Dinitrophenylhydrazine

Manufactured by Merck Group

Sourced in United States, India

Dinitrophenylhydrazine is a chemical reagent commonly used in analytical chemistry and organic synthesis. It is a pale yellow crystalline solid that reacts with carbonyl compounds, such as aldehydes and ketones, to form colored hydrazone derivatives. The primary function of dinitrophenylhydrazine is to detect and identify the presence of carbonyl groups in organic compounds.

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2,4-dinitrophenylhydrazine (119 citations) 2,4-dinitrophenylhydrazine (DNPH), (37 citations) Dinitrophenylhydrazine (DNPH) (5 citations) 2,4-dinitrophenylhydrazine (2,4-DNP), (2 citations) Trichloroacetic acid (TCA) dinitrophenylhydrazine (2 citations) 2,4-dinitrophenylhydrazine (2,4-DNPH), (2 citations)

18 protocols using dinitrophenylhydrazine

Antioxidant and Enzymatic Assays Protocol

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Folin‐Ciocalteu's reagent, thiobarbituric acid, 1,10‐phenanthroline, deoxyribose, gallic acid, trichloroacetic acid (TCA), dinitrophenyl hydrazine (DNPH), 1,1‐diphenyl‐2 picrylhydrazyl (DPPH) radical, 2,2‐azino‐bis(3‐ethyl‐benzothia‐zoline‐6‐sulfonic acid) (ABTS), catechin, epicatechin, quercetin, quercitrin, isoquercitrin, rutin, kaempferol, α‐amylase and α‐glucosidase were sourced from Sigma‐Aldrich, Inc. (St Louis, MO). Methanol, formic acid, gallic acid, chlorogenic acid, caffeic acid and ellagic acids were purchased from Merck (Darmstadt, Germany). All other chemicals and reagents used were of analytical grade and glass‐distilled water was used.

Ademiluyi A.O., Aladeselu O.H., Oboh G, & Boligon A.A. (2018). Drying alters the phenolic constituents, antioxidant properties, α‐amylase, and α‐glucosidase inhibitory properties of Moringa (Moringa oleifera) leaf. Food Science & Nutrition, 6(8), 2123-2133.

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Biochemical Reagents Acquisition Protocol

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Angiotensin II (Ang II), dichloromethane, diallyl disulphide, hydrogen peroxide, 4-nitrophenol, tetramethoxypropane, dinitrophenylhydrazine, methanesulfonic acid, and 1-methyl-2-phenylindole were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used were of the highest purity available.

García-Trejo E.M., Arellano-Buendía A.S., Argüello-García R., Loredo-Mendoza M.L., García-Arroyo F.E., Arellano-Mendoza M.G., Castillo-Hernández M.C., Guevara-Balcázar G., Tapia E., Sánchez-Lozada L.G, & Osorio-Alonso H. (2016). Effects of Allicin on Hypertension and Cardiac Function in Chronic Kidney Disease. Oxidative Medicine and Cellular Longevity, 2016, 3850402.

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Chitosan-Based Biochemical Assay Protocol

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Chitosan, medium molecular weight, deacetylation value of 75–85%, and viscosity of 200–800 cP, was purchased from Aldrich Chemical Co.
Bovine serum albumin, acrylamide, N,N′-methylenebisacrylamide, trichloroacetic acid (TCA), FeSO₄, sulfuric acid, cumene hydroperoxide (CHP), butylhydroxytoluene, m ethanol, xylenol orange, di-nitrophenylhydrazine (DNPH), guanidine, ethanol, ethyl acetate, hydrochloric acid, Coomassie® Brilliant Blue R-250, thioglycolic acid, NBD-Cl, and o-phthalaldehyde (OPA) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).
Thiobarbituric acid was purchased (TBA) from Fluka (Sigma-Aldrich, Toluca, MX); sodium chloride, EDTA disodium salt, N,N′,N′- tetramethylethylenediamine (TEMED), Tris (base), urea, β-mercaptoethanol, glycine, acetic acid glacial, sodium phosphate monobasic, sodium phosphate dibasic, and copper sulfate pentahydrate were purchased from J.T. Baker (Pennsylvania, USA); sodium carbonate and lactic acid were purchased from Fermont (Monterrey, MX); sodium dodecyl sulfate (SDS) and bromophenol blue were obtained from Hycel (Mexico, MX); and plate count agar was purchased from Bioxon, Becton and Dickinson (Mexico, MX). All reagents used were of analytical grade.

Morachis-Valdez A.G., Gómez-Oliván L.M., García-Argueta I., Hernández-Navarro M.D., Díaz-Bandera D, & Dublán-García O. (2017). Effect of Chitosan Edible Coating on the Biochemical and Physical Characteristics of Carp Fillet (Cyprinus carpio) Stored at −18°C. International Journal of Food Science, 2017, 2812483.

