PURIFICATION AND CHARACTERIZATION OF A FUC-ALPHA-1-]2GAL-BETA-1-] AND GALNAC-BETA-1-]-SPECIFIC LECTIN IN ROOT TUBERS OF TRICHOSANTHES-JAPONICA

A prominent lectin in the root tubers of Trichosanthes japonica was purified by affinity chromatography on a porcine stomach mucin-Sepharose column and termed TJA-II. The molecular mass of the native lectin was determined to be 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TJA-II was separated into two different subunits of 33 and 29 kDa in the presence of 2-mercaptoethanol. The respective subunits contained mannose, N-acetylglucosamine, fucose, and xylose. It was determined by equilibrium dialysis to have two equal binding sites per molecule, the association constant toward tritium-labeled Fucalpha1-->2Galbeta1-->3GlcNAcbeta1-->3Galbeta1-->4Glc(OT) being K(alpha) = 3.05 x 10(5) M-1. The precise carbohydrate binding specificity of immobilized TJA-II was studied using various tritium-labeled oligosaccharides. A series of oligosaccharides possessing Fucalpha1-->2Galbeta1--> or GalNAcbeta1--> groups at their nonreducing terminals showed stronger binding ability than ones with Galbeta1-->GlcNAc (Glc) groups, indicating that TJA-II fundamentally recognizes a beta-galactosyl residue and the binding strength increases on substitution of the hydroxyl group at the C-2 position with a fucosyl or acetylamino group. This lectin column is useful for fractionating oligosaccharides or glycoproteins containing blood group type 1H, type 2H, and Sd antigenic determinants.