The prolyl-4-hydroxylase proteins regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylation of proline residues targeting HIF-1alpha for proteasomal degradation. Using the purified catalytic domain of prolyl hydroxylase 2 (PHD2(181-417)), an enzymatic assay has been developed to test inhibitors of the enzyme in vitro. Because PHD2 hydroxylates HIF-1alpha, with succinic acid produced as an end product, radiolabeled [5-(14)C]-2-oxoglutaric acid was used and formation of [14C]-succinic acid was measured to quantify PHD2(181-417) enzymatic activity. Comparison of the separation of 2-oxoglutaric acid and succinic acid by either ion exchange chromatography or precipitation with phenylhydrazine showed similar results, but the quantification and throughput were vastly increased using the latter method. The PHD2 reaction was substrate and concentration dependent. The addition of iron to the enzyme reaction mix resulted in an increase in enzymatic activity. The Km value for 2-oxoglutaric acid was determined to be 0.9 microM, and known PHD2 inhibitors were used to validate the assay. In addition, the authors demonstrate that this assay can be applied to other 2-oxoglutaric acid-dependent enzymes, including the asparaginyl hydroxylase, factor-inhibiting HIF-1alpha (FIH). A concentration-dependent increase in succinic acid production using recombinant FIH enzyme with a synthetic peptide substrate was observed. The authors conclude that a by-product enzyme assay measuring the conversion of 2-oxoglutaric acid to succinic acid using the catalytic domain of the human PHD2 provides a convenient method for the biochemical evaluation of inhibitors of the 2-oxoglutaric acid-dependent hydroxylases.