Kinetics of the Inhibition of Sarcoplasmic Reticulum Ca2+ ATPase by Hypochlorite

Myeloperoxidase (MPO), a heme protein secreted by activated phagocytes, is present and enzymatically active in human atherosclerotic lesions. The major reactive species generated by MPO is hypochlorous acid (HOCl) known to irreversibly damage Ca 2+ -ATPase. In the current study the kinetics of the impact of HOCl on sarcoplasmic Ca 2+ -ATPase activity was explored. We used a continuous activity assay based on the hydrolysis of para-nitrophenyl phosphate and compared it with a coupled-enzyme assay of ATP hydrolysis. Over longer periods of measurement, the coupled-enzyme system became highly affected by HOCl leading to inconsistent results. In contrast, the p-NPP assay was more precise and unaffected by HOCl. Even low-micromolar concentrations of HOCl led to a significant loss of activity by a process with two distinctly different kinetics. During the first phase a small loss of activity showed hardly any dependence on HOCl concentration. In the subsequent phase the oxidant lowered the activity less than proportionally. This process was only weakly delayed by the HOCl scavenger taurine. Collectively, these results suggest that the loss of Ca 2+ -ATPase activity due to HOCl is both time- and dose-dependent, and that non-essential labile thiol groups act as