Biotinylated derivative of a human brain lectin: Synthesis and use in affinoblotting for endogenous ligand studies☆

Abstract Coupling of biotin to an endogenous lectin yields a probe which can be used for selective nonradioactive detection of complementary endogenous ligands. To exemplify practical applications of this type of compounds, we have synthesized and characterized a biotinylated derivative of a β-galactoside-specific human brain lectin. Proteins which bind this lectin can be located on nitrocellulose sheets after electrophoretic transfer from gradient polyacrylamide gels, by sequential incubation with biotinylated probes and streptavidin-peroxidase, with visualization by an insoluble reaction product (affinoblotting). Biotinylated galactoside-binding plant lectins were used in the same way to visualize human brain glycoproteins, and their binding specificity was compared with that of human brain lectin. The results obtained by means of these different probes showed the usefulness of the endogenous lectin derivative to actually identify its endogenous partners. Thus this approach may find extended applications in the study of biological activities of vertebrate lectins in homologous systems, i.e., with lectins and ligands coming from the same tissue origin.