Fig. 2
In vitro characterization of purified D2HGlo. (A) Diagram of the Citric Acid Cycle, highlighting key intermediates. (B) Selectivity of D2HGlo for D-2-HG compared with seven Citric Acid Cycle intermediates (citrate, isocitrate, α-ketoglutarate, succinate, fumarate, oxaloacetate and malate). In addition, pyruvate, lactate and glutamate were tested. R-Rmin was calculated by subtracting the FRET ratio in the presence of 100 nM metabolite (Rmin) from the FRET ratio in the presence of 100 µM metabolite (R). The average ± standard deviation for each condition is shown for three independent experiments. (C) The influence of pH on in vitro fluorescence measurements. Purified D2HGlo was diluted in three different buffers (pH 6.5, pH 7.4 and pH 8), and the representative binding curves are shown. (D) The impact of temperature on in vitro fluorescence measurements. D2HGlo was incubated at 30ºC or 37ºC in the presence of D-2-HG for 15 min prior to fluorescent measurements and the representative binding curves are shown