Derivatization of carbonyl compounds with 2,4-dinitrophenylhydrazine and their subsequent determination by high-performance liquid chromatography

Derivatization of carbonyl compounds with 2,4-dinitrophenylhydrazine (DNPH) is one of the most widely used analytical methods. In this article, we highlight recent advances using DNPH provided by our studies over past seven years. DNPH reacts with carbonyls to form corresponding stable 2,4-DNPhydrazone derivatives (DNPhydrazones). This method may result in analytical error because DNPhydrazones have both E- and Z-stereoisomers caused by the CN double bond. Purified aldehyde-2,4-DNPhydrazone demonstrated only the E-isomer, but under UV irradiation and the addition of acid, both E- and Z-isomers were seen. In order to resolve the isometric problem, a method for transforming the CN double bond of carbonyl-2,4-DNPhydrazone into a C-N single bond, by reductive amination using 2-picoline borane, has been developed. The amination reactions of C1-C10 aldehyde DNPhydrazones are completely converted into the reduced forms and can be analyzed with high-performance liquid chromatography. As a new application using DNPH derivatization, the simultaneous measurement of carbonyls with carboxylic acids or ozone is described in this review.     

Hydrazone formation of 2,4-dinitrophenylhydrazine with pyrroloquinoline quinone in porcine kidney diamine oxidase

Homogeneous diamine oxidase (EC 1.4.3.6) from porcine kidney was treated with the inhibitor 2,4-dinitrophenylhydrazine (DNPH). The coloured compounds formed were detached with pronase and purified to homogeneity. When the reaction with DNPH was conducted under an O2 atmosphere, the product (obtained in a yield of 55%) was the C(5)-hydrazone of pyrroloquinoline quinone (PQQ) and DNPH, as revealed by its chromatographic behaviour, absorption spectrum and 1H-NMR spectrum. Only 6% of this hydrazone was formed under air, the main product isolated being an unidentified reaction product of DNPH with the enzyme. Porcine kidney diamine oxidase is the second mammalian enzyme shown to have PQQ as its prosthetic group. In view of the requirements for hydrazone formation with DNPH, it is incorrect to assume that inhibition of this type of enzymes with common hydrazines is simply due to blocking of the carbonyl group of its cofactor.     

Ratiometric Raman spectroscopy for quantification of protein oxidative damage

A novel ratiometric Raman spectroscopic (RMRS) method has been developed for quantitative determination of protein carbonyl levels. Oxidized bovine serum albumin (BSA) and oxidized lysozyme were used as model proteins to demonstrate this method. The technique involves conjugation of protein carbonyls with dinitrophenyl hydrazine (DNPH), followed by drop coating deposition Raman spectral acquisition (DCDR). The RMRS method is easy to implement because it requires only one conjugation reaction, uses a single spectral acquisition, and does not require sample calibration. Characteristic peaks from both protein and DNPH moieties are obtained in a single spectral acquisition, allowing the protein carbonyl level to be calculated from the peak intensity ratio. Detection sensitivity for the RMRS method is approximately 0.33 pmol carbonyl per measurement. Fluorescence and/or immunoassay-based techniques only detect a signal from the labeling molecule and, thus, yield no structural or quantitative information for the modified protein, whereas the RMRS technique allows protein identification and protein carbonyl quantification in a single experiment.     

2,4-dinitrophenylhydrazine carbonyl assay in metal-catalysed protein glycoxidation

Using an experimental in vitro glycation model, long-term incubations of bovine serum albumin with glucose (fructose) resulted in a significant increase in protein content of 2,4-dinitrophenylhydrazine (DNPH)-reactive carbonyl groups, which could be strongly inhibited by anaerobiosis and metal chelation. The pattern of yields of the protein-bound DNPH was not in accordance with that of the sugar-derived carbonyls determined as the ketoamine Amadori product. In spite of the fact that the contribution of the final advanced glycation end-products to the total DNPH-reactivity of glycation-altered protein remains unclear, the present results stress the need of oxidative steps in formation of most of the DNPH-reactive carbonyl compounds generated by glycation. The results provide evidence that, in protein glycoxidation, the DNPH assay is selective enough to discriminate between protein-bound carbonyls produced by metal-catalysed oxidations and those formed in the early glycation steps.     

