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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 [14C]O2 release from labeled [14C]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-dinitrophenylhydrazine (2,4-DNPH) followed by treatment with concentrated base. We extensively optimized this 2,4-DNPH α-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 α-KG assay may be adaptable to explore non-HIF substrates of PHDs and potentially to high-throughput assays.
Highlights
Prolyl hydroxylase domain (PHD) proteins are a family of three (PHD1-3) evolutionarily conserved oxygen, iron- and α-ketoglutarate-dependent dioxygenases best known for their role in metazoan oxygen homeostasis [1,2,3]
We describe a new in vitro colorimetric α-ketoglutarate detection assay optimized for monitoring PHD activity by measuring the amount of derivatized residual α-ketoglutarate with 2,4-DNPH
One key feature of 2,4-DNPH α-KG assay is the addition of concentrated sodium hydroxide as a developer reagent, which enhances the sensitivity of the assay by increasing the peak wavelength shift between unreacted 2,4-DNPH and the α-ketoglutarate 2,4-DNP-hydrazone product
Summary
PHD proteins hydroxylate target proteins on specific proline residues, using oxygen and α-ketoglutarate as cosubstrates. We noticed that formation of the hydrazone product shifted the peak absorption wavelength for 2,4-DNPH by only 10 nm, from 360 nm to 370 nm (Fig. S1, C and D). The addition of concentrated sodium hydroxide solution to α-ketoglutarate 2,4-DNP-hydrazone produced a dark red solution with a maximum absorption of 425 nm (Fig. 2, B and C, Fig. S1F). We observed the reverse effect on 2,4-DNPH upon the addition of base, where it caused the peak absorption to shift to a shorter wavelength of 260 nm (Fig. 2D), thereby removing all potential interference of unreacted 2,4-. C, shift in maximum wavelength absorption from 370 nm to 425 nm of 1 mM α-ketoglutarate 2,4-DNP-hydrazone in the presence of 2 M NaOH. 500 μM Ascorbic Acid 150 μM FeSO4 0.5 mM α-ketoglutarate Peptide substrate PHD enzyme
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