FIG. 1.
Purified SuperNova protein generates superoxide in response to light, in vitro. (A) Schematic depicting ∼580 nm light-dependent superoxide (O2˙−) generation by purified SuperNova protein in vitro that is detected with the probe DHE, with HPLC used to separate and measure the superoxide-specific reaction product, 2-OH-E+ that can be prevented by SOD. (B) SuperNova (0.25 mg/mL) induces 2-OH-E+ formation from DHE in response to light (5 min ∼580 nm irradiation, 6.7 mW/mm2), is attenuated in the presence of SOD, occurs in a (C) time-dependent manner (y = 448x + 636; r2 = 0.999; p < 0.001); and is (D) unaffected by buffer pH levels corresponding to either the mitochondrial IMS (pH ∼6.8), cytosol (pH ∼7.3), or mitochondrial matrix (pH ∼7.8), as depicted by wedge shape from left to right. Values are expressed as mean ± SEM for n = 3 independent experiments; *p < 0.05 versus all others; n.s., not significant. 2-OH-E+, 2-hydroxyethidium; DHE, dihydroethidium; HPLC, high-performance liquid chromatography; IMS, intermembrane space; SOD, superoxide dismutase. Color images are available online.