2,4-Dinitrophenylhydrazine carbonyl assay in metal-catalysed protein glycoxidation

Abstract

Using an experimental in vitro glycation model, long-term incubations of bovine serum albumin with glucose )fructose) resulted in a significant increase in protein content of 2,4-dinitrophenyl-hydrazine (DNPH)-reactive carbonyl groups, which could be strongly inhibited by anaerobiosis and metal chelation. The pattern of yields of the protein-bound DNPH was not in accordance with that of the sugar-derived carbonyls determined as the ketoamine Amadori product. In spite of the fact that the contribution of the final advanced glycation end-products to the total DNPH-reactivity of glycation-altered protein remains unclear, the present results stress the need of oxidative steps in formation of most of the DNPH-reactive carbonyl compounds generated by glycation. The results provide evidence that, in protein glycoxidation, the DNPH assay is selective enough to discriminate between protein-bound carbonyls produced by metal-catalysed oxidations and those formed in the early glycation steps.     

Quantitation of oxidative damage to tissue proteins.

Active oxygen species are thought to be involved in many physiological and pathological processes and are known to oxidatively modify DNA, lipids and proteins. One such modification is the addition of carbonyl groups to amino acid residues in proteins. The number of carbonyl groups on proteins can be quantitated spectrophotometrically using 2,4-dinitrophenylhydrazine (DNPH). The DNPH assay described in the literature was found to be unreliable in samples containing high amounts of chromophore (e.g. hemoglobin, myoglobin, retinoids). By using an HCl-acetone wash, hemes from the chromophores could be extracted, enabling the determination of carbonyl content to be made even in highly colored tissue extracts. Residual DNPH, which was also found to interfere with the assay, was removed by additional washes with trichloroacetic acid and ethanol-ethylacetate. These improvements are known to remove lipids, do not lengthen the time required to do the assay, permit quantification of carbonyl content in 1-4 mg protein from a variety of tissue types and provide a sensitive and reliable method for assessing oxidative damage to tissue proteins.     

Denuder sampling techniques for the determination of gas-phase carbonyl compounds: a comparison and characterisation of in situ and ex situ derivatisation methods

Two denuder sampling techniques have been compared for the analysis of gaseous carbonyl compounds. One type of denuder was coated with XAD-4 resin and the other type of denuder was coated with XAD-4 and 2,4-dinitrophenylhydrazine (DNPH) to derivatise gaseous carbonyl compounds to their hydrazone forms simultaneously. A detailed protocol for the denuder coating procedure is described. The collection efficiency under dry (RH <3%) and humid conditions (RH 50%) as well as filter positive artefacts were evaluated. The XAD-4/DNPH coated denuders showed significantly less break-through potential and hence collection than the XAD-4-only coated denuders. The performance of the XAD-4/DNPH denuder was better under humid conditions with no detected break-through for hydroxyacetone, methacrolein, methylglyoxal, campholenic aldehyde and nopinone. Calibration experiments were performed in a simulation chamber and carbonyl-hydrazone concentrations determined in the extracts of both the denuder types were related to the mixing ratios of gaseous carbonyl compounds in the chamber to overcome losses and errors associating with the denuder sampling, extraction and sample preparation. The application of on-tube conversion for the XAD-4/DNPH denuders resulted in higher R(2) values than the XAD-4 denuder, ranging up to 0.991 for nopinone. The XAD-4-only coated denuders showed acceptable calibration curves only for lower vapour pressure carbonyl compounds though larger relative standard deviations (RSD) were observed. Carbonyl compounds that were formed during the oxidation of nopinone were collected using the XAD-4/DNPH denuders. The results showed that the denuder sampling device was able to provide reproducible nopinone mixing ratios that remained in the chamber after about 1h of the oxidation. One isomer of oxo-nopinones was tentatively identified from off-line HPLC/(-)ESI-TOFMS analysis. Based on the TOFMS response of the nopinone-DNPH derivative, the oxo-nopinone molar yield of 0.7±0.1% (n=3) was determined from the reaction of nopinone with OH radicals. Depending on target analytes, accuracy and sensitivity requirements, the present method can be employed for the determination of gaseous carbonyl compounds that are formed during the oxidation of monoterpenes.     

