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Zhou 1997 Anal Biochem

From Bioblast
Publications in the MiPMap
Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP (1997) 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. Anal Biochem 253:162-8.

ยป PMID:9367498 Open access

Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP (1997) Anal Biochem

Abstract: The enzymatic determination of hydrogen peroxide can be accomplished with high sensitivity and specificity using N-acetyl-3, 7-dihydroxyphenoxazine (Amplex Red), a highly sensitive and chemically stable fluorogenic probe for the enzymatic determination of H2O2. Enzyme-catalyzed oxidation of Amplex Red, which is a colorless and nonfluorescent derivative of dihydroresorufin, produces highly fluorescent resorufin, which has an excitation maximum at 563 nm and emission maximum at 587 nm. The 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 fluorescence microplate assay. When applied to the measurement of NADPH oxidase activation, the Amplex Red assay can detect H2O2 release from as few as 2000 phorbol myristate acetate-stimulated neutrophils with a sensitivity 5- to 20-fold greater than that attained in the scopoletin assay under the same experimental conditions. Furthermore, the oxidase-catalyzed 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 assays for the detection of monoamine oxidase and glucose oxidase, this probe is also found to be more sensitive. Given its high sensitivity and specificity, Amplex Red should have a broad application for the measurement of H2O2 in a variety of oxidase-mediated reactions and very low levels of H2O2 in food, environmental waters, and consumer products.

Cited by

  • Komlรณdi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi:10.26124/BEC:2021-0004

Labels: MiParea: Instruments;methods 

Stress:Oxidative stress;RONS 

MitoFit 2021 AmR-O2