A sensitive fluorescence assay based on Amplex Red (AR) oxidation by horseradish peroxidase (AR/HRP) is described which continuously monitor rates of H2O2 production by microsomal enzymes in the presence of relatively high concentrations of NADPH. NADPH and NADH are known to interact with HRP and generate significant quantities of superoxide anion, a radical that spontaneously dismutates to form H2O2 which interferes with the AR/HRP assay. Microsomal enzymes generate H2O2 as a consequence of electron transfer from NADPH to cytochrome P450 hemoproteins with subsequent oxygen activation. We found that superoxide anion formation via the interaction of NADPH with HRP was inhibited by superoxide dismutase (SOD) without affecting H2O2 generation by microsomal enzymes. Using SOD in enzyme assays, we consistently detected rates of H2O2 production using microgram quantities of microsomal proteins (2.62 ± 0.20 picomol/min/µg protein for liver microsomes from naïve female rats, 12.27 ± 1.29 for liver microsomes from dexamethasone induced male rats, and 2.17 ± 0.25 picomol/min/µg protein for human liver microsomes). This method can also be applied to quantify rates of H2O2 production by oxidases where superoxide anion generation by NADH or NADPH and HRP can interfere with enzyme assays.
The Amplex Red/horseradish peroxidase assay requires superoxide dismutase to measure hydrogen peroxide in the presence of NAD(P)H