Proteomic approaches to identifying carbonylated proteins in brain tissue

Oxidative stress plays a critical role in the pathogenesis of a number of diseases. The carbonyl end products of protein oxidation are among the most commonly measured markers of oxidation in biological samples. Protein carbonyl functional groups may be derivatized with 2,4-dinitrophenylhydrazine (DNPH) to render a stable 2,4-dinitrophenylhydrazone-protein (DNP-protein) and the carbonyl contents of individual proteins then determined by two-dimensional electrophoresis followed by immunoblotting using specific anti-DNP antibodies. Unfortunately, derivatization of proteins with DNPH could affect their mass spectrometry (MS) identification. This problem can be overcome using nontreated samples for protein identification. Nevertheless, derivatization could also affect their mobility, which might be solved by performing the derivatization step after the initial electrophoresis. Here, we compare two-dimensional redox proteome maps of mouse cerebellum acquired by performing the DNPH derivatization step before or after electrophoresis and detect differences in protein patterns. When the same approach is used for protein detection and identification, both methods were found to be useful to identify carbonylated proteins. However, whereas pre-DNPH derivatized proteins were successfully analyzed, high background staining complicated the analysis when the DNPH reaction was performed after transblotting. Comparative data on protein identification using both methods are provided.     

Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins

This work proposes a modification of the 2,4-dinitrophenylhydrazine (DNPH) spectrophotometric assay commonly used to evaluate the concentration of carbonyl groups in oxidized proteins. In this approach NaOH is added to the protein solution after the addition of DNPH, shifting the maximum absorbance wavelength of the derivatized protein from 370 to 450 nm. This reduces the interference of DNPH and allows the direct quantification in the sample solution without the need for the precipitation, washing, and resuspension steps that are carried out in the traditional DNPH method. The two methods were compared under various conditions and are statistically equivalent.     

Fluorimetric screening assay for protein carbonyl evaluation in biological samples

Many assays are available for the detection of protein carbonyls (PCs). Currently, the measurement of PC groups after their derivatization with 2,4-dinitrophenol hydrazine (DNPH) is widely used for measuring protein oxidation in biological samples. However, this method includes several washing steps. In this context, we have developed a rapid, sensitive, and accurate fluorimetric method adapted to 96-well microplates for the convenient assessment of protein carbonyl level in biological samples. The method reported here is based on the reaction of carbonyl content in proteins with 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH) to form highly fluorescent derivatives via hydrazone formation. PCs were determined using the DNPH and NBDH assays in fully reduced bovine serum albumin (BSA) and plasma and liver homogenates obtained from healthy control rats up the addition of various amounts of HOCl-oxidized BSA (OxBSA). Using the NBDH assay, PC concentrations as low as 0.2 nmol/mg were detected with precision as low as 5%. Matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectroscopy was used to successfully identify the formation of the NBDH adducts after derivatization with standard oxidized peptides. Finally, the two methods were further used for PC determination in plasma and liver samples from diabetic and normal rats, showing that the NBDH assay can be reliably used in biological experiments.     

