Kinetics properties of Cu,Zn-superoxide dismutase as a function of metal content

The kinetics of bovine Cu,Zn superoxide dismutase were studied by pulse radiolysis. To ensure the absence of catalytically active free copper, commercially obtained holo-superoxide dismutase was demetallated, and the apo-superoxide dismutase concentrations were determined by isothermal titration calorimetry prior to reconstitution with defined amounts of copper and zinc. The catalytic rate constant was determined as a function of ionic strength over the range of 4-154 mM, and of the copper and zinc content. The catalytic rate constant increases with ionic strength up to (1.5 ± 0.2) × 109 M−1 s−1 at an ionic strength of 15 mM, and then decreases. At pH 7 and 50 mM ionic strength, k = (1.2 ± 0.2) × 109 M−1 s−1, and at a physiologically relevant ionic strength of 150 mM, it is (0.7 ± 0.1) × 109 M−1 s−1. The effect of ionic strength is ascribed to the inhomogeneous electric field generated by the surface charges of superoxide dismutase. The value of the catalytic rate constant at 50 mM is ca. 2-fold smaller than earlier values reported in the literature. The relationship between copper content and the catalytic rate constant shows that addition of more than a stoichiometric amount of copper cannot be masked efficiently by edta. The possibility exists that earlier reported values were based on experiments contaminated with trace amounts of copper.