The carbohydrate binding specificity of the seed lectin from Artocarpus integrifolia, artocarpin, has been elucidated by the enzyme-linked lectin absorbent assay, wherein it was demonstrated to be a Man/Glc specific lectin with high affinity for the trisaccharide present in the core of all N-linked oligosaccharide chains of glycoproteins. As a consequence of this characterization, the binding epitopes of this trisaccharide, 3,6-di(a-D-mannopyranosyl)-D-mannose, for artocarpin were investigated by isothermal titrn. calorimetry using its monodeoxy as well as Glc and Gal analogs. The thermodn. data presented here implicate 2-, 3-, 4-, and 6-hydroxyl groups of the a(1-3) Man and a(1-6) Man residues, and the 2- and 4-OH groups of the central Man residue, in binding to artocarpin. Nevertheless, a(1-3) Man is the primary contributor to the binding affinity, unlike other Man/Glc binding lectins which exhibit a preference for a(1-6) Man. In addn., unlike the binding reactions of most lectins reported so far, the interaction of mannotriose involves all of its hydroxyl groups with the combining site of the lectin. Moreover, the free energy and enthalpy contributions to binding of individual hydroxyl groups of the trimannoside estd. from the corresponding monodeoxy analogs show nonlinearity, suggesting differential contributions of the solvent and protein to the thermodn. of binding of the analogs. Thus, this study not only provides evidence for the extended site recognition of artocarpin for the trimannoside epitope but also suggests that its combining site is best described as a deep cleft as opposed to shallow indentations implicated in other lectins. [on SciFinder (R)]