Pulse radiolysis and steady-state analyses of the reaction between hydroethidine and superoxide and other oxidants

Abstract

Hydroethidine (HE) is a cell-permeable probe used for the intracellular detection of superoxide. Here we report the direct measurement of the rate constant between hydroethidine and superoxide radical anion using the pulse radiolysis technique. This reaction rate constant was calculated to be ca. 2·10 6 M -1 s -1 in water:ethanol (1:1) mixture. The spectral characteristics of the intermediates indicated that the one-electron oxidation product of HE was different from the one-electron reduction product of ethidium (E + ). The HPLC-electrochemical measurements of incubation mixtures containing HE and the oxygenated Fenton's reagent (Fe 2+ /DTPA/H 2 O 2 ) in the presence of aliphatic alcohols or formate as a superoxide generating system revealed 2-OH-E + as a major product. Formation of 2-OH-E + by the Fenton's reagent without additives was shown to be superoxide dismutase-sensitive and we attribute the formation of superoxide radical anion to the one-electron reduction of oxygen by the DTPA-derived radical. Addition of tert-butanol, DMSO, and potassium bromide to the Fenton's system caused inhibition of 2-OH-E + formation. Results indicate that reducing and oxidizing radicals have differential effects on the formation of 2-OH-E + .

Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers

“…Addition of hydroxyl radical scavengers (ethanol or formate) to the Fenton's system containing Mito-HE increased the formation of 2-OH-Mito-E + which was inhibited by the superoxide dismutase, consistent with the notion that hydroxyethyl and carbon dioxide anion radicals formed from ethanol and formate enhanced superoxide generation (not shown). These findings are very similar to those observed with HE [3]. Using large amounts of iron (0.5 mM) and hydrogen peroxide (0.5 mM), investigators claimed that hydroxyl radicals also could oxidize HE to form 2-OH-E + [20].…”

“…The effect of SOD on 2-OH-E + formed under these conditions was not, however, determined [20]. By reacting HE with Fenton's reagent, we detected only low levels of 2-OH-E + (that was inhibited by SOD) formed from the reaction between HE and superoxide radicals [3].…”

“…The solutions were prepared as described previously for HE [3,6,14]. Because of the low solubility of Mito-HE in 0.1 M HClO 4 we used the stock solutions in DMSO.…”

“…The HPLC-EC system for analysis of HE and its oxidation products has been described previously [3,6]. For analysis of mito-HE, and its oxidation products we used the same system (equipped with Synergi Polar RP, Phenomenex column) but using a gradient elution with ratios of A:B mobile phases changing from 35:65 to pure B phase over a period of 30 min followed by 20 min elution using a pure mobile phase B.…”

“…1) is a selective probe for detecting superoxide [1,2]. HE reacts fairly rapidly with (k = 2 × 10 6 M −1 s −1 ) forming a unique red fluorescent marker product, 2-hydroxyethidium cation (2-OH-E + ) [3,4]. Results from these studies dispelled the long-held notion that superoxide reacts with HE to form ethidium (E + ) as a product.…”