PEROXIDASE-ACTIVITY OF AN ANTIBODY-FERRIC PORPHYRIN COMPLEX

The catalytic antibody 2B4 which catalyzes insertion of a cupric ion i nto porphyrin also combines with ferric porphyrin to form an antibody- ferric porphyrin complex. The antibody has distinct amino acid sequenc es in complementarity-determining regions compared to other anti-porph yrin 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, b ut did not oxidize pyrogallol nor hydroquinone. The peroxidase reactio n of the complex was examined kinetically for o-dianisidine, and compa red with that of ferric porphyrin. With increasing concentrations of o -dianisidine, the reaction rate obtained for ferric porphyrin increase d gradually, in contrast, that for the complex increased steeply and t hen saturated. These results indicated that the interaction of the com plex with o-dianisidine was much higher than that of ferric porphyrin, At a constant concentration of o-dianisidine, the reaction rates obta ined for the complex and for ferric porphyrin both showed saturation b ehavior against hydrogen peroxide concentration. The K-m value for hyd rogen peroxide of the complex was similar to that of ferric porphyrin but much larger than that of natural peroxidase, suggesting that the a ntibody did not have a residue facilitating the binding of hydrogen pe roxide as in natural peroxidase. In the reaction of the complex with h ydrogen peroxide, active intermediates were not observed. Based on the results, a scheme for the peroxidase reaction by the complex was prop osed. It was considered that the enhancement of the peroxidase activit y by the antibody was mainly attributed to an increase in the interact ion with o-dianisidine, and that the substrate specificity of the comp lex resulted from the difference in the interaction. (C) 1998 Elsevier Science B.V. All rights reserved.