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Quantifying Oxidative Protein Damage

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Protein carbonyls were analyzed as described previously (Escobales et al., 2014 (link)). Briefly, an aliquot of perfused or intact heart, or intact liver mitochondrial protein was derivatized with dinitrophenylhydrazine (Sigma–Aldrich) under acid denaturing conditions. Proteins were separated by SDS-PAGE and subjected to western blotting with anti-dinitrophenyl primary antibodies (Sigma–Aldrich) at 1:1000 dilution followed by by IRDye^®(LI-COR Biosciences) secondary antibodies. Bands were visualized using ODYSSEY^®CLx (LI-COR Biosciences) infrared scanner. In order to correct for non-specific binding of antibodies, separate aliquots of the mitochondrial proteins that had been acid-denatured but not treated with dinitrophenylhydrazine were run in parallel.

Parodi-Rullán R.M., Chapa-Dubocq X., Rullán P.J., Jang S, & Javadov S. (2017). High Sensitivity of SIRT3 Deficient Hearts to Ischemia-Reperfusion Is Associated with Mitochondrial Abnormalities. Frontiers in Pharmacology, 8, 275.

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Protein Oxidation Determination via Carbonyl Assay

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The oxidative damage to proteins was assessed by the determination of carbonyl groups based on the reaction with dinitrophenylhydrazine (Sigma-Aldrich, Saint Louis, MO, USA) as previously described (Levine et al., 1990 (link)). Briefly, proteins were precipitated by the addition of 20 % trichloroacetic acid and redissolved in dinitrophenylhydrazine, with absorbance being read at 370 nm. The results were expressed as protein carbonyls per mg of protein.

Abelaira H.M., Veron D.C., de Moura A.B., Carlessi A.S., Borba L.A., Botelho M.E., Andrade N.M., Martinello N.S., Zabot G.C., Joaquim L., Biehl E., Bonfante S., Budni J., Petronilho F., Quevedo J, & Réus G.Z. (2021). Sex differences on the behavior and oxidative stress after ketamine treatment in adult rats subjected to early life stress. Brain research bulletin, 172, 129-138.

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Oxidative Stress Biomarker Analysis

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Horse heart myoglobin, 30% H₂O₂,
3,3’,5,5’-tetramethylbenzidine (TMB),
2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS),
(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox C),
caffeic acid, resveratrol, L-tyrosine, nitro-tyrosine, chloro-tyrosine,
indole-3-carbinol, saliciylic acid, levodopa (L-DOPA), dopamine, lipoic acid,
methanol, NaOCl, acetaldehyde, oleic acid, linoleic acid, dinitrophenylhydrazine
(DNPH), horseradish peroxidase, sodium cyanide, tris(2-carboxyethyl)phosphine
(TCEP), sodium hydrosulfite, 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic
acid (HEPES), NaF, sodium cyanide (NaCN), leupeptin, aprotinin, pepstatin,
phenylmethylsulfonyl fluoride (PMSF), reduced β-Nicotinamide adenine
dinucleotide (NADH), Triton X-100, catalase, and bovine glutathione peroxidase
(GPx) were purchased from Sigma Aldrich Corporation (St. Louis, MO). Ascorbic
acid and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were from ICN
Biomedicals Inc. (Aurora, OH). Perchloric acid was from Fisher Scientific
(Waltham, MA). Reduced β-Nicotinamide adenine dinucleotide phosphate
(NADPH) was from Enzo Life Sciences (Farmingdale, NY). Dihydrolipoic acid was
from Calbiochem (San Diego, CA). Ethanol was from Pharmco-AAPER (Shelbyville,
KY). Sodium azide was from Acros (New Jersey, USA). Ethyl acetate was from
BioQuip (Rancho Dominguez, CA).

Mannino M.H., Patel R.S., Eccardt A.M., Perez Magnelli R.A., Robinson C.L., Janowiak B.E., Warren D.E, & Fisher J.S. (2019). Myoglobin as a versatile peroxidase: Implications for a more important role for vertebrate striated muscle in antioxidant defense. Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology, 234, 9-17.

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Comprehensive Food Analysis Protocols

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Different chemicals, reagents and enzymes of food or analytical grade were supplied by standard firms and used during the study. The media used during microbiological assays such as violet-red bile agar, potato-dextrose agar, and total plate agar and the enzymes used during digestion simulation viz. pepsin (1000 NF U/mg) and pancreatin (protease activity of ≥75 USP U/mg, amylase activity of ≥75 USP U/mg and lipase activity of ≥6 USP U/mg) were bought from the HI-Media Company (India, Mumbai). The chemicals such as dinitrophenylhydrazine (DNPH), aluminium chloride (AlCl₃), 1,1,3,3 tetra-ethoxypropane, ABTS (2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), quercetin, Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), Folin-Ciocalteu's phenol reagent, DPPH (2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl), silver nitrate, and gallic acid were purchased from Sigma-Aldrich (Bangalore, India).

