A nonradioactive 96-well plate assay for the detection of hypoxia-inducible factor prolyl hydroxylase activity

The transcriptional activation of hypoxia-inducible genes is essential for the adaptation of mammalian tissues to oxygen deficiency. The hypoxia-inducible transcription factor (HIF) is a cellular switch for the up-regulation of these genes during hypoxia. Under normoxia, HIFs are hydroxylated on conserved prolyl residues by a recently discovered family of HIF prolyl hydroxylases (HIF-PHD1-3). Hydroxylated HIF specifically interacts with the von Hippel–Lindau protein–elongin B–elongin C complex (VBC) which leads to ubiquitination and subsequent proteasomal degradation of HIF. We developed a nonradioactive microtiter plate assay based on the interaction of hydroxylated HIF with VBC which enabled us to detect hydroxylated HIF in the nanomolar concentration range. A biotinylated HIF peptide substrate was bound to a streptavidin-coated microtiter plate and hydroxylated with the HIF-PHD3 isoenzyme. Recombinant VBC complex with a thioredoxin (Trx) tag was purified from Escherichia coli and bound to the hydroxylated HIF peptide. The interaction between VBC and hydroxylated HIF was detected by using an anti-thioredoxin antibody.