Quantification of Phospholipids, Proteins, and Q10 in Samples

Total phospholipid contents were determined using an assay for the quantification of phosphate described previously.²⁸ Total protein contents were determined using the amido black method to minimize interference by phospholipids.^{62 (link),63} Total Q₁₀ content was measured using a previously published method with modifications.^{35 (link)} 1−2 μL of PL sample was added to 100 μL of HPLC-grade ethanol, vortexed for ~30 s, and then the Q₁₀ was reduced by addition of 1 mM KBH₄ from a 1 M aqueous stock solution. After 10 min, the precipitated protein was removed by a 2 min spin at 16,300 × g, and samples placed on dry ice until required. For analysis, 50 μL was injected onto a Nucleosil 100−5C18 column attached to an Agilent 1100 series HPLC system equipped with a Thermo Scientific Dionex Ultimate 3000RS Electrochemical Detector (ECD) and eluted in a mobile phase of 70% ethanol, 30% methanol, 0.7% NaClO₄, and 0.07% HClO₄ flowing at 800 μL min⁻¹. Standard ECD potentials of +1000, −500, and +300 mV were used to detect the Q₁₀H₂. The first cell (+1000 mV) conditions the buffer, while the second and third cells allow quantification of Q₁₀ and Q₁₀H₂. Because no Q₁₀ is present (it has been reduced chemically prior to injection), we observe only the Q₁₀H₂ peak detected using the third cell. Mito-CI content and orientation were determined using the NADH:A-PAD⁺ oxidoreduction assay and by comparison to standard samples as described previously,²⁸ in the presence and absence of 20 μg mL⁻¹ alamethicin. Ec-F₁F₀ orientation was determined by measuring ATP hydrolysis as described previously,²⁸ in the presence and absence of 20 μg mL⁻¹ alamethicin. Ec-F₁F₀ contents were calculated by subtracting the mito-CI content from the total protein content.