Development of a colorimetric α-ketoglutarate detection assay for prolyl hydroxylase domain (PHD) proteins
| Autor: | William Yuan, Marcia C. Haigis, Alison E. Ringel, Samantha J. Wong, Joao A. Paulo, Haejin Yoon, Mark A. Currie |
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| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Bcl-2 B-cell lymphoma 2 Proteomics Biochemistry Substrate Specificity Hydroxylation chemistry.chemical_compound Maltose-binding protein NAD nicotinamide adenine dinucleotide α-ketoglutarate-dependent dioxygenases enzyme kinetics ATF4 activating transcription factor-4 GSH glutathione PHD prolyl hydroxylases domain biology AGC automatic gain control Phenylhydrazines HIF hypoxia-inducible factor Hypoxia-inducible factors prolyl hydroxylase NOG N-oxalylglycine ACC2 acetyl-CoA carboxylase 2 TCEP Ketoglutaric Acids Colorimetry Research Article Gene isoform TCEP tris(2-carboxyethyl)phosphine MBP maltose-binding protein GDH glutamate dehydrogenase 2 4-DNPH 2 4-dinitrophenylhydrazine TCA trichloroacetic acid IVH in vitro hydroxylation Hypoxia-Inducible Factor-Proline Dioxygenases in vitro hydroxylation 03 medical and health sciences Humans 2 4-dinitrophenylhydrazine Enzyme kinetics N-Oxalylglycine Molecular Biology Enzyme Assays high-throughput assay DMOG dimethyloxalylglycine 030102 biochemistry & molecular biology CKD chronic kidney disease DDT dithiothreitol Cell Biology PKM2 pyruvate kinase M2 Hypoxia-Inducible Factor 1 alpha Subunit VHL von Hippel–Lindau Kinetics 030104 developmental biology chemistry biology.protein OPD o-phenylenediamine HA hemagglutinin |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| DOI: | 10.1016/j.jbc.2021.100397 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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