Enzymatic Assay for Fumarate

The 500 μl assay solution contains 100 mM Tris-HCl (pH 7.5), 500 pmol CmFUM, and 200 mM disodium fumarate. The assay solution before adding fumarate was pre-incubated for 5 min at 52°C. Thereafter, fumarate which was also pre-incubated for 5 min at 52°C was added to the assay solution and the reaction was started at 52°C. After the reaction for 5, 10, 20, 30, 40, 50, and 60 min, 50 μl of the assay solution was collected and the reaction was stopped by 100 mM HCl. The samples were analyzed by high-performance liquid chromatography (HPLC) using an LC-2000Plus System (JASCO, Tokyo, Japan). Organic acids were quantified using 0.2 mM bromothymol blue in 15 mM sodium phosphate buffer; peaks were detected at 445 nm, as described previously (Osanai et al., 2015 (link)).