Determination of Catalase Activity at Physiological Hydrogen Peroxide Concentrations

Abstract

A method for the determination of catalase activity (EC 1.11.1.6.) in homogenates and cell suspensions is described by following the decomposition of H2O2at physiological H2O2levels. This first chemiluminescence assay for catalase activity is based on the reaction of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and NaOCl. The chemiluminescence of this reaction specifically depends on the H2O2concentration and shows fast kinetics of less than 2 s. Using a flow technique, the exponential decay of H2O2in the presence of catalase is followed down to 10−8mH2O2at pH 7.4 over three orders of magnitude. At these very low H2O2concentrations neither oxygen is liberated in gaseous form nor enzyme inactivation or loss of cell viability is observed. Addition of the catalase inhibitor NaN3completely inhibits H2O2decomposition. Since the method is not influenced by sulfhydryl and amino group containing compounds, it is especially suited for crude tissue homogenates and suspensions of intact cells. Interestingly, application to cell suspensions shows that intact human erythrocytes and rat hepatocytes exhibit only 5.8 and 1.9% of catalase activity when compared to homogenized cells. These data suggest that the diffusion of H2O2through membranes is lower than that assumed so far.