Affinity purified concanavalin A (Con A) is composed of two identical subunits of 237 amino acid residues (MW: 26,000 without any cystine residues). While above pH 7 it is predominantly tetrameric, at pH 4.5 – 5.6, Con A binds two metal ions per monomer: transition metals, Mn2+ and Ca2+. Both ions are required for binding with optimum activity at a pH 7.0. Con A binds with non-reducing α-D-glucose, α-D-mannose and α-methyl-D-glucopyranoside acts as a competitive inhibitor. Con A does not have a blood group specificity. It exhibits mitogenic activity with lymphocytes and cancer cells. While lymphocytes and cancer aggregate by Con A; normal white cells do not. Normal cells react to Con A after proteolytic treatment which suggests that trypsinization causes clustering of the reactive glycan residues on the membrane. Con A interacts with cell types include locust muscle fibers, adipocytes, and rat liver plasma membrane components. This lectin induces endoreduplication in mammalian cells and it reacts with E. coli, Dictyostelium discoideum and Bacillus lipopolysaccharides. Immobilized Con A has been used in affinity chromatography purifications of a wide variety of glycoproteins and cellular structures.
Alkaline phosphatase (AP) is conjugated to Concanavalin A (Jackbean) Lectin (Con A) to show the binding of Con A in many applications including Western blotting and ELISA. Alkaline phosphatase is a large protein (140 kDa) that catalyzes the hydrolysis of phosphate groups from a substrate resulting in a colored or fluorescent product. The optimal enzymatic activity of this protein is between pH 8 and 10, and its reaction rate remains linear, improving sensitivity over time.