Peroxidase activity of an antibody–ferric porphyrin complex

Abstract The catalytic antibody 2B4 which catalyzes insertion of a cupric ion into porphyrin also combines with ferric porphyrin to form an antibody–ferric porphyrin complex. The antibody has distinct amino acid sequences in complementarity-determining regions compared to other anti-porphyrin antibodies reported. The 2B4-ferric porphyrin complex oxidized o -dianisidine and 2,2′-azino- bis (3-ethylbenz-thiazoline-6-sulfonic acid) utilizing hydrogen peroxide more efficiently than ferric porphyrin, but did not oxidize pyrogallol nor hydroquinone. The peroxidase reaction of the complex was examined kinetically for o -dianisidine, and compared with that of ferric porphyrin. With increasing concentrations of o -dianisidine, the reaction rate obtained for ferric porphyrin increased gradually, in contrast, that for the complex increased steeply and then saturated. These results indicated that the interaction of the complex with o -dianisidine was much higher than that of ferric porphyrin. At a constant concentration of o -dianisidine, the reaction rates obtained for the complex and for ferric porphyrin both showed saturation behavior against hydrogen peroxide concentration. The K m value for hydrogen peroxide of the complex was similar to that of ferric porphyrin but much larger than that of natural peroxidase, suggesting that the antibody did not have a residue facilitating the binding of hydrogen peroxide as in natural peroxidase. In the reaction of the complex with hydrogen peroxide, active intermediates were not observed. Based on the results, a scheme for the peroxidase reaction by the complex was proposed. It was considered that the enhancement of the peroxidase activity by the antibody was mainly attributed to an increase in the interaction with o -dianisidine, and that the substrate specificity of the complex resulted from the difference in the interaction.