A neutral diphosphate mimic crosslinks the active site of human O-GlcNAc transferase

Glycosyltransferases (Gtfs) catalyze the formation of a diverse array of glycoconjugates. Small-molecule inhibitors to manipulate Gtf activity in cells have long been sought as tools for understanding Gtf function. Success has been limited because of challenges in designing inhibitors that mimic the negatively charged diphosphate substrates. Here we report the mechanism of action of a small molecule that inhibits O-linked N-acetylglucosamine transferase (OGT), an essential human enzyme that modulates cell signaling pathways by catalyzing a unique intracellular post-translational modification, β-O-GlcNAcylation. The molecule contains a five-heteroatom dicarbamate core that functions as a neutral diphosphate mimic. One dicarbamate carbonyl reacts with an essential active site lysine that anchors the diphosphate of the nucleotide-sugar substrate. A nearby cysteine then reacts with the lysine adduct to form a carbonyl crosslink in the OGT active site. Though this unprecedented double-displacement mechanism reflects the unique architecture of the OGT active site, related dicarbamate scaffolds may inhibit other enzymes that bind nucleotide-containing substrates.     

Differential rapid analysis of ascorbic acid and ascorbic acid 2-sulfate by dinitrophenylhydrazine method

Ascorbic acid 2-sulfate (AAS) has recently been detected in animals (1,2), and the suggestion has been made that it is involved in some physiological functions (3–5).AAS has been determined by spectrophotometer measurements at 254 nm, ? = 17,000, after isolation from animal tissues (1,6,7). This procedure, however, is complicated and time consuming. Baker et al. (8) reported a rapid assay for AAS from biological samples based on a dinitrophenylhydrazine (DNPH) method which is a standard method for ascorbic acid (AA) determination in biological materials. In this method, AA is oxidized to the osazone by incubation with DNPH in a dilute sulfric acid solution. According to Baker et al. (8), AAS reacts with DNPH during a 3-hr incubation at 60°C but not during a 1-hr incubation at 37°C, and the difference in the reading at 540 nm between the two temperatures corresponds to AAS.This report is concerned with a more rapid and specific method with DNPH for differential measurement of AA and AAS, that is, the differential oxidation of AA and AAS with 2,6-dinitrophenolindophenol (2,6-Dye) and KBrO₃ and the determination of the osazone produced with the original method by Roe and Kuether (9).     

Cholesterol ozonation products as biomarkers for ozone exposure in rats.

Cholesterol, 1, which is present in both the lung lining fluid and cell membranes of lung tissue, reacts with ozone in aqueous systems to give 3 beta-hydroxy-5-oxo-5,6-secocholestan-6-al (2) as the major product. Reaction of 2 with 2,4-dinitrophenyl hydrazine (DNPH) in aqueous solutions, liposomes or lung extracts affords the anti and syn DNPH derivatives of 2 (3b and 3c) and of the rearrangement product 3,5-dihydroxy-B-norcholestane-6-carboxaldehyde (3a). These derivatives also are detected in lung tissue extracts from rats exposed to 1.3 ppm ozone for 12 hr.     

High sensitivity enzyme-linked immunosorbent assay (ELISA) method for measuring protein carbonyl in samples with low amounts of protein

The oxidative modification of proteins has been shown to play a major role in a number of pathological processes. One such modification is the addition of the carbonyl groups to the amino acid residue in proteins. For the measurement of the carbonyl groups in low concentration protein samples, we have modified the ELISA (enzyme-linked immunosorbent assay) method that was developed by Buss et al. [Buss, I. H; Chan, T. P.; Sluis, K. B.; Domigan, N. M.; Winterbourn, C. C. Protein carbonyl measurement by a sensitive ELISA method. Free Radic. Biol. Med.23:361-366; 1997 ]. In the modified method, protein samples diluted in phosphate-buffered saline were adsorbed to wells of an ELISA plate and then reacted with dinitrophenylhydrazine (DNPH). The protein-conjugated DNPH was probed by a commercial anti-DNPH antibody, and then a second antibody conjugated with horseradish peroxidase was added for quantification. The method was calibrated using oxidized albumin, and required only 5 mug protein. This obviated the need to concentrate protein in experimental and clinical samples with low amounts of protein. In addition the effect of TCA on carbonyl measurement is eliminated. The standard curve was linear in the range of 0-3.36 nmol carbonyls/mg protein, which is the range within which clinical samples fell. The results correlated well with the colorimetric carbonyl assay. The method was used to analyze the amount of protein carbonyl in aqueous humor and diluted plasma samples.     

