Figure 4.

Optimizing detection of α-ketoglutarate. A, in total, 2 mg/ml of PHD3 was quenched in increasing concentrations of trichloroacetic acid (TCA) followed by a bicinchoninic acid (BCA) assay to verify efficacy of TCA protein precipitation. Normalized to No PHD3 control (n.s. with Student's t test, p < 0.05, n = 3). B, in vitro hydroxylation assays were run with increasing concentrations of ascorbic acid to determine the concentration of ascorbic acid that produces the highest signal-to-noise ratio. Absorbance was read at T = 30 min (n.s. with Student's t test, p < 0.05, n = 3). C, final 2,4-DNPH α-KG assay schematic. The enzyme reaction was first prepared with everything except the enzyme and commenced by adding the PHD enzyme. The in vitro hydroxylation assay was allowed to proceed for 30 min before quenching with TCA to a final concentration of 5%. In total, 100 μl of the supernatant was then transferred to a 96-well plate, to which 100 μl of 50 mM 2,4-DNPH was added (final concentration 25 mM) and incubated for 20 min. Thereafter, 50 μl of 10 M NaOH (final concentration 2 M) was added and left for 5 min before reading at 425 nm. Image created with BioRender.com. α-KG, α-ketoglutarate; TCA, trichloroacetic acid.