2600 uv vis recording spectrophotometer

Optical spectra were measured on a Shimadzu 2600 UV/Vis recording spectrophotometer. The concentrations of Q₁ and Q₂ were determined by absorbance spectra from NaBH₄ reduction using a millimolar extinction coefficient ε_{(275nm-290nm)} = 12.25 mM⁻¹cm⁻¹ [30 (link)]. The electron transfer activities of Complexes I-IV from the heart mitochondrial preparation were assayed by our published methods [31 (link)]. The enzymatic activity of aconitase in mitochondria was assayed by measuring the conversion of citrate to α-ketoglutarate in the presence of isocitrate dehydrogenase by the absorbance increase at 340 nm at 37 °C. An appropriate amount of mitochondrial preparation (permeabilized by alamethicin at a 40 µg mitochondrial protein: 1 µg alamethicin ratio) was added to the assay mixture (to a final volume of 1 mL, containing in mM: Tris-HCl 50, cysteine 1, sodium citrate 1, MnCl₂ 0.5, NADP 0.2, pH 7.4), and the reaction was initiated with isocitrate dehydrogenase (2 units/mL).

Optical spectra were measured on a Shimadzu 2600 UV/VIS recording spectrophotometer. The protein concentrations of mitochondrial preparations were determined by the Lowry method using BSA (bovine serum albumin) as a standard. The concentration of Q₁ was determined by absorbance spectra from NaBH₄ reduction using a millimolar extinction coefficient of ε_{(275nm-290nm)} = 12.25 mM⁻¹cm^-1.
Measurements of mitochondrial NADH-linked ^•O₂⁻ production and redox activity of converting cyclic hydroxylamine (CM-H) to stable nitroxide by EPR spin trapping and direct EPR were carried out on a Bruker EMX Micro spectrometer operating at 9.43 GHz with 100 kHz modulation frequency at room temperature by following the established protocol [24 (link)]. The spectral simulations for spin quantitation were performed using the WinSim program developed at NIEHS by Duling [27 (link)].