Immunochemical studies of the combining sites of the two isolectins, A₄ and B₄, isolated from Bandeiraea simplicifolia

The specificity of two isolectins, A₄ and B₄, of Bandeiraea simplicifolia lectin I (BS-I) was studied by quantitative precipitin, precipitin inhibition, as well as by competitive binding assays using various blood group substances and tritium-labeled human B substance. A₄ precipitated well with A₁, A₂, B, and precursor substances, with A₂ precipitating less strongly than did A₁ substance; H, Le^a and Le^b substances did not react. Precipitin inhibition and competitive binding assays confirmed the precipitin data that A₄ is most specific for terminal nonreducing α-linked 2-acetamido-2-deoxy-d-galactopyranose (dGalNAc) but also reacts with oligosaccharides with terminal nonreducing α-linked dGal, thus accounting for its blood group A and B specificities. Of the oligosaccharides tested, A₄ reacted best with dGalNAcα1 → 3dGal and a trisaccharide dGalNAcα1 → 3dGalβ1 → 3dGlcNAc (A₅II) was equally active, suggesting that the A₄ site is no larger than a disaccharide. B₄ precipitated well with B substances and with a precursor substance to a lesser extent, while A₁ A₂, H, Le^a, and Le^b substances were inactive. Precipitin and competitive binding assays showed that it reacted well with oligosaccharides with terminal α-linked dGal with dGalα1 → 3dGal being most active, while lFucαl → 2 dGalα1 → 3dGalβ1 → 4dGlcNAcβ1 → 6-R (BR_L 0.44) was much less active, indicating a substitution at the subterminal residue affects the binding substantially and indicating that the B₄ site involves at least the subterminal α1 → 3 linked dGal. The B₄ site was found to be strictly B specific.