Kinetics of oxidation of o-dianisidine by hydrogen peroxide in the presence of antibody complexes of iron(III) coproporphyrin

The complex of iron(III) coproporphyrinl (FeCPI) with antibody D5E3 was studied as an artificial peroxidase, usingo-dianisidine as a substrate. At saturation with respect to antibody, the initial rates ofo-dianisidine oxidation are practically the same for free and bound FeCPI at a concentration 5 × 10^-9M, but the catalytic rate constant (k_c) for bound FeCPI exceed (k_c) for free FeCPI by two-to threefold. This difference can be explained by a real enhancement of (k_c) at the antibody-active site. The dependence of initial rates of the reaction on substrate concentrations obeyed Michaelis-Menten kinetics and revealed substrate activation at high concentrations ofo-dianisidine. A comparison of the Stern-Volmer constants foro-dianisidineinduced quenching of the porphyrin fluorescence proves that antibody-bound coproporphyrin is equivalently accessible to the substrate as protoporphyrin bound to apoperoxidase from horseradish peroxidase (HRP). Based on analysis of the (k_c) dependence on H₂O₂ concentrations in the FeCPI-antibody system, we suggest that interaction with hydrogen peroxide is the rate-limiting step for the oxidation reaction.