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

Abstract

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-14]O-2 release from labeled [C-14]alpha-ketoglutarate. Here, we report an alternative rapid, cost-effective assay in which the consumption of a-ketoglutarate is monitored by its derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by treatment with concentrated base. We extensively optimized this 2,4-DNPH alpha-ketoglutarate assay to maximize the signal-to-noise ratio and demonstrated that it is robust enough to obtain kinetic parameters of the well-characterized PHD2 isoform comparable with those in published literature. We further showed that it is also sensitive enough to detect and measure the IC50 values of pan-PHD inhibitors and several PHD2 inhibitors in clinical trials for chronic kidney disease (CKD)induced anemia. Given the efficiency of this assay coupled with its multiwell format, the 2,4-DNPH alpha-KG assay may be adaptable to explore non-HIF substrates of PHDs and potentially to high-throughput assays.