NDLI: Analysis of Kinetics of Dihydroethidium Fluorescence with Superoxide Using Xanthine Oxidase and Hypoxanthine Assay

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Analysis of Kinetics of Dihydroethidium Fluorescence with Superoxide Using Xanthine Oxidase and Hypoxanthine Assay

Content Provider	Springer Nature Link
Author	Chen, Juan Rogers, Steven C. Kavdia, Mahendra
Copyright Year	2012
Abstract	Superoxide (O2 −) is an important reactive oxygen species (ROS), and has an essential role in physiology and pathophysiology. An accurate detection of O2 − is needed to better understand numerous vascular pathologies. In this study, we performed a mechanistic study by using the xanthine oxidase (XOD)/hypoxanthine (HX) assay for O2 − generation and a O2 − sensitive fluorescent dye dihydroethidium (DHE) for O2 − measurement. To quantify O2 − and DHE interactions, we measured fluorescence using a microplate reader. We conducted a detailed reaction kinetic analysis for DHE–O2 − interaction to understand the effect of O2 − self-dismutation and to quantify DHE–O2 − reaction rate. Fluorescence of DHE and 2-hydroethidium (EOH), a product of DHE and O2 − interaction, were dependent on reaction conditions. Kinetic analysis resulted in a reaction rate constant of 2.169 ± 0.059 × 103 M−1 s−1 for DHE–O2 − reaction that is ~100× slower than the reported value of 2.6 ± 0.6 × 105 M−1 s−1. In addition, the O2 − self-dismutation has significant effect on DHE–O2 − interaction. A slower reaction rate of DHE with O2 − is more reasonable for O2 − measurements. In this manner, the DHE is not competing with superoxide dismutase and NO for O2 −. Results suggest that an accurate measurement of O2 − production rate may be difficult due to competitive interference for many factors; however O2 − concentration may be quantified.
Starting Page	327
Ending Page	337
Page Count	11
File Format	PDF
ISSN	00906964
Journal	Annals of Biomedical Engineering
Volume Number	41
Issue Number	2
e-ISSN	15739686
Language	English
Publisher	Springer US
Publisher Date	2012-09-11
Publisher Place	Boston
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	DHE Reaction rate constant Kinetic analysis Oxidative stress Cytochrome c Biomedicine general Biomedical Engineering Biophysics and Biological Physics Mechanics Biochemistry
Content Type	Text
Resource Type	Article
Subject	Biomedical Engineering

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Analysis of Kinetics of Dihydroethidium Fluorescence with Superoxide Using Xanthine Oxidase and Hypoxanthine Assay

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