Fig. 3. CysOx1 and CysOx2 are reaction-based fluorogenic probes for sulfenic acid.

a Structures of CysOx1 and CysOx2. b, c Intact MS analysis of the reaction products of reduced or sulfenic acid forms of C64,82 S Gpx3 (10 µM) with CysOx1 or CysOx2 (1 mM) after 1 h in 50 mM HEPES pH 7.4. d, e Emission spectra of CysOx1 or CysOx2 alone (10 µM) compared to purified Gpx3-CysOx1 or Gpx3-CysOx2 (10 µM). Reactions with CysOx1 or CysOx2 were excited at 357 nm and 394 nm, respectively. Spectra were recorded in 50 mM HEPES pH 7.4. f, g Emission spectra of CysOx1 or CysOx2 alone (5 µM) or in combination with H₂O₂ (15 µM) or Gpx3 (5 µM) or H₂O₂ (15 µM) and Gpx3 (5 µM). Only the reaction containing all three components gives a fluorogenic product. Reactions with CysOx1 or CysOx2 were excited at 357 and 394 nm, respectively. Spectra were recorded in 50 mM HEPES pH 7.4. Analysis by intact MS indicates that ~75% Gpx3-SOH is labeled by CysOx probes under these conditions. h, i, In-gel fluorescence analysis of reaction products between Gpx3 (10 µM) and CysOx1 or CysOx2 (1 mM) with or without H₂O₂ (15 µM) or TCEP (1 mM) after 1 h in 50 mM HEPES pH 7.4. N = 2 independent experiments for each probe.