Determination of H 2 O 2 Generation by pHPA Assay.

Authors :: Larson-Casey JL
Carter AB
Source :: Bio-protocol [Bio Protoc] 2016 Nov 20; Vol. 6 (22).
Publication Year :: 2016
Abstract: The production of reactive oxygen species, including H <subscript>2</subscript> O <subscript>2</subscript> , is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H <subscript>2</subscript> O <subscript>2</subscript> release from cells (1-5). This assay takes advantage of H <subscript>2</subscript> O <subscript>2</subscript> -mediated oxidation of horseradish peroxidase (HRP) to Complex I, which, in turn, oxidizes p-hydroxyphenylacetic acid (pHPA) to a stable, fluorescent pHPA dimer (2,2'-dihydroxy-biphenyl-5,5' diacetate [(pHPA) <subscript>2</subscript> ]). The H <subscript>2</subscript> O <subscript>2</subscript> -dependent HRP-mediated oxidation of pHPA is a sensitive and specific assay for quantifying H <subscript>2</subscript> O <subscript>2</subscript> release from cells. This assay can measure H <subscript>2</subscript> O <subscript>2</subscript> release from whole cells, mitochondria, or the NADPH oxidase.