A STABLE NONFLUORESCENT DERIVATIVE OF RESORUFIN FOR THE FLUOROMETRIC-DETERMINATION OF TRACE HYDROGEN-PEROXIDE - APPLICATIONS IN DETECTING THE ACTIVITY OF PHAGOCYTE NADPH OXIDASE AND OTHER OXIDASES

The enzymatic determination of hydrogen peroxide can be accomplished w ith high sensitivity and specificity using N-acetyl-3,7-dihydroxypheno xazine (Amplex Red), a highly sensitive and chemically stable fluoroge nic probe for the enzymatic determination of H2O2. Enzyme-catalyzed ox idation of Amplex Red, which is a colorless and nonfluorescent derivat ive of dihydroresorufin, produces highly fluorescent resorufin, which has an excitation maximum at 563 nm and emission maximum at 587 nn. Th e reaction stoichiometry of Amplex Red and H2O2 was determined to be 1 :1. This probe allows detection of 5 pmol H2O2 in a 96-well fluorescen ce microplate assay. When applied to the measurement of NADPH oxidase activation, the Amplex Red assay can detect H2O2 release from as few a s 2000 phorbol myristate acetate-stimulated neutrophils with a sensiti vity 5- to 20-fold greater than that attained in the scopoletin assay under the same experimental conditions. Furthermore, the oxidase-catal yzed assay using Amplex Red results in an increase in fluorescence on oxidation rather than a decrease in fluorescence as in the scopoletin assay. In comparison with other fluorometric and spectrophotometric as says for the detection of monoamine oxidase and glucose oxidase, this probe is also found to be more sensitive. Given its high sensitivity a nd specificity, Amplex Red should have a broad application for the mea surement of H2O2 in a variety of oxidase-mediated reactions and very l ow levels of H2O2 in food, environmental waters, and consumer products . (C) 1997 Academic Press.