates in the present study showed positive deviations in -T Delta S for the complex type carbohydrate, a s well as alpha(1,2)-di- and trimannosyl oligosaccharides which are pa rt of the structures of oligomannose type carbohydrates. The relative favorable changes in binding entropies of these compounds are attribut ed to the presence of multiple internal and terminal residues in each molecule which can independently bind to the monosaccharide binding si te of the lectin. The Delta H values for the complex type carbohydrate and the alpha(1,2) mannose oligosaccharides were also approximately - 2.5 kcal mol(-1) greater than that of methyl alpha-D-mannopyranoside, indicating some extended binding site interactions. The thermodynamics of binding of N-linked oligomannose type carbohydrates to dimeric Con A and its succinyl and acetyl derivatives were determined since these carbohydrates are bivalent and precipitate with the native tetrameric lectin but not with the dimeric protein and its two derivatives. Titr ation of succinyl-Con A with a Man5 oligomannose type oligosaccharide gave a Delta H of -14.5 kcal mol(-1), which is similar to that of the branch chain trimannoside. This indicates that the alpha(1,6) core arm of Man5 which contains the trimannosyl moiety is the primary binding epitope for Con A. A fraction of quail ovalbumin containing a mixture of Man7 and Man8 chains at a single glycosylation site showed univalen t binding to succinyl-Con A and a Delta H of -13.6 kcal mol(-1). These results indicate that the trimannoside moiety on the alpha(1,6) arm(s ) of the carbohydrate chains is the primary binding epitope and that i ts interactions with the lectin are relatively unaffected by the prote in matrix of ovalbumin.