Shotgun Redox Proteomics: Identification and Quantitation of Carbonylated Proteins in the UVB-Resistant Marine Bacterium,Photobacterium angustum S14

UVB oxidizes proteins through the generation of reactive oxygen species. One consequence of UVB irradiation is carbonylation, the irreversible formation of a carbonyl group on proline, lysine, arginine or threonine residues. In this study, redox proteomics was performed to identify carbonylated proteins in the UVB resistant marine bacterium Photobacterium angustum. Mass-spectrometry was performed with either biotin-labeled or dinitrophenylhydrazide (DNPH) derivatized proteins. The DNPH redox proteomics method enabled the identification of 62 carbonylated proteins (5% of 1221 identified proteins) in cells exposed to UVB or darkness. Eleven carbonylated proteins were quantified and the UVB/dark abundance ratio was determined at both the protein and peptide levels. As a result we determined which functional classes of proteins were carbonylated, which residues were preferentially modified, and what the implications of the carbonylation were for protein function. As the first large scale, shotgun redox proteomics analysis examining carbonylation to be performed on bacteria, our study provides a new level of understanding about the effects of UVB on cellular proteins, and provides a methodology for advancing studies in other biological systems.     

Protein carbonyl formation on mucosal proteins in vitro and in dextran sulfate-induced colitis.

Reactive oxygen and nitrogen species have been implicated as mediators of mucosal injury in inflammatory bowel disease, but few studies have investigated protein oxidation in the inflamed mucosa. In this study, protein carbonyl formation on colonic mucosal proteins from mice was investigated following in vitro exposure of homogenates to iron/ascorbate, hydrogen peroxide, hypochloric acid (HOCl), or nitric oxide (*NO). Total carbonyl content was measured spectrophotometrically by derivatization with dinitrophenylhydrazine (DNPH), and oxidation of component proteins within the tissue was examined by Western blotting for DNPH-derivatized proteins using anti-dinitrophenyl DNP antibodies. These results were compared with protein carbonyl formation found in the acutely inflamed mucosa from mice with colitis induced by dextran sulfate sodium (DSS) administered at 5% w/v in the drinking water for 7 d. In vitro, carbonyl formation was observed after exposure to iron/ascorbate, HOCl and *NO. Iron/ascorbate (20 microM/20 mM) exposure for 5 h increased carbonyl groups by 80%, particularly on proteins of 48, 75-100, 116, 131, and 142 kDa. Oxidation by 0.1 and 0.5 mM HOCl did not increase total carbonyl levels, but Western blotting revealed carbonyl formation on many proteins, particularly in the 49-95 kDa region. After exposure to 1-10 mM HOCl, total carbonyl levels were increased by 0.5 to 12 times control levels with extensive cross-linking and fragmentation of proteins rich in carbonyl groups observed by Western blotting. In mice with acute colitis induced by DSS, protein carbonyl content of the inflamed mucosa was not significantly different from control mucosa, (7.80 +/- 1.05 vs. 8.43 +/- 0.59 nmo/mg protein respectively, p = .16 n = 8, 10); however, Western blotting analysis indicated several proteins of molecular weight 48, 79, 95, and 131 kDa that exhibited increased carbonyl content in the inflamed mucosa. These proteins corresponded to those observed after in vitro oxidation of normal intestinal mucosa with iron/ ascorbate and HOCl, suggesting that both HOCl and metal ions may be involved in protein oxidation in DSS-induced colitis. Identification and further analysis of the mucosal proteins susceptible to carbonyl modification may lead to a better understanding of the contribution of oxidants to the colonic mucosa tissue injury in inflammatory bowel disease.     

