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

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• 作者：Jacek Zielonka ; Satish Srinivasan ; Micael Hardy ; Olivier Ouari ; Marcos Lopez ; Jeannette Vasquez-Vivar ; Narayan G. Avadhani ; B. Kalyanaraman
• 关键词：Hydroethidine ; Cytochrome c ; Mitochondria ; HPLC ; Superoxide ; Dimerization
• 刊名：Free Radical Biology and Medicine
• 出版年：2008
• 出版时间：1 March 2008
• 年：2008
• 卷：44
• 期：5
• 页码：835-846
• 全文大小：883 K

文摘

Here we report that ferricytochrome c (cyt c³⁺) induces oxidation of hydroethidine (HE) and mitochondria-targeted hydroethidine (Mito-HE or MitoSOX Red) forming highly characteristic homo- and heterodimeric products. Using an HPLC-electrochemical (EC) method, several products were detected from cyt c³⁺-catalyzed oxidation of HE and Mito-HE and characterized by mass spectrometry and NMR techniques as follows: homodimers (HE-HE, E⁺-E⁺, Mito-HE-Mito-HE, and Mito-E⁺-Mito-E⁺) and heterodimers (HE-E⁺ and Mito-HE-Mito-E⁺), as well as the monomeric ethidium (E⁺) and mito-ethidium (Mito-E⁺). Similar products were detected when HE and Mito-HE were incubated with mitochondria. In contrast, mitochondria depleted of cyt c³⁺ were much less effective in oxidizing HE or Mito-HE to corresponding dimeric products. Unlike E⁺ or Mito-E⁺, the dimeric analogs (E⁺-E⁺ and Mito-E⁺-Mito-E⁺) were not fluorescent. Superoxide (O₂^•−) or Fremy's salt reacts with Mito-HE to form a product, 2-hydroxy-mito-ethidium (2-OH-Mito-E⁺) that was detected by HPLC. We conclude that HPLC-EC but not the confocal and fluorescence microscopy is a viable technique for measuring superoxide and cyt c³⁺-dependent oxidation products of HE and Mito-HE in cells. Superoxide detection using HE and Mito-HE could be severely compromised due to their propensity to undergo oxidation.