Figure 5. Radiochemical assay measuring the relative amount of 4-Hydroxy[³H]proline formed after incubation of reported substrate with PHD enzyme.

Substrates were produced by IVTT in the presence of L-[2,3,4,5-³H]proline and incubated with and without the indicated recombinant PHD enzyme. Conversion to 4-hydroxy[³H]proline was measured by radiochemical assay with data expressed as DPM Hyp/1 × 10⁶ DPM Pro, DPM of the reaction without the PHD being subtracted. Assay efficacy was confirmed with a positive HIF-1α (WT) control. Background DPM range was determined with a negative PP/AG HIF-1α (P402A, P564G proline mutant) control. The hydroxylation level observed in PHD-reacted non-HIF substrates was not above background. Data are from two independent assays with the following (n = 1) exceptions: PKM (PHD3); TELO2 (PHD3); TRPA1 (PHD2).