Development of a colorimetric α-ketoglutarate detection assay for prolyl hydroxylase domain (PHD) proteins
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100397
ISSN1083-351X
AutoresSamantha J. Wong, Alison E. Ringel, William Yuan, João A. Paulo, Haejin Yoon, Mark A. Currie, Marcia C. Haigis,
Tópico(s)Mitochondrial Function and Pathology
ResumoSince 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. 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. 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 (1Bruick R.K. McKnight S.L. A conserved family of prolyl-4-hydroxylases that modify HIF.Science. 2001; 294: 1337-1340Crossref PubMed Scopus (1982) Google Scholar, 2Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. Characterization of the human prolyl 4-hydroxylases that modify the hypoxia-inducible factor.J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar, 3Berra E. Benizri E. Ginouvès A. Volmat V. Roux D. Pouysségur J. HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1α in normoxia.EMBO J. 2003; 22: 4082-4090Crossref PubMed Scopus (1003) Google Scholar). PHDs consume molecular oxygen (O2) in a reaction that couples proline hydroxylation to the oxidative decarboxylation of α-ketoglutarate to succinate (Fig. 1A). The hypoxia-inducible factor transcription factor (HIF) was the first PHD substrate identified, which revealed how PHD enzymes play key roles in the cellular response to hypoxia (4Semenza G.L. Nejfelt M.K. Chi S.M. Antonarakis S.E. Hypoxia-inducible nuclear factors bind to an enhancer element located 3′ to the human erythropoietin gene.Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5680-5684Crossref PubMed Scopus (648) Google Scholar). These early studies demonstrated that oxygen availability modulates the catalytic activity of PHDs (1Bruick R.K. McKnight S.L. A conserved family of prolyl-4-hydroxylases that modify HIF.Science. 2001; 294: 1337-1340Crossref PubMed Scopus (1982) Google Scholar, 5Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin J. HIFα targeted for VHL-mediated destruction by proline hydroxylation: Implications for O2 sensing.Science. 2001; 292: 464-468Crossref PubMed Scopus (3572) Google Scholar, 6Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Von Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.Science. 2001; 292: 468-472Crossref PubMed Scopus (4104) Google Scholar, 7Epstein A.C.R. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. et al.C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2380) Google Scholar), since PHD enzymes require molecular oxygen as an obligate cosubstrate. Under normoxia, PHDs hydroxylate two conserved proline residues in the oxygen degradation domains of HIF-1α and HIF-2α. Interestingly, HIF-2α appears to be less active than HIF-1α and has a threefold higher affinity for PHD3 compared with PHD2 (8Smirnova N.A. Hushpulian D.M. Speer R.E. Gaisina I.N. Ratan R.R. Gazaryan I.G. Catalytic mechanism and substrate specificity of HIF prolyl hydroxylases.Biochem. 2012; 77: 1108-1119PubMed Google Scholar). This posttranslational modification facilitates the interaction between HIF and von Hippel–Lindau (VHL), an E3 ubiquitin ligase, which promotes HIF turnover through proteasomal degradation (6Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Von Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.Science. 