Cadmium causes the oxidative modification of proteins in pea plants

In pea (Pisum sativum L.) leaves from plants grown in the presence of 50 µm CdCl₂ the oxidative production of carbonyl groups in proteins, the rate of protein degradation and the proteolytic activity were investigated. In leaf extracts the content of carbonyl groups measured by derivatization with 2,4‐dinitrophenylhydrazine (DNPH), was two‐fold higher in plants treated with Cd than in control plants. The identification of oxidized proteins was carried out by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of proteins derivatized with DNPH and immunochemical detection with an antibody against DNPH. The intensity of the reactive bands was higher in plants exposed to Cd than in controls. By using different antibodies some of the oxidized proteins were identified as Rubisco, glutathione reductase, manganese superoxide dismutase, and catalase. The incubation of leaf crude extracts with increasing H₂O₂ concentrations showed a progressive enhancement in carbonyl content and the pattern of oxidized proteins was similar to that found in Cd‐treated plants. Oxidized proteins were more efficiently degraded, and the proteolytic activity increased 20% due to the metal treatment. In peroxisomes purified from pea leaves a rise in the carbonyl content similar to that obtained in crude extracts from Cd‐treated plants was observed, but the functionality of the peroxisomal membrane was not apparently affected by Cd. Results obtained demonstrate the participation of both oxidative stress, probably mediated by H₂O₂, and proteolytic degradation in the mechanism of Cd toxicity in leaves of pea plants, and they appear to be involved in the Cd‐induced senescence previously reported in these plants.     

Oxidatively Modified Plasma Phospholipids Containing Reactive Carbonyl Functions Measured by HPLC: Evidence for Phosphatidylcholine-Bound Aldehydes in Plasma of Burn Patients

A HPLC method has been developed to measure phosphatidylcholine (PC) containing reactive carbonyl functions in the sn-acyl residue in order to study processes in which such reactive carbonyls can be formed due to e.g. oxidative fragmentation. The method has been applied to determine PC-bound carbonyls as 2, 4-dinitrophenylhydrazones (DNPH) in plasma of burn patients. Plasma from healthy volunteers served as controls. Additionally, in vitro oxidation experiments (A: plasma, buffer diluted; B: plasma + iron-EDTA complex and C: plasma + iron-EDTA complex + H₂O₂) have been performed to obtain and to identify 2, 4-dinitrophenylhydrazine derivatizable carbonyl functions in plasma PC. Both, the PC-aldehydes and PC-aldehyde DNPH derivatives were cleavable with phospholipase C. Quantification was based on thin-layer chromatography purified soybean phosphatidylcholine, which was identically oxidized and derivatized as the plasma lipids in vitro.     

Overexpression of the rhodanese PspE, a single cysteine-containing protein, restores disulphide bond formation to an Escherichia coli strain lacking DsbA

Escherichia coli uses the DsbA/DsbB system for introducing disulphide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulphide bond formation but does not entirely eliminate it. Whether such background disulphide bond forming activity is enzyme-catalysed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulphide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulphide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulphide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulphenic acid form and reacts with substrate proteins to form mixed disulphide adducts. DsbC either prevents the formation of these mixed disulphides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyse disulphide bond formation. Although this PspE/DsbC system is not responsible for the background disulphide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system.     

Modification of hepatic proteins in rats exposed to high oxygen concentration

The effect of high concentrations of oxygen on cellular proteins was examined in hepatocytes isolated from rats exposed to 100% oxygen for 0, 24, 48, or 54 h. Previous studies have demonstrated that protein oxidation mediated by mixed-function oxidase systems is accompanied by the formation of protein carbonyl derivatives that can be detected by the formation of protein hydrazone derivatives on treatment with 2,4-dinitrophenylhydrazine (DNPH). In these studies the levels of DNPH-reactive proteins increased steadily during 48 h of continuous oxygen treatment and then decreased to control levels by 54 h. Although the specific activity of two hepatic enzymes, glutamine synthetase and glucose-6-phosphate dehydrogenase, decreased during oxygen treatment, antibody titration of each enzyme indicated that the levels of immunological cross-reactive protein either remained constant or increased slightly during 48 h of oxygen treatment. After 54 h of oxygen exposure the levels of immunologically cross-reactive material were significantly reduced. These results suggest that exposure of rats to high concentrations of oxygen leads to the oxidative inactivation of enzymes and the accumulation of oxidized proteins that are subsequently degraded.