V 570 double beam spectrophotometer

The midpoint redox potential (E°) was measured as described^{13 (link)} using 2,6-dichloroindophenol (DCIP, E°′ = 237 mV) as a reference compound, which has an absorption band at 600 nm in its oxidized state. Briefly, 1 mL of 2 mg/mL Cytc solution was added into a cuvette with 50 µL of 1 mM potassium ferricyanide, 2 mL of 50 mM citrate buffer, pH = 6.5, and 0.1 mL of 1 mM DCIP. Absorbance corresponding to fully oxidized Cytc (A₅₅₀-A₅₇₀) and DCIP (A₆₀₀) were recorded using a Jasco V-570 double beam spectrophotometer. Then sequential 1 µL additions of 5 mM ascorbate were used to reduce Cytc, and absorbance values were measured for each step in one-minute intervals. When the readings were constant, a few grains of sodium dithionite (Na₂S₂O₄) were added at the end of the titration to fully reduce Cytc and DCIP. The ratios of oxidized and reduced forms of Cytc and DCIP were plotted as log (DCIP_ox/DCIP_red) versus log (Cytc_ox/Cytc_red), which yielded a linear line with a slope, n_DCIP/n_Cytc, and a y-intercept, n_Cytc/59.2 (E_Cytc-E_DCIP). These values were used to calculate the E°_Cytc with the Nernst equation.

The midpoint redox potential (E^0′) was analyzed spectrophotometrically by the equilibration method [24 ] using 2,6-dichloroindophenol (DCIP, E^0′ = 237 mV) as a reference compound, which has an absorption band at 600 nm in its oxidized state. One hundred μL of Cytc solution (2 mg/mL) were mixed in a spectrophotometric cuvette with 200 μL of 50 mM citrate buffer (pH 6.5), 10 μL of 1 mM DCIP, and 2.5 μL of 1 mM K₃Fe(CN)₆ to fully oxidize Cytc. Absorbance values corresponding to fully oxidized Cytc (A550-A570) and DCIP (A600) were recorded using a Jasco V-570 double beam spectrophotometer. The mixture was then sequentially reduced by adding 0.5 μL of 1 mM ascorbate (pH 6.5), and absorbance values were acquired at each step. When readings became constant, a few grains of Na₂S₂O₄ were added to fully reduce Cytc and DCIP. For each step, ratios of oxidized and reduced forms of both compounds were calculated. Data obtained were plotted as log(DCIP_OX/DCIP_RED) versus log(Cytc_OX/Cytc_RED), yielding a linear graph with a slope of n_DCIP /n_Cytc and a y-axis intercept of n_Cytc/59.2 (E_Cytc − E_DCIP) as previously described [19 (link)]. These values were used to calculate the E^0′ (mV) of Cytc from the Nernst equation.