Interaction of dimethyl-p-phenylenediamine with ceruloplasmin

The stoichiometric interaction of dimethyl-p-phenylenediamine (DPD) with ceruloplasmin-Cu²⁺ has been investigated by spectrophotometric and electron paramagnetic resonance technique. Intermediates in the reaction sequence, DPD → (DPD)⁺ → (DPD)²⁺ → further oxidation products, were characterized. Cerulo-plasmin-Cu²⁺ was rapidly reduced by DPD as well as by the monoradical ion (DPD)⁺ but not by (DPD)²⁺. The two oxidation products (DPD)⁺ and (DPD)²⁺ were reversibly reduced by NADH₂. The stoichiometry of the reactions confirmed that DPD is oxidized by one electron transfer to ceruloplasmin-Cu²⁺ in two subsequent steps. Calculations of some of the rate constants were done. The “association constant” k₁ for DPD with ceruloplasmin was 750m⁻¹ sec⁻¹, and k′₁ for (DPD)⁺ 800m⁻¹ sec⁻¹ at 10 °. The rate constant for the reoxidation of ceruloplasmin-Cu⁺ was 0.037 sec⁻¹. When DPD was oxidized by ceruloplasmin, the formation of a relatively stable protein-bound radical was indicated. Kinetic studies of the oxidation of DPD by ceruloplasmin in catalytical amounts were done. The results suggested two different types of active sites or oxidizing units in ceruloplasmin.