A Fluorometric Assay for Hydrogen Peroxide, Suitable for NAD(P)H-Dependent Superoxide Generating Redox Systems

We report a simple and sensitive fluorimetric method for quantitative assay of the production rate of hydrogen peroxide, and indirectly of superoxide, during electron transfer reactions. The assay requires the inclusion of superoxide dismutase, catalase, and 6% methanol in the tested reaction system, to stochiometrically produce formaldehyde per molecule of H₂O₂ generated. The reaction is terminated by adding 2 vol of Nash reagent and heating at 60°C for 10 min, to convert accumulated formaldehyde to diacetyldihydrolutidine (DDL). The standard curve for formaldehyde, based on the fluorescence of DDL, is highly reproducible and allows measurement of 1 μM amounts in the reaction sample (coefficient of variation <15%). The excitation and emission wavelengths of DDL at 412 and 505 nm are distant from those of NAD(P)H. Thus, the method can be used in NAD(P)H-dependent enzymatic systems to measure both NAD(P)H oxidation and superoxide production in the same sample. We validated the assay in a mitochondrial P450 system determining the fraction of total electron flow that is channeled to oxy-radical formation. The assay should be useful in the study of this and other superoxide/H₂O₂ generating systems.