Kinetics of oxidation of o-dianisidine by hydrogen peroxide in the presence of antibody complexes of iron(III) coproporphyrin |
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Authors: | Alexander P Savitsky Mary I Nelen Anatoly K Yatsmirsky Mary V Demcheva Gely V Ponomarev Igor V Sinikov |
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Institution: | 1. Laboratory of Molecular Immunology, A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, 117071, Moscow, Russia 2. Laboratory of Chemical Enzymology, Department of Chemistry, Moscow State University, Moscow, Russia
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Abstract: | 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 (kc) for bound FeCPI exceed (kc) for free FeCPI by two-to threefold. This difference can be explained by a real enhancement of (kc) 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 (kc) 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. |
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