2.6. Cuvette-based anaerobic ETF fluorescence reduction assay

Recombinant His-tagged human MCAD, LCAD, and VLCAD proteins were purified from E coli as described (21,22). Substrates were purchased from Sigma (palmitoyl-CoA, stearoyl-CoA) or Toronto Research Chemicals (2,6-dimethylheptanoyl-CoA). For each assay, 700 ul of reaction buffer (50 mM Tris-HCl, pH 8.0, 0.5% w/v glucose) was stoppered into a quartz cuvette and degassed by 10 cycles of argon/vacuum applied by a needle inserted through the rubber stopper. Each cycle consisted of 30 seconds of argon gas and 30 seconds of vacuum. Then, glucose oxidase (Sigma G2133, ~20U/mL final concentration) and catalase suspension (Sigma C30, 0.5 μl/ml final) were added with a Hamilton syringe and the cuvettes kept warm in a 32°C water bath. For each assay, a cuvette was transferred from the water bath into a Jasco FP-6300 spectrofluorometer fitted with a heated cuvette holder (32°C) and fluorescence was zeroed (Ex₃₄₀/Em₄₉₀). Then, enzyme sample (250 ng recombinant ACAD, 200 μg human fibroblast lysate, 40 μg liver mitochondrial lysate, or 15 μg mouse heart homogenate) and pig ETF (2 μM final protein concentration) were added and background fluorescence recorded for one minute. Finally, the reaction was initiated by addition of degassed acyl-CoA substrate at a saturating concentration (25 μM final) and fluorescence was recorded for another minute. The slope and Y-intercept was determined using Jasco Spectra Manager software. Activity in milliunits (mU)— where a mU represents the amount of activity needed to reduce 1 nmole of ETF-bound FAD in 1 minute— is calculated from the following equation:

The nmoles of ETF-bound FAD can be accurately calculated from the A₄₃₆ absorbance peak using the published extinction coefficient of 13,400 (23), or when the preparation is virtually pure as with the recombinant ETF, a final ETF protein concentration of 2 μM in the reaction corresponds to 1 μM of FAD (1 FAD per heterodimer), which is equivalent to 0.75 nmoles of FAD when using a 750 μl reaction volume. The time component in the calculation (60 seconds) is adjusted by the factor of 0.91 to reflect the observation that maximum amount of fluorescence reduction that can be achieved is 91% of the starting fluorescence (10).