*2.2. CO-Release Assay*

All reagents were of analytical grade and purchased from Merck. Gaseous CO was obtained from Rivoira (Milan, Italy). A Shimadzu UV1900 UV-Vis Spectrophotometer from 275 to 700 nm at the scanning rate of 200 nm/min was used to record UV-Vis absorption spectra in a disposable plastic cuvette (path length 0.44 cm). An Origin Lab software generated second derivative spectra, and the Savitzky–Golay method was applied using 25 data points for the differentiation process. Neither an increase nor a decrease in the number of points caused changes in the wavelength or in the bandwidth. Lyophilized horse heart Mb was dissolved in phosphate buffered saline flushed with N2 (PBS, 0.01 M, pH 7.4 to a 20–22 μM final concentration). Two milliliters of this freshly prepared stock solution were placed in a cuvette to record the UV-Vis absorption spectrum of met-Mb. Next, the solutions were divided into two: 10 μL of sodium dithionite (30 mg/mL) were added to the first half (reference) and the UV-Vis spectrum of deoxy-Mb was registered. After that, the solution was flushed with CO gas, and the Mb-CO spectrum was acquired. Sodium dithionite was added to the second half (sample), and a spectrum was recorded. Afterwards, a CORM DMSO solution was added to a final CORM concentration of 3.33 μM and gently mixed. The solution was covered with 300 μL of light mineral oil to avoid CO escaping and oxygenation of Mb, and the absorption spectrum was recorded at t = 0. Spectra were acquired every 30 min for 210 min, keeping the sample at 37 ◦C. When necessary, a freshly prepared sodium dithionite solution was added. After 210 min, the total Mb concentration at the end of the assay was determined by flushing the sample with CO gas. Mb-CO concentration at each time point was determined as previously reported [23]. Each experiment was replicated three times, and the data were expressed as mean ± SEM.
