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 s uperoxide, during electron transfer reactions. The assay requires the inclusion of superoxide dismutase, catalase, and 6% methanol in the te sted reaction system, to stochiometrically produce formaldehyde per mo lecule of H2O2 generated. The reaction is terminated by adding 2 vol o f Nash reagent and heating at 60 degrees C for 10 min, to convert accu mulated formaldehyde to diacetyldihydrolutidine (DDL). The standard cu rve for formaldehyde, based on the fluorescence of DDL, is highly repr oducible and allows measurement of 1 mu M amounts in the reaction samp le (coefficient of variation <15%). The excitation and emission wavele ngths 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 mea sure both NAD(P)H oxidation and superoxide production in the same samp le. We validated the assay in a mitochondrial P450 system determining the fraction of total electron flow that is channeled to oxy-radical f ormation. The assay should be useful in the study of this and other su peroxide/H2O2 generating systems. (C) 1994 Academic Press, Inc.