4.6. Myoglobin Assay for Quantification of CO

For quantification of CO we used a well-validated method of myoglobin assay. The assay was performed as described by Fayad-Kobeissi [38] with minor changes. Heart myoglobin was dissolved in phosphate-buffered saline (PBS, pH 7.4) at a concentration of 100 µM, converted to deoxymyoglobin (dMb) with sodium dithionite (0.2%) and maintained at 37 °C. PBS saturated with gaseous CO was mixed with fresh PBS giving 60% of CO–saturated buffer; CORM-A1 was added to PBS to produce 30 or 300 µM concentrations; as a control PBS without CO was used. PBS (control) or PBS containing CO (gaseous or delivered by CORM-A1) were mixed with dMB in a ratio of 1:2 (vol:vol), incubated at 37 °C and taken after 1, 2, 5 or 8 min of incubation for recordings of dMb (without CO) and carboxymyoglobin (MbCO) spectra (500–600 nm) using UV-VIS spectrometer (Perkin Elmer) at room temperature. A fraction of carboxymyoglobin [MbCO] of total myoglobin ([MbCO]+[dMb]) was calculated as described by Hasegawa et al. [39].