Involvement of carboxyl and phenoxyl groups in Cajanus cajan lectin-sugar interaction: interpretation of titration curves.

Hydrogen ion titration of an affinity-purified mannose/glucose-specific lectin from Cajanus cajan seeds at 30 degrees C and ionic strength of 0.15 was reported earlier by our lab (Salahuddin and Khan, 1998). Further work has been carried out on the systematic study of H+ titration of Cajanus cajan lectin at three different temperatures, i.e., 10 degrees, 20 degrees, and 30 degrees C at ionic strength of 0.15. In all, 88 protons were dissociated reversibly in pH range 2-12. Different ionizable groups present in lectin were also determined with respect to their heat of ionization. Heat of ionization, deltaH, for 43 carboxyl groups was 1.92 Kcal/mol, and for 7 phenoxyl groups, 6.84 Kcal/mol. Spectrophotometric titration was performed in the presence and absence of 0.1 M methyl alpha-D-mannopyranoside to characterize the role of phenoxyl groups present at the active site. One phenoxyl group was found to be involved in carbohydrate binding. The presence of two carboxyl groups per subunit at the active site was confirmed by H+ titration in the presence and absence of 0.1 M methyl alpha-D-mannopyranoside. Hemagglutinating and IgM precipitating activity was checked for the chemically modified carboxyl and phenoxyl group preparation. Loss of activity further confirmed the participation of carboxyl as well as phenoxyl groups at the active site. It is interesting to note the near-UV CD changes in the aromatic environment/conformation of the lectin in the presence of specific sugar, further indicating the possible participation of phenoxyl groups in the carbohydrate-binding pocket.