FIGURE 3.

Mechanism of Orp1-mediated roGFP2 oxidation. A, dimedone attenuates the response of both roGFP2-Orp1(wt) and roGFP2-Orp1(CS). Reduced fusion proteins were treated with 1 μm H₂O₂ in the presence or absence of 50 mm dimedone. B, roGFP2-Orp1(CS), but not roGFP2-Orp1(wt), is susceptible to overoxidation (overox.). Reduced fusion proteins were exposed to 5 mm H₂O₂ for 30 min, desalted, exposed to DTT (10 mm, 20 min), and desalted again. The fusion proteins (1 μm) were then treated with 10 μm H₂O₂. Untreated fusion proteins served as controls. C, the disulfide form of Orp1 is capable of mediating roGFP2 oxidation. Wild type and mutant Orp1 proteins were oxidized with a 5-fold molar excess of H₂O₂ or blocked with N-ethyl maleimide (NEM) followed by desalting. 1 μm reduced roGFP2 was incubated with 50 μm pretreated Orp1(wt) or Orp1(CS). D, thioredoxin competes thiol-disulfide exchange between roGFP2 and wild type Orp1. roGFP2-Orp1(wt) was exposed to 1 or 10 μm H₂O₂ in the presence or absence of a functional TrxS consisting of Trx1, Trx reductase, and NADPH. In A, B, and D, H₂O₂ was injected after 50 s.