Inhibtion of PHD activity by CuCl₂ in vitro. PHD activity was measured in the prescence of oxygen, 2-oxoglutarate, and ascorbate by the binding of a purified pVHL-elonginB-elonginC complex to a HIF-1α-derived peptide bound onto microtiter plates as described in “Materials and methods.” (A) PHD enzyme-free assays using a nonhydroxylated peptide or 40 nM of a hydroxyproline-containing peptide served as negative and positive controls (black bars labeled with HIF-P₅₆₄ and HIF-P₅₆₄OH, respectively). PHD1 and PHD3 were obtained from insect cells as 6His fusion proteins and PHD2 was expressed in bacteria as MBP fusion protein. Representative experiments performed in triplicate are shown as mean values ± SD. (B) PHD3-dependent HIF-1α Pro₅₆₄ hydroxylation activity was determined in the presence of either 1 or 100 μM FeSO₄. Shown are mean values ± SD of n = 3 independent experiments. OD indicates optical density.