Simfonia

The EPR spectra of Cu-containing zeolites were recorded as the first derivative of the absorption signal of a EMXplus EPR spectrometer (Bruker, Karlsruhe, Germany) in the X-band (9.4 GHz). A variable temperature unit ER4141VTM was used for temperature variation. The EPR spectra were simulated by the program SimFonia (Bruker) and the quantitative analysis was performed by the licensed program SpinCount (Bruker).

EPR spectra were obtained at X-band and at room temperature on a Bruker EMX-8/2.7 (9.86 GHz) equipped with a high-sensitivity cavity (4119/HS 0205) and a gaussmeter (Bruker, Wissembourg, France). A flat quartz cell (FZKI160–5 × 0.3 mm, Magnettech, Berlin, Germany) was used to measure radical formation. WINEPR and SIMFONIA software (Bruker, Wissembourg, France) were used for data processing and spectrum computer simulation. Typical scanning parameters were: scan rate, 1.2 G/s; scan number, 5; modulation amplitude, 1 G; modulation frequency, 100 kHz; microwave power, 20 mW; sweep width, 100 G; sweep time, 83.88 s; time constant, 40.96 ms; magnetic field 3460–3560 G.
EPR experiments were carried out using the spin-trapping agent DMPO (5,5-dimethyl-1-pyrroline N-oxide). The analyses were performed by adding DMPO (final concentration 50 mM) on 10⁶ CHO cells in PBS buffer. When needed, H₂O contained in the PBS buffer was replaced by D₂0. The illumination was performed directly though the quartz flat cell containing the sample.

X-band EPR spectra were recorded at 295 K on a Bruker EMX spectrometer (Billerica, MA, USA). The instrument parameters were frequency, 9.30 GHz; MW power, 1 or 4 mW; range, 20 G; modulation frequency, 100 kHz; modulation amplitude, 0.1 or 0.2 G; and time constant, 0.17 s.
EPR samples were prepared by diluting 16 mM 1,4-NQ DMSO stocks in PBS buffer (pH 7.4, final 1,4-NQ concentration 4 mM) and reacted with GSH, Cys, or H₂S prepared in PBS buffer. H₂S stock was prepared by dissolving Na₂S in PBS. For hypoxia reactions, 1,4-NQs were diluted into N₂-sparged PBS buffer and the solutions were further sparged with N₂ while H₂S was prepared by dissolving Na₂S into N₂-sparged PBS. Reaction solutions were then transferred into capillary tubes for EPR measurements. To prepare 1,4-NQ semiquinone radical, 4 mM 1,4-NQs were reacted with 0.15–0.25× sodium borohydride (NaBH₄).
The simulation of EPR spectra was conducted using Simfonia (Bruker). The parameters used in the simulation were g = 2.0044 and hyperfine splitting of 3.21 G for protons 2 and 3 and 0.63 G for protons 5, 6, 7, and 8. The parameters for simulating the spectrum of the SH-substituted 1,4-NQ were g = 2.0044 and hyperfine splitting of 0.69 G for protons 5, 6, 7, and 8.

All experiments were performed with hQR2 (20 µg/mL), DMPO (125 mM), the indolone substrates (125 µM), and BNAH (3 mM); for some assays, we added S29434 (5 µM). EPR spectra were acquired with a Bruker EMX-8/2.7 (9.86 GHz) equipped with a high-sensitivity cavity (4119/HS 0205) and a gaussmeter (Bruker, Wissembourg, France), at X-band and at room temperature. For analyses, we used a flat quartz cell, FZKI160-5 × 0.3 mm (Magnettech, Berlin, Germany). We performed data processing and spectrum simulation with WINEPR and SIMFONIA software (Bruker). Scanning parameters were: microwave power, 20 mW; sweep width, 100 G; sweep time, 83.88 s; time constant, 40.96 ms; magnetic field, 3460–3560 G; scan rate, 1.2 G/s; scan number, 5; modulation amplitude, 1 G; and modulation frequency, 100 kHz. We conducted the EPR spectra analysis as reported previously [20 (link)]. Each EPR spectrum comprises the intensity of the peaks on the y-axis in arbitrary units (a.u.), and the magnetic field on the x-axis in gauss (g). The scale of the magnetic field is not included for reasons of simplicity because it is not used in this study. The features used are the signal intensity measured by integrating the area under all peaks and the signal shape with the number of peaks.