Development of a colorimetric α-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 [ 14 C]O 2 release from labeled [ 14 C]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 IC 50 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.

Spectroscopic and in vitro Investigations of Fe2+/α‐Ketoglutarate‐Dependent Enzymes Involved in Nucleic Acid Repair and Modification

“…However, it should be noted that for the Fe 2+ /α‐KG‐dependent oxygenase TfdA (2,4‐dichlorophenoxyacetate/α‐KG dioxygenase) it was reported that addition of dithionite remediated the effects of self‐hydroxylation to some extent [70] . Recently, Haigis and co‐workers have established a colorimetric α‐KG detection assay for the prolyl hydroxylases domain protein family [88] . The α‐KG consumption assay, relying on the derivatization of α‐KG with 2,4‐dinitrophenylhydrazine (2,4‐DNPH, Scheme 5) in the presence of concentrated base (Scheme 4), could also find use for the enzymes described in Table 1.…”

“…[70] Recently, Haigis and co-workers have established a colorimetric α-KG detection assay for the prolyl hydroxylases domain protein family. [88] The α-KG consumption assay, relying on the derivatization of α-KG with 2,4-dinitrophenylhydrazine (2,4-DNPH, Scheme 5) in the presence of concentrated base (Scheme 4), could also find use for the enzymes described in Table 1. Succinate does not form the colored product.…”

Colorimetric Detection of Furfural with Enhanced Visible Absorption of Furfural-DNPH in Basic Conditions

“…The DNPH colorimetry assay was performed following a modified method from previous reports. , 0.5 mM DNPH solution in a mixture of acetonitrile and 2 N HCl (1/1) was freshly prepared. Furfural solutions of 0, 1 (0.01 mM), 5 (0.05 mM), 10 (0.1 mM), 20 (0.2 mM), and 100 ppm (1.0 mM) and formaldehyde at 0, 1 (0.03 mM), 5 (0.17 mM), 10 (0.33 mM), 20 (0.67 mM), and 100 ppm (3.3 mM) were prepared, respectively.…”

“…Meanwhile, colorimetric methods for the in situ detection of aldehydes have been developed with many important applications, including for chemical toxin detection, security screening, food inspection, and disease monitoring, serving as alternatives to traditional analytical methods for rapid and inexpensive determination. − ,− Various methods have been reported to enhance the sensitivity and selectivity of aliphatic and aromatic aldehyde detection, such as fluorescent supramolecular polymers, chemiluminescence (CL) detection of aldehydes derivatized with 2,4-dinitrophenylhydrazine (DNPH), MnO 2 nanosheets, and supramolecular complex. − Generally, the colorimetric detection of aldehydes is based on a nucleophilic addition to a carbonyl group by, for example, an amine such as aniline, DNPH, or 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald), which results in the formation of an imine, which gives a different UV–vis absorption band. However, these methods for the detection of aldehydes are not applicable to furfurals due to the absence of imine in the product.…”

“…In the case of the furfural-DNPH derivative, the extended conjugation likely imparts an orange color to the solution, and in a basic medium, DNPH may undergo deprotonation and the resulting species may have a different color compared to the protonated form. This response amplification in a basic aqueous medium is inspired by the colorimetric detection of α-ketoglutarate with DNPH by forming DNP-hydrazone. , Our method provides a relatively short response time for coloration. Moreover, high linearity between the absorbance and the concentration of furfural enables quantitative detection.…”