2001; 292: 468-472Crossref PubMed Scopus (4104) Google Scholar). Oxygen levels under hypoxia are not high enough to sustain PHD activity, and the resulting HIF stabilization induces the expression of an array of HIF-regulated genes involved in angiogenesis, erythropoiesis, and anaerobic metabolism (2Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. Characterization of the human prolyl 4-hydroxylases that modify the hypoxia-inducible factor.J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). Current PHD activity assays can be divided broadly into two categories: substrate-independent (indirect) and substrate-dependent (direct). Substrate-independent in vitro hydroxylation (IVH) assays track concentrations of reactants (α-ketoglutarate, O2) or products (succinate, CO2) of the PHD-catalyzed reaction over time. The most commonly used indirect assay utilizes radioactive [14C]O2 capture, where [14C] α-ketoglutarate is used as a substrate and gaseous radioactive [14C]O2 provides a readout of PHD activity (9Koivunen P. Myllyharju J. Kinetic analysis of HIF prolyl hydroxylases.Methods Mol. Biol. 2018; 1742: 15-25Crossref PubMed Scopus (3) Google Scholar). Other indirect IVH assays rely on the chemical derivatization of α-ketoglutarate by o-phenylenediamine (OPD) to generate a fluorescent derivative, oxygen consumption, and HPLC [14C]–succinic acid fractionation (9Koivunen P. Myllyharju J. Kinetic analysis of HIF prolyl hydroxylases.Methods Mol. Biol. 2018; 1742: 15-25Crossref PubMed Scopus (3) Google Scholar, 10McNeill L.A. Bethge L. Hewitson K.S. Schofield C.J. A fluorescence-based assay for 2-oxoglutarate-dependent oxygenases.Anal. Biochem. 2005; 336: 125-131Crossref PubMed Scopus (43) Google Scholar, 11Ehrismann D. Flashman E. Genn D.N. Mathioudakis N. Hewitson K.S. Ratcliffe P.J. Schofield C.J. Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay.Biochem. J. 2007; 401: 227-234Crossref PubMed Scopus (161) Google Scholar, 12Kanelakis K.C. Palomino H.L. Li L. Wu J. Yan W. Rosen M.D. Rizzolio M.C. Trivedi M. Morton M.F. Yang Y. Venkatesan H. Rabinowitz M.H. Shankley N.P. Barrett T.D. Characterization of a robust enzymatic assay for inhibitors of 2-oxoglutarate-dependent hydroxylases.J. Biomol. Screen. 2009; 14: 627-635Crossref PubMed Scopus (12) Google Scholar). By contrast, direct measures of HIF-1α hydroxylation are substrate-dependent, in that they rely on detecting the posttranslational modification of a proline residue on HIF-1α. Radioactive methods in this class include the [35S]HIF/HA-tagged VHL and the [35S]VHL and biotinylated hyProHIF scintillation proximity pull-down assays (1Bruick R.K. McKnight S.L. A conserved family of prolyl-4-hydroxylases that modify HIF.Science. 2001; 294: 1337-1340Crossref PubMed Scopus (1982) Google Scholar, 6Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Von Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.Science. 2001; 292: 468-472Crossref PubMed Scopus (4104) Google Scholar). Nonradioactive methods include LC/MS and MALDI-TOF proteomics of the HIF-1α substrate, the bead-based AlphaScreen biotin-streptavidin assay, fluorescence polarization assays that measure increased fluorescence of fluorescein-labeled HIF upon VHL binding and the use of (2S,4S)-4-fluoroproline analogs, which release fluoride ions during the PHD catalytic cycle (13Cho H. Park H. Yang E.G. A fluorescence polarization-based interaction assay for hypoxia-inducible factor prolyl hydroxylases.Biochem. Biophys. Res. Commun. 2005; 337: 275-280Crossref PubMed Scopus (25) Google Scholar, 14Hewitson K.S. Schofield C.J. Ratcliffe P.J. Hypoxia-inducible factor prolyl-hydroxylase : Purification and assays of PHD2.Methods Enzymol. 2007; 435: 25-42Crossref PubMed Scopus (40) Google Scholar, 15Tuckerman J.R. Zhao Y. Hewitson K.S. Tian Y.M. Pugh C.W. Ratcliffe P.J. Mole D.R. 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Januszyk M. Brownlee M. Gurtner G.C. The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 13505-13510Crossref PubMed Scopus (276) Google Scholar, 22McMahon G.M. Singh A.K. Prolyl-hydroxylase inhibitors for the treatment of anemia in chronic kidney disease.Curr. Opin. Nephrol. Hypertens. 