Decylubiquinol

Assessment
of complex III activity was
completed spectrophotometrically, modified from the methods of Spinazzi et al.(46 (link)) To a 1 mL cuvette, 50
μL of 0.5 M potassium phosphate buffer (pH 7.5), 75 μL
of 1 mM oxidized cytochrome c, 50 μL of 10
mM KCN, 20 μL of 5 mM EDTA, and 10 μL of 2.5% Tween 20
(all Sigma-Aldrich) were added; ddH₂O was added to 985
μL. To this assay buffer, 20 μg of mitochondrial rich
lysate was added. Next, 5 μL of the desired treatment (1 μM
M1, 2 μM Avo B, 1 μM M1 + 2 μM Avo B, or DMSO) was
added. The cuvette was then placed in a Genesys 10 spectrophotometer
(Fisher Scientific) for a blank reading and absorbance was measured
at 550 nM. After 1 min, 10 μL of 10 mM decylubiquinol (reduced
decylubiquinone, Sigma-Aldrich) was added to the cuvette and absorbance
measurements at 550 nM were taken for another 4 min. The assay was
repeated in triplicate for each treatment.

Mitochondrial complex III activity was assayed as described previously [24] (link). To prepare decylubiquinol, 10 mg decylubiquinone (Sigma, D7911) dissolved in 400 µl of nitrogen-saturated hexane was mixed with 400 µl of 1.15 M sodium dithionite, and vortexed until colorless. The organic phase was collected, and the decylubiquinol was recovered by evaporating the hexane under nitrogen. The decylubiquinol was dissolved in 1 ml ethanol (acidified with 10 mM HCl) and stored in aliquots at −80 °C. Oxidized cytochrome c was from Sigma (C3131). 300 µl reaction mixture (15 µg mitochondrial protein and 60 µM cytochrome c) was added to a cuvette. After the addition of 3 µl decylubiquinol (stock concentration 15 mM, final concentration about 150 µM), reduction of cytochrome c was monitored at 550 nm once every second for 1 min with a SpectraMax M5 Microplate Reader (the chamber temperature was set at 30 °C). The assay was repeated with the addition of 1 µg Antimycin A (Sigma, A8674). Antimycin A-sensitive activity was calculated for the complex III activity. The extinction coefficient of cytochrome c is 21 mM⁻¹ cm⁻¹.

Streptozotocin (STZ), NADH, NAD⁺, ATP, cytochrome c, antimycin A, sodium cyanide, decylubiquinol, n-dodecyl-beta-D-maltoside, sodium succinate, nitro blue tetrazolium (NBT) tablets, EDTA, dimethyl sulfoxide, dichlorophenolindophenol (DCPIP), NADPH, glucose-6-phosphate, and metformin hydrochloride were purchased form Sigma (St. Louis, MO). Lead nitrate, tricine, amino- N- caproic acid, Tris base, and Bis-tris were obtained from VWR International. Serva blue G-250 was purchased from Serva (Heidelberg, Germany). Coomassie brilliant blue G-250, SDS-PAGE protein standard markers, ECL Western blot detection kit, and immunoblotting membrane were purchased from Bio-Rad (Richmond, CA).