The 'mitoflash' probe cpYFP does not respond to superoxide/Cheng et al. reply

• Published in: Nature (London) Vol. 514; no. 7523; p. E12
• Main Authors: Schwarzländer, Markus, Wagner, Stephan, Ermakova, Yulia G, Belousov, Vsevolod V, Radi, Rafael, Beckman, Joseph S, Buettner, Garry R, Demaurex, Nicolas, Duchen, Michael R, man, Henry J, Fricker, Mark D, Gems, David, Halestrap, Andrew P, Halliwell, Barry, Jakob, Ursula, Johnston, Iain G, Jones, Nick S, Logan, David C, Morgan, Bruce, Müller, Florian L, Nicholls, David G, Remington, S James, Schumacker, Paul T, Winterbourn, Christine C, Sweetlove, Lee J, Meyer, Andreas J, Dick, Tobias P, Murphy, Michael P, Cheng, Heping, Wang, Wang, Wang, Xianhua, Sheu, Shey-Shing, Dirksen, Robert T, Dong, Meng-Qiu
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 23-10-2014
• Subjects: E coli; Experiments; Free radicals; Medicine; Mitochondria; Oxidizing agents; Physiology; Potash; Potassium; Proteins; Sensors
• ISSN: 0028-0836; 1476-4687

Exposure of cpYFP to a superoxide-generating system (xanthine (X) and xanthine oxidase (XO)) slightly changed the excitation and emission spectra.However, the same change occurred when cpYFP was incubated with the individual assay constituents in the absence of superoxide production, or when Cu/Zn superoxide dismutase (SOD) was added to degrade superoxide (Fig. 1a, b). Extended reductive or oxidative treatment with thiol redox agents (the reducing agent dithiothreitol (DTT)and oxidizing agent 2,2'-dipyridyl disulphide (DPS)) did not alter the spectral behaviour (Fig. 1f-h), consistent with structural information suggesting that both Cys residues are buried inside the mature protein and are unlikely to be accessible for thiol redox chemistry (Fig. 1i).