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Determination of H2O2 Generation by pHPA Assay
Jennifer L Larson-Casey1, A Brent Carter2
1Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA 35294.
Bio-Protocol
|March 11, 2017
Summary
This study details a fluorescence assay to measure hydrogen peroxide (H2O2) release from cells. The method uses horseradish peroxidase (HRP) and p-hydroxyphenylacetic acid (pHPA) for sensitive oxidative stress detection.
Area of Science:
- Biochemistry
- Cell Biology
- Analytical Chemistry
Background:
- Reactive oxygen species, including hydrogen peroxide (H2O2), play critical roles in cellular signaling, immune responses, and cell death.
- Quantifying oxidative stress is essential for understanding cellular function and disease pathogenesis.
Purpose of the Study:
- To present a modified fluorescence-based assay for the sensitive and specific quantification of H2O2 release from biological samples.
- To enable the measurement of H2O2 production in various cellular contexts, including whole cells and subcellular components.
Main Methods:
- The assay utilizes the H2O2-dependent oxidation of horseradish peroxidase (HRP) to Complex I.
- Complex I then catalyzes the oxidation of p-hydroxyphenylacetic acid (pHPA) to a fluorescent dimer, (pHPA)2.
- This fluorescence signal is directly proportional to the amount of H2O2 present.
Main Results:
- The described HRP-mediated oxidation of pHPA provides a sensitive and specific method for H2O2 detection.
- The assay is adaptable for measuring H2O2 release from whole cells, isolated mitochondria, and NADPH oxidase activity.
Conclusions:
- This modified fluorescence assay offers a robust tool for quantifying oxidative stress via H2O2 measurement.
- The technique's versatility allows for application across diverse biological research areas investigating oxidative processes.
