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Development of a colorimetric α-ketoglutarate detection assay for prolyl hydroxylase domain (PHD) proteins

J Biol Chem. 2021-02; 
Samantha J Wong, Alison E Ringel, William Yuan, Joao A Paulo, Haejin Yoon, Mark A Currie, Marcia C Haigis
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Mutant Libraries its corresponding P450A mutant (ERIGFALMIKASEGGGGK) (GenScript) were used as substrates for the respective IVH assays. Get A Quote

摘要

Since the discovery of the prolyl hydroxylases domain (PHD) proteins and their canonical hypoxia-inducible factor (HIF) substrate two decades ago, a number of in vitro hydroxylation (IVH) assays for PHD activity have been developed to measure the PHD-HIF interaction. However, most of these assays either require complex proteomics mass spectrometry methods that rely on the specific PHD-HIF interaction or require the handling of radioactive material, as seen in the most commonly used assay measuring [C]O release from labeled [C]α-ketoglutarate. Here, we report an alternative rapid, cost-effective assay in which the consumption of α-ketoglutarate is monitored by its derivatization with 2,4-dinitrophenylhydraz... More

关键词

2,4-dinitrophenylhydrazine, enzyme kinetics, high-throughput assay, in vitro hydroxylation, prolyl hydroxylase, α-ketoglutarate-dependent dioxygenases