Decay kinetics of O2.- studied by direct spectrophotometry. Interaction with catalytic and non-catalytic substances

The kinetic behaviour of O2.- during spontaneous dismutation and in the presence of Cu,Zn-superoxide dismutase and other compounds, was studied by monitoring the decrease in absorbance (A250nm-A360nm) on a time-scale of > or = 1 min, at pH 9.5. O2.- was generated from KO2, and calculations were performed between 25 and 4 microM of O2.-. An algorithm for the simultaneous calculation of the 1st and 2nd-order rate constants from the decay curve, was evolved. The respective fractions of O2.- which interacted with catalysts or disappeared spontaneously, in various experimental situations, could be estimated. Substances could be classified as inert, catalysts or scavengers. The high assay pH excluded examination of the effect of alkali sensitive substances, e.g., Mn-superoxide dismutase. However, the high pH minimized the interfering effect of trace amounts of Cu(II). Therefore a metal chelator was superfluous and even the effect of metals and metal complexes could be tested. The extremely high sensitivity of the method allowed minute concentrations of reagents to be used, including proteins absorbing in the UV-region. The rate constants found by this simple method, agreed with those obtained by more sophisticated and inaccessible techniques like pulse radiolysis and stopped-flow spectrophotometry.