Binding affinity to human P4H expressed in Escherichia coli using dansyl-Gly-phe-pro-Gly-OEt as substrate at 356 mM incubated up to 5 mins followed by substrate addition by competition assay in presence of alpha-ketoglutarate
Irreversible inhibition of human P4H expressed in Escherichia coli using dansyl-Gly-phe-pro-Gly-OEt and alpha-ketoglutarate as substrate incubated up to 15 mins followed by substrate addition by Kitz-Wilson plot analysis
In Vitro Inhibition Assay: In vitro tests for determination of the activity and selectivity of HIF prolyl 4-hydroxylase inhibitors. Hydroxylated HIF bonds specifically to the von Hippel-Lindau protein-elongin B-elongin C complex (VBC complex). This interaction occurs only if HIF is hydroxylated on a conserved prolyl radical. It is the basis for the biochemical determination of HIF prolyl hydroxylase activity. The test is carried out as described [Oehme F., Jonghaus W., Narouz-Ott L., Huetter J., Flamme I., Anal. Biochem. 330 (1), 74-80 (2004)].
Inhibition Assay: Inhibition of the Activity of HIF Prolyl Hydroxylase is carried out as described [Oehme F., Jonghaus W., Narouz-Ott L., Huetter J., Flamme I., Anal. Biochem. 330 (1), 74-80 (2004)].