Singh S., Bhat H.F., Kumar S., Aadil R.M., Abdi G, & Bhat Z.F. (2024). Insect protein-based composite film incorporated with E. purpurea-based nanoparticles augmented the storage stability of parmesan cheese. Heliyon, 10(7), e29036.

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Assay for Oxidative Stress Markers

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(R)-α-Lipoic acid, myoglobin, nitro blue tetrazolium (NBT), hydroxylamine hydrochloride, ferrozine, dinitrophenylhydrazine (DNPH), guanidine hydrochloride, ethyl acetate, ethanol, trichloroacetic acid (TCA), dimethyl sulfoxide (DMSO), fructose, 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB), L-cysteine and aminoguanidine (AG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fructosamine and iron standards were obtained from PM Separations (Capalaba DC, QLD, Australia). All other chemicals and reagents used were of analytical grade.

Ghelani H., Razmovski-Naumovski V., Pragada R.R, & Nammi S. (2018). (R)-α-Lipoic acid inhibits fructose-induced myoglobin fructation and the formation of advanced glycation end products (AGEs) in vitro. BMC Complementary and Alternative Medicine, 18, 13.

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Evaluation of Antioxidant Properties

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All solvents and chemicals were of analytical grade. Nutrient Broth (NB), Nutrient Agar (NA), Mueller Hinton Broth (MHB), Mueller Hinton Agar (MHA), McFarland standard were purchased from Oxoid (Basingstone, UK). 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), dinitrophenylhydrazine (DNPH), trichloroacetic acid (TCA), perchloric acid (PCA), thiobarbituric acid (TBA), 1,1,3,3-tetramethoxypropane (TEP), guanidine hydrochloride, ortho-phthalaldehyde (OPA), reduced glutathione (GSH), phosphoric acid, potassium dihydrogen phosphate (KH₂PO₄), hydrochloric acid (HCl), streptozotocin (STZ), ethanol, ethyl acetate, and methanol from Sigma-Aldrich (St. Louis, MO, USA). All HPLC analytical standards, including protocatechuic acid, syringic acid, rutin, ellagic acid, cynaroside, daidzein, neo chlorogenic acid, chlorogenic acid, vitexin, trans p-coumaric acid, trans-sinapic acid, trans ferulic acid, rosmarinic acid, resveratrol, apigenin, myricetin, quercetin, and kaempferol, were bought from Sigma-Aldrich (St. Louis, MO, USA). Phosphate buffer saline (PBS) was bought from VWR (Radnor, PA, USA).

Pozzo L., Russo R., Frassinetti S., Vizzarri F., Árvay J., Vornoli A., Casamassima D., Palazzo M., Della Croce C.M, & Longo V. (2019). Wild Italian Prunus spinosa L. Fruit Exerts In Vitro Antimicrobial Activity and Protects Against In Vitro and In Vivo Oxidative Stress. Foods, 9(1), 5.

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Comprehensive Biochemical Assay Protocol

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Glutathione (GSH), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), trichloroacetic acid, malondialdehyde, 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, 2,4,6-tripyridyl-s-triazine, ferric chloride hexahydrate (FeCl₃.6H₂O), D-mannitol, thiobarbituric acid, 3-(N-morpholino) propane sulfonic acid, fatty acid-free bovine serum albumin fraction V, coomassie brilliant blue, Rhodamine123, dinitrophenylhydrazine (DNPH), dithiothreitol (DTT), ethylene glycol-bis(β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA), Ethylenediaminetetraacetic acid (EDTA), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), tramadol, bovine serum albumin (BSA), and sucrose were obtained from Sigma (Sigma-Aldrich, St. Louis, MO). Kits for assessing biomarkers of renal injury were obtained from Parsazmoon® (Tehran, Iran). High-performance liquid chromatography (HPLC) grade methanol, potassium chloride (KCl), 3-(N-morpholino) propanesulfonic acid (MOPS), iodoacetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), acetonitrile HPLC grade, meta-phosphoric acid, dinitro fluoro benzene, n-butanol, and 2-amino-2-hydroxymethyl-propane-1,3-diol-hydrochloride (Tris-HCl), were purchased from Merck (Darmstadt, Germany).

Mousavi K., Manthari R.K., Najibi A., Jia Z., Ommati M.M, & Heidari R. (2021). Mitochondrial dysfunction and oxidative stress are involved in the mechanism of tramadol-induced renal injury. Current Research in Pharmacology and Drug Discovery, 2, 100049.

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