Micro method for determination of reactive carbonyl groups in proteins and peptides, using 2,4-dinitrophenylhydrazine

Fields, Robert; Dixon, H. B. F.

doi:10.1042/bj1210587

Cited by 69 publications

(47 citation statements)

References 8 publications

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“…Microsomal membrane lipids were separated by sonicating for 5 min in either (i) 0.1 m-KCl/0-1 M-Na citrate, (ii) 3-5 M sucrose, (iii) 3-5 M sucrose/glycerol. Mitochondrial suspensions were sonicated in K citrate/ dithiothreitol/glycerol for 30 sec. Qualitative and quantitative estimations of acetaldehyde were made by (i) Rimini-Simon reaction, (ii) Fields & Dixon (1971) 2:4 dinitrophenyl hydrazine, and (iii) gas-liquid chromatography employing a 2 m column containing Chromosorb 102, 100-120 mesh at 1100 C with a flow rate of 60 ml./min. Acetaldehyde is retained for 4 min.…”

Section: Experimental Methods and Reagentsmentioning

confidence: 99%

An alkyl etherase in rat liver.

Peters¹,

Shorthouse²

1975

The Journal of Physiology

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SUMMARY1. Diethyl ether, which is known to be partly metabolized in vivo, has been found to show an 02 uptake with the rat liver microsomal membranes; a similar reaction is given with other short chain aliphatic ethers, isopropyl and n-butyl ether.2. The 'etherase' reaction is optimal at pH 7-2-7-4 and is not accompanied by an increased formation of malondialdehyde.3. When CoA is added to the microsomes together with a source of oxaloacetate and the condensing enzyme synthase, the etherase present forms citrate from diethyl ether, indicating an acetylation of CoA, which then enters the citric acid cycle.4. Similarly to 3, fluorocitrate is formed from methyl fluoroethyl ether.5. Differing from plasmalogens, a tetrahydropteridine does not have to be added as a co-factor.

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Section: Experimental Methods and Reagentsmentioning

confidence: 99%

An alkyl etherase in rat liver.

Peters¹,

Shorthouse²

1975

The Journal of Physiology

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“…Red cell ghost preparation 30,36 and red cell membrane carbonyl group determination were carried out as previously reported…”

Section: Methodsmentioning

confidence: 99%

“…Red cell ghost preparation 30,36 and red cell membrane carbonyl group determination were carried out as previously reported 36 (Online Supplementary Appendix). FOXO-PRDX2 alignment analysis is described in the Online Supplementary Appendix, along with details of the statistical analysis carried out.…”

Section: In Vitro Erythropoiesis From Cd34mentioning

confidence: 99%

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Resveratrol accelerates erythroid maturation by activation of FoxO3 and ameliorates anemia in beta-thalassemic mice

Franco

Falco²,

Ghaffari

et al. 2013

Haematologica

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© F e r r a t a S t o r t i F o u n d a t i o ncules might represent complementary therapeutic strategies to counteract the toxic effects of ROS in β-thalassemia. However, few of them have been shown to beneficially affect in vivo β-thalassemic red cell features and/or thalassemic ineffective erythropoiesis in vivo. 16,21 MethodsIn vitro erythropoiesis from CD34 + cells from peripheral circulation of normal and β-thalassemia-intermedia subjects Regarding cell culture, phenotypic analysis and cell sorting strategy, peripheral blood from adult normal volunteers and from transfusion independent β-thalassemia patients (β-thalassemia intermedia) was collected, after obtaining informed consent according to the guidelines established by the Ethic Committee for human subject studies of the University of Milan and the principles of the Declaration of Helsinki. Approval by the Ethic Committee of the University of Milan for human erythroid precursors studies was obtained. We analyzed 20 erythroid cultures from the peripheral blood of different normal subjects and 20 erythroid cultures from 10 homozygous β-thalassemic intermedia patients (β 0cod39 ). 11,17 Details on cell cultures are reported in the Online Supplementary Appendix. The Resveratrol concentration (5 mM; Sigma Aldrich, St. Louis, MO, USA) used in this study was selected from dose-response studies (Online Supplementary Figure 1SA) and a review of the literature. 3,5,6,[8][9][10]22 The erythroid cell antigen profile and the sorting of erythroid precursors were carried out as reported by Merry-Weather Clarke et al. 23 Details are reported in the Online Supplementary Appendix and Figure S1B).Quantitative real-time PCR was carried out as previously described. 24 Details are reported in the Online Supplementary Appendix. The primers used are listed in Online Supplementary Tables S1 and S2.For immunoblot-analysis of sorted human erythroid precursors and immunofluorescence assay for FoxO3a, 1 x 10 6 sorted CFU-E cells from both normal and β-thalassemic were solubilized as previously described. 11,20 Details on immunoblot and immunofluorescence analysis are reported in the Online Supplementary Appendix. Whenever indicated, sorted CFU-Es were separated into cytosol and nuclear fractions as previously reported. 25 The study was carried out in accordance with the Scientific Committee for Animal Experimentation (CIRSAL, University of Verona, Italy). C57B6/2J mice, wild-type controls (WT) and Hbb th3/+ mice were used as β-thalassemia models. Age-and sexmatched 2-month old mice (weight 20 g) were studied. The female/male ratio in the different groups was 1:1. Based on previous studies on resveratrol bioavailability in vivo, [26][27][28] the mice were placed either on resveratrol (2.5 mg/kg incorporated into AIN-93G diet) or standard diet (AIN-93G diet). The mice were fed with resveratrol diet for six months. Hematologic parameters and red cell indices were determined as previously reported. 16,[29][30][31][32] For cytofluorimetric analysis of mouse bone-marrow p...

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“…The content of carbonyl groups in oxidatively modified proteins was measured by determining the amount of 2,4-dinitrophenylhydrazone formed upon reaction with DNPH (8). After precipitation of nucleic acids with 1% streptomycin sulfate, samples (Ͼ1 mg of protein per ml) were treated with 2 mM DNPH at room temperature, usually for 1 h. In some experiments, a 15-min derivatization time was used, which gave results indistinguishable from those obtained with the longer incubation (14).…”

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confidence: 99%

Homeostatic regulation of intracellular hydrogen peroxide concentration in aerobically growing Escherichia coli

1997

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The exponential phase of aerobic growth is associated with risk of endogenous oxidative stress in which cells need to cope with an ϳ10-fold increase in the rate of H 2 O 2 generation. We addressed this issue by studying the regulation of the intracellular concentration of H 2 O 2 in aerobically growing Escherichia coli. Intracellular H 2 O 2 was kept at an almost constant steady-state value of ϳ0.2 M (variation, less than twofold) over a broad range of cell densities in rich medium. This regulation was achieved in part by a transient increase in the OxyRdependent transcription of the catalase gene katG (monitored by using a katG::lacZ operon fusion) during exponential growth, directly correlated with the increased rate of H 2 O 2 generation. The OxyR-regulated alkyl hydroperoxide reductase encoded by ahpFC did not detectably affect H 2 O 2 or catalase activity levels. Induction of katG, ahpFC, and perhaps other genes prevented the accumulation of oxidatively modified lipids but may not have protected DNA: the spontaneous mutation rate was significantly increased in both wild-type and ⌬oxyR strains during exponential growth compared to that in these strains during lag or stationary phases. Strains lacking