Hydropropidine: A novel, cell-impermeant fluorogenic probe for detecting extracellular superoxide

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• 作者：Radoslaw Michalski^a ; ¹ ; ^{rmichalski@mcw.edu} ; ^{radoslaw.michalski@p.lodz.pl} ; Jacek Zielonka^a ; ^{jzielonk@mcw.edu} ; Micael Hardy^b ; ^{micael.hardy@univ-provence.fr} ; Joy Joseph^a ; ^{jjoseph@mcw.edu} ; Balaraman Kalyanaraman^a ; ^{balarama@mcw.edu}
• 关键词：HPr+ ; hydropropidine ; Pr2+ ; propidium ; 2-OH-Pr2+ ; 2-hydroxypropidium ; HE ; hydroethidine ; NDS ; nitrosodisulfonate radical dianion ; Mito-HE (or MitoSOX Red) ; mitochondria-targeted HE analog ; DPBS ; Dulbecco's PBS ; DPBS-GP ; Dulbecco's PBS supplemented with py
• 刊名：Free Radical Biology and Medicine
• 出版年：2013
• 出版时间：January, 2013
• 年：2013
• 卷：54
• 期：Complete
• 页码：135-147
• 全文大小：1848 K

文摘

Here we report the synthesis and characterization of a membrane-impermeant fluorogenic probe, hydropropidine (HPr⁺), the reduction product of propidium iodide, for detecting extracellular superoxide (O₂^??). HPr⁺ is a positively charged water-soluble analog of hydroethidine (HE), a fluorogenic probe commonly used for monitoring intracellular O₂^??. We hypothesized that the presence of a highly localized positive charge on the nitrogen atom would impede cellular uptake of HPr⁺ and allow for exclusive detection of extracellular O₂^??. Our results indicate that O₂^?? reacts with HPr⁺ (k=1.2¡Á10⁴ M^?1 s^?1) to form exclusively 2-hydroxypropidium (2-OH-Pr²⁺) in cell-free and cell-based systems. This reaction is analogous to the reaction between HE and O₂^?? (Zhao et al., Free Radic. Biol. Med. 34:1359-1368; 2003). During the course of this investigation, we also reassessed the rate constants for the reactions of O₂^?? with HE and its mitochondria targeted analog (Mito-HE or MitoSOX Red) and addressed the discrepancies between the present values and those reported previously by us. Our results indicate that the rate constant between O₂^?? and HPr⁺ is slightly higher than that of HE and O₂^?? and is closer to that of Mito-HE and O₂^??. Similar to HE, HPr⁺ undergoes oxidation in the presence of various oxidants (peroxynitrite-derived radicals, Fenton's reagent, and ferricytochrome c) forming the corresponding propidium dication (Pr²⁺) and the dimeric products (e.g., Pr²⁺-Pr²⁺). In contrast to HE, there was very little intracellular uptake of HPr⁺. We conclude that HPr⁺ is a useful probe for detecting O₂^?? and other one-electron oxidizing species in an extracellular milieu.