2019; 28: 600-606Crossref PubMed Scopus (4) Google Scholar). While all three PHD isoforms can hydroxylate and destabilize HIF-1α, PHD2 appears to be the dominant physiological sensor in this oxygen-sensing program (3Berra E. Benizri E. Ginouvès A. Volmat V. Roux D. Pouysségur J. HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1α in normoxia.EMBO J. 2003; 22: 4082-4090Crossref PubMed Scopus (1003) Google Scholar). It is therefore unsurprising that the majority of drug discovery efforts have focused on identifying inhibitors against PHD2, although the structural conservation of the active site suggests that these inhibitors may also inhibit PHD1 and PHD3 (1Bruick R.K. McKnight S.L. A conserved family of prolyl-4-hydroxylases that modify HIF.Science. 2001; 294: 1337-1340Crossref PubMed Scopus (1982) Google Scholar, 23Joharapurkar A.A. Pandya V.B. Patel V.J. Desai R.C. Jain M.R. Prolyl hydroxylase inhibitors: A breakthrough in the therapy of anemia associated with chronic diseases.J. Med. Chem. 2018; 61: 6964-6982Crossref PubMed Scopus (51) Google Scholar). The most widely used high-throughput PHD inhibitor screening assays are substrate-dependent and rely on the specific interaction between hydroxylated HIF-1α and VHL for readouts (1Bruick R.K. McKnight S.L. A conserved family of prolyl-4-hydroxylases that modify HIF.Science. 2001; 294: 1337-1340Crossref PubMed Scopus (1982) Google Scholar, 6Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Von Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.Science. 2001; 292: 468-472Crossref PubMed Scopus (4104) Google Scholar, 13Cho H. Park H. Yang E.G. A fluorescence polarization-based interaction assay for hypoxia-inducible factor prolyl hydroxylases.Biochem. Biophys. Res. Commun. 2005; 337: 275-280Crossref PubMed Scopus (25) Google Scholar, 24Rabinowitz M.H. Inhibition of hypoxia-inducible factor prolyl hydroxylase domain oxygen sensors: Tricking the body into mounting orchestrated survival and repair responses.J. Med. Chem. 2013; 56: 9369-9402Crossref PubMed Scopus (108) Google Scholar, 25Oehme F. Jonghaus W. Narouz-ott L. 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Tian Y.M. Pegg H.B. Figg W.D. Abboud M.I. Heilig R. Fischer R. Myllyharju J. Schofield C.J. Ratcliffe P.J. Lack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates.Elife. 2019; 8: 1-27Crossref Scopus (29) Google Scholar) in 2019 reported a lack of detectable PHD-catalyzed prolyl hydroxylation for non-HIF substrates under specific conditions where robust HIF hydroxylation was observed. This finding highlights the emerging need for facile methods to assess non-HIF substrates as bona fide targets of the PHDs, particularly as reported putative substrates display diverse cellular roles that may translate into significant implications for PHD inhibitor discovery. However, the majority of non-HIF substrates are not known to bind VHL, thereby rendering HIF- and VHL-dependent assays inapplicable. Hence, validation and subsequent characterization rely on HIF-independent enzymatic assays that are ideally also scalable to a high-throughput format for future inhibitor discovery—conditions that the 2,4-DNPH α-KG assay fulfills. In this work, we describe a novel PHD assay based on the reactivity of the universal PHD cosubstrate, α-ketoglutarate, with 2,4-dinitrophenylhydrazine (2,4-DNPH), henceforth referred to as the 2,4-DNPH α-KG assay. In this colorimetric assay, the reaction between α-ketoglutarate and 2,4-DNPH produces a colored derivative, 2,4-dinitrophenylhydrazone (2,4-DNP-hydrazone). We detect 2,4-DNP-hydrazone spectrophotometrically after the addition of base, which shifts the wavelength of maximal absorption away from unreacted 2,4-DNPH. We performed extensive protocol optimization to maximize its signal-to-noise ratio, then validated its use for kinetic analyses using PHD2 and PHD3 with a synthetic 19-mer HIF-1α peptide substrate. The kinetics of proline hydroxylation using this assay are comparable with those determined in other publications. As a key advantage of this assay lies in its amenability to a prospective high-throughput screening format, we first validated its sensitivity in detecting reductions in PHD activity by performing inhibition studies with PHD2 using pan-PHD inhibitors (N-oxalylglycine (NOG) and cobalt (II) chloride), as well as PHD2 inhibitors in clinical trials (Daprodustat, Roxadustat, and Vadadustat). Finally, we probed the potential applications of this assay beyond PHDs by characterizing the activity of glutamate dehydrogenase (GDH) as an example of its use in a non-PHD, α-ketoglutarate-dependent enzyme system. PHD proteins hydroxylate target proteins on specific proline residues, using oxygen and α-ketoglutarate as cosubstrates. Since the PHD reaction oxidatively decarboxylates α-ketoglutarate to succinate and carbon dioxide during this process, we reasoned that the decrease in α-ketoglutarate over time could be used as a proxy measure of PHD enzyme activity (Fig. 1A), which forms the basis of our described method. Since α-ketoglutarate (but not succinate) has a carbonyl functional group, the addition of 2,4-DNPH results in a condensation reaction between α-ketoglutarate and 2,4-DNPH to form a colorimetric product, α-ketoglutarate 2,4-DNP-hydrazone (Fig. 1B). To confirm the identity of the α-ketoglutarate 2,4-DNP-hydrazone product, we reacted increasing concentrations of α-ketoglutarate with a fixed concentration of 2,4-DNPH (and vice versa) and subjected the resulting mixture to LC-MS analysis. As expected, we detected a molecular ion at m/z 325.0, consistent with the α-ketoglutarate 2,4-DNP-hydrazone, which only appeared in the presence of both α-ketoglutarate and 2,4-DNPH at intensities that tracked with increasing concentrations of α-ketoglutarate or 2,4-DNPH (Fig. 1C, Fig. S1, A and B). It is important to note that the addition of 2,4-DNPH to a carbonyl-containing compound typically results in the formation of a yellow precipitate. However, the low concentrations of α-ketoglutarate used in this work produced a clear yellow solution amenable to spectrophotometric characterization (Fig. S1, C and D). Since α-ketoglutarate 2,4-DNP-hydrazone absorbs light in the visible spectrum, the difference in peak absorbance wavelength between unreacted 2,4-DNPH and the hydrazone product has a large impact on assay sensitivity. However, 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). As the difference in peak absorption between 2,4-DNPH and α-ketoglutarate 2,4-DNP-hydrazone is effectively indistinguishable, we reasoned that the addition of a base theoretically could increase the delocalization of electrons in α-ketoglutarate 2,4-DNP-hydrazone by redistributing the pi election system over the two nitrogen groups, the aromatic ring, and the carboxyl group of the hydrazone compound (Fig. 2A), which should shift its peak absorption to longer wavelengths. Indeed, 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). Interestingly, 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-DNPH when measuring absorption of the hydrazone at 425 nm. To optimize the 2,4-DNPH α-KG assay for quantifying PHD kinetic parameters, our next steps aimed to systematically investigate assay conditions that produce the strongest detection signal. We first confirmed that 2,4-DNPH reacts specifically with α-ketoglutarate, but not succinate, and that absorbance with respect to α-ketoglutarate concentration was linear up to 1 mM (Fig. 3A). As published PHD enzyme Km values for α-ketoglutarate range from 1 μM to 60 μM (2Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. Characterization of the human prolyl 4-hydroxylases that modify the hypoxia-inducible factor.J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar, 10McNeill L.A. Bethge L. Hewitson K.S. Schofield C.J. A fluorescence-based assay for 2-oxoglutarate-dependent oxygenases.Anal. Biochem. 2005; 336: 125-131Crossref PubMed Scopus (43) Google Scholar, 11Ehrismann D. Flashman E. Genn D.N. Mathioudakis N. Hewitson K.S. Ratcliffe P.J. Schofield C.J. Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay.Biochem. J. 2007; 401: 227-234Crossref PubMed Scopus (161) Google Scholar, 35Koivunen P. Hirsila M. Remes A.M. Hassinen I.E. Kivirikko K.I. