Journal
FREE RADICAL BIOLOGY AND MEDICINE
Volume 39, Issue 7, Pages 853-863Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.freeradbiomed.2005.05.001
Keywords
hydroethidine; Fremy's salt; nitrosodisulfonate radical dianion; superoxide radical anion; 2-hydroxyethidium; fluorescent probes; stopped flow; HPLC
Funding
- NHLBI NIH HHS [5P01HL68769-01, 5R01HL067244] Funding Source: Medline
- NINDS NIH HHS [2R01NS39958] Funding Source: Medline
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We have previously shown that superoxide radical anion (O-2(center dot-)) reacts with hydroethidine (HE) to form a product that is distinctly different from ethidium (E+) (Zhao et al., Free Radic. Biol Med. 34:1359; 2003). The structure of this product was recently determined as the 2-hydroxyethidium cation (2-OH-E+) (Zhao, et al., Proc. Natl. Acad. Sci. USA 102:5727; 2005). In this study, using HPLC and mass spectrometry techniques, we show that 2-OH-E+ is formed from the reaction between HE and nitrosodisulfonate radical dianion (NDS) or Fremy's salt. The reaction kinetics and mechanism were determined using steady-state and time-resolved optical and EPR techniques. Within the first 50 ins, an intermediate was detected. Another intermediate absorbing strongly at 460 nm and weakly at 670 nm was detected within a second. The structure of this species was assigned to an imino quinone derivative of HE. The stoichiometry of the reaction indicates that two molecules of NDS were needed to oxidize a molecule of HE. We postulate that the first step of the reaction involves the hydrogen atom abstraction from HE to form an aminyl radical that reacts with another molecule of NDS to form an adduct that decomposes to an imino quinone derivative of HE. A similar mechanism has been proposed for the reaction between HE and O2(center dot-). The reaction between HE and the Fremy's salt should provide a facile route for the synthesis of 2-OH-E+, a diagnostic marker product of the HE/O-2(center dot-) reaction. (c) 2005 Elsevier Inc. All rights reserved.
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