Figure 1.

FreSHtracer Is a Reversible and Ratiometric Probe for Glutathione

(A) Changes in the absorption and fluorescence spectra of FreSHtracer when reacted with increasing concentrations of glutathione (GSH).

(B) Fluorescence ratio (F₅₁₀/F₅₈₀; FR) of FreSHtracer plotted as a function of the GSH concentration.

(C) Reversible reaction of FreSHtracer with GSH following treatment with diamide.

(D and E) GSH-specific reaction of FreSHtracer. FreSHtracer equilibrated with GSH (5 mM; 15 min) was treated with H₂O₂ (0–2 mM) (D), and FreSHtracer equilibrated with oxidized GSH (GSSG, 5 mM) was treated with GSH reductase (5 U/mL) and 0.5 mM NADPH (E).

(F and G) Fluorescence properties of FreSHtracer in dialyzed cell lysates. The FR change was monitored in dialyzed HeLa cell lysates (F) and then plotted against the protein concentration (G).

(H) Effect of oxidants on the PSH-induced FR. Dialyzed cell lysates (25 mg/mL protein) were incubated with FreSHtracer (2 hr) and treated with diamide or H₂O₂ for 1 hr.

(I and J) Fluorescence properties of FreSHtracer in PSH-GSH mixtures. FreSHtracer was added to dialyzed cell lysates (25 mg/mL protein) spiked with various GSH concentrations. The FR change was monitored for 20 min (I). The final FR value was plotted against the GSH concentration (J).

(K) Effect of oxidants on the FR of the PSH-GSH mixture. PSH-GSH mixtures (25 mg/mL protein and 2 mM GSH) were incubated with FreSHtracer (2 hr) and treated with diamide or H₂O₂ for 1 hr.

See also Figure S1.