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates possible links between cell metabolism and stabilization of HIF.J. Biol. Chem. 2007; 282: 4524-4532Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), most PHD in vitro hydroxylation (IVH) assays employ between 100 and 500 μM of α-ketoglutarate (14Hewitson K.S. Schofield C.J. Ratcliffe P.J. Hypoxia-inducible factor prolyl-hydroxylase : Purification and assays of PHD2.Methods Enzymol. 2007; 435: 25-42Crossref PubMed Scopus (40) Google Scholar). Thus, the 2,4-DNPH α-KG assay can access substrate concentration ranges suitable for kinetic analysis of PHD enzymes. We then determined the time required for 1 mM of α-ketoglutarate to react with an excess of 2,4-DNPH. We observe that 50 mM of 2,4-DNPH requires at least 10 min of incubation at room temperature to completely react with 1 mM of α-ketoglutarate (Fig. 3B). As the absorption at 370 nm plateaus from 10 min onward, we allocated 20 min for all subsequent derivatization steps to allow for possible variation in derivatization time. Next, we ascertained the molar ratio of 2,4-DNPH to α-ketoglutarate required for complete derivatization of α-ketoglutarate. We found that 25 mM of 2,4-DNPH was required to fully derivatize 1 mM of α-ketoglutarate (Fig. 3C). Our parallel assessment using LC-MS to assess residual α-ketoglutarate levels confirmed this observation, as α-ketoglutarate was fully derivatized with at least 20 mM of 2,4-DNPH (Fig. S2A). We further determined that at least 2 M sodium hydroxide (final concentration) was required for full color development of α-ketoglutarate 2,4-DNP-hydrazone (Fig. 3D). As past reports noted the instability of dinitrophenylhydrazones in alkaline media (36Jones L.A. Holmes J.C. Seligman R.B. Spectrophotometric studies of some 2,4-dinitrophenylhydrazones.Anal. Chem. 1956; 28: 191-198Crossref Scopus (88) Google Scholar), we assessed the signal stability of the hydrazone derivative by tracking the change in absorption over 60 min at room temperature. The spectrophotometric properties of the hydrazone adduct were unstable only at high concentrations (>0.5 mM) (Fig. S2B). Hence, we capped the concentration of α-ketoglutarate at 0.5 mM for all subsequent experiments. As 2,4-DNPH reacts nonspecifically with carbonyl moieties to create background signal that might interfere with our analysis, we sought to optimize the signal-to-noise ratio by accounting for each carbonyl-containing component of the 2,4-DNPH α-KG assay. Indeed, protein carbonylation has been reported as a biomarker of oxidative stress that is detectable by reaction with 2,4-DNPH (37Dalle-Donne I. Rossi R. Giustarini D. Milzani A. Colombo R. Protein carbonyl groups as biomarkers of oxidative stress.Clin. Chim. Acta. 2003; 329: 23-38Crossref PubMed Scopus (1584) Google Scholar, 38Mesquita C.S. Oliveira R. Bento F. Geraldo D. Rodrigues J.V. Marcos J.C. Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins.Anal. Biochem. 2014; 458: 69-71Crossref PubMed Scopus (135) Google Scholar). Hence, the purpose of adding 10% TCA solution at reaction end points is twofold. First, TCA precipitates proteins in the reaction (catalase and MBP-HA-PHD3), which can be removed by subsequent centrifugation (39Koontz L. TCA precipitation.Methods Enzymol. 2014; 541: 3-10Crossref PubMed Scopus (57) Google Scholar). Second, the TCA solution serves as a quenching agent for the 2,4-DNPH α-KG reaction. We found that a final concentration of 5% TCA was sufficient to completely remove the protein in a reaction, as verified by a standard BCA assay (Fig. 4A). Interestingly, we noticed that the efficacy of TCA precipitation started to decrease at higher concentrations of TCA (>25% TCA). Hence, we utilized 5% TCA as a precipitant for subsequent experiments. While most TCA-based protein precipitation methods are carried out at 4 °C (39Koontz L. 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