1. Academic Validation
  2. Development of a colorimetric α-ketoglutarate detection assay for prolyl hydroxylase domain (PHD) proteins

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

  • J Biol Chem. 2021 Jan-Jun;296:100397. doi: 10.1016/j.jbc.2021.100397.
Samantha J Wong 1 Alison E Ringel 1 William Yuan 2 Joao A Paulo 1 Haejin Yoon 1 Mark A Currie 3 Marcia C Haigis 4
Affiliations

Affiliations

  • 1 Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
  • 2 Department of Biomedical Informatics, Harvard Medical School, Boston, Massachusetts, USA.
  • 3 Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA; Department of Biology, University of Toronto Mississauga, Mississauga, Ontario, Canada; Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada.
  • 4 Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA. Electronic address: marcia_haigis@hms.harvard.edu.
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.

Keywords

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

Figures
Products