MitoA and MitoN stock solutions (5 mm in ethanol (EtOH)) were stable for at least 6 months at −20 °C. Absorbance spectra were carried out in 1-ml cuvettes in KCl buffer (120 mm KCl, 10 mm Hepes and 1 mm EGTA, pH 7.2 (KOH)) using a Shimadzu UV-2501PC UV-visible spectrophotometer. The molar extinction coefficient for MitoA at 267 nm in KCl buffer was calculated gravimetrically: 20.4 ± 0.1 × 103m −1 cm−1.
Reversed phase-high performance liquid chromatography (RP-HPLC) was done using a Gilson 321 pump with attached UV-visible 151 system (Gilson). Samples were filtered through a 0.22-μm polyvinylidene difluoride (PVDF) filter (Millex, Millipore), loaded, and run over a C18 column (Jupiter 300 Å, Phenomenex) with a Widepore C18 guard column (Phenomenex). HPLC buffer A (0.1% (v/v) trifluoroacetic acid (TFA) in water) and HPLC buffer B (0.1% (v/v) TFA/acetonitrile) were used with the gradient: 0–2 min, 5% B; 2–17 min, 5–100% B; 17–19 min, 100% B; 19–22 min 100–5% B.
Reversed phase-high performance liquid chromatography (RP-HPLC) was done using a Gilson 321 pump with attached UV-visible 151 system (Gilson). Samples were filtered through a 0.22-μm polyvinylidene difluoride (PVDF) filter (Millex, Millipore), loaded, and run over a C18 column (Jupiter 300 Å, Phenomenex) with a Widepore C18 guard column (Phenomenex). HPLC buffer A (0.1% (v/v) trifluoroacetic acid (TFA) in water) and HPLC buffer B (0.1% (v/v) TFA/acetonitrile) were used with the gradient: 0–2 min, 5% B; 2–17 min, 5–100% B; 17–19 min, 100% B; 19–22 min 100–5% B.