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

作者：Chen Juan; Rogers Steven C; Kavdia Mahendra^*

来源：Annals of Biomedical Engineering, 2013, 41(2): 327-337.

DOI：10.1007/s10439-012-0653-x

• DHE
• Reaction rate constant
• Kinetic analysis
• Oxidative stress
• Cytochrome c.

Superoxide (O-2 (-)) is an important reactive oxygen species (ROS), and has an essential role in physiology and pathophysiology. An accurate detection of O-2 (-) 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 O-2 (-) generation and a O-2 (-) sensitive fluorescent dye dihydroethidium (DHE) for O-2 (-) measurement. To quantify O-2 (-) and DHE interactions, we measured fluorescence using a microplate reader. We conducted a detailed reaction kinetic analysis for DHE-O-2 (-) interaction to understand the effect of O-2 (-) self-dismutation and to quantify DHE-O-2 (-) reaction rate. Fluorescence of DHE and 2-hydroethidium (EOH), a product of DHE and O-2 (-) interaction, were dependent on reaction conditions. Kinetic analysis resulted in a reaction rate constant of 2.169 +/- A 0.059 x 10(3) M-1 s(-1) for DHE-O-2 (-) reaction that is similar to 100x slower than the reported value of 2.6 +/- A 0.6 x 10(5) M-1 s(-1). In addition, the O-2 (-) self-dismutation has significant effect on DHE-O-2 (-) interaction. A slower reaction rate of DHE with O-2 (-) is more reasonable for O-2 (-) measurements. In this manner, the DHE is not competing with superoxide dismutase and NO for O-2 (-). Results suggest that an accurate measurement of O-2 (-) production rate may be difficult due to competitive interference for many factors; however O-2 (-) concentration may be quantified.

• 出版日期2013-2