Stopped Flow Kinetic Analysis A Direct Assay for Superoxide Dismutase Activity

Stopped Flow Kinetic Analysis A Direct Assay for Superoxide Dismutase Activity 

Numerous indirect assays, such as the cytochrome c assay, have been used in attempts to measure the SOD activity of putative SOD mimics,However, these assays, which typically rely on a spectrophotometric change of a redox indicator to measure superoxide levels, cannot kinetically distinguish between a catalytic dismutation of superoxide and a stoichiometric interaction of superoxide with the putative SOD mimic. Moreover, the indirect assays are prone to false positives or false negatives respectively, when the putative SOD mimic oxidizes or reduces the spectrophotometric indicator. 

We recognized the need for methodology to measure SOD activity directly that would be more accessible to the bench-top scientist than is the method of pulse radiolysis, another direct measure. Consequently, we developed methodology to measure the catalytic dismutation of superoxide by stopped-flow kinetic analysis.By this technique, we directly monitor the decay of superoxide spectrophotometrically in the presence or absence of a putative SOD mimic at a given pH. Kinetic analysis of this decay can determine whether the complex is a SOD mimic (decay of superoxide becomes first-order in superoxide and first-order in complex see equations 1 and 2), or is inactive (decay of superoxide remains second-order for its self-dismutation see equation 3). At least a tenfold excess of superoxide over the putative SOD mimic is used in the stopped-flow assay, to eliminate contributions due to a stoichiometric reaction of the complex with superoxide. A catalytic rate constant for the dismutation of superoxide by the complex can be determined from the observed rate constants of superoxide decay as a function of catalyst concentration.  

Design of Manganese-based Superoxide Dismutase Mimics 

Manganese-based complexes are particularly attractive as potential SOD mimics because they have a reduced capacity to react with the dismutation 

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