FIGURE 1.

Orp1 mediates the oxidation of roGFP2. A, reduced roGFP2 (1 μm) was incubated with either reduced Orp1(wt) or Orp1(CS) (5 μm). H₂O₂ (1 mm) was injected after 50 s. The response of roGFP2 in the absence of Orp1 was used as a negative control. B, schematic representation of roGFP2-Orp1 fusion proteins. C, response of reduced roGFP2-Orp1(wt) to increasing concentrations of H₂O₂ (0.1–10 μm). D, response of reduced roGFP2-Orp1(CS) to increasing concentrations of H₂O₂ (0.1–10 μm). In C and D, dotted horizontal lines indicate the dynamic range as defined by complete reduction (10 mm DTT, lower dotted line, set to 0.1) and oxidation (1 mm diamide, upper dotted line). The ratiometric dynamic range of roGFP2-Orp1(CS) is lower (7-fold rather than 8-fold) because of its susceptibility to overoxidation. In each experiment, the ratio of roGFP2 emissions (510–530 nm) after excitation at 390 and 480 nm was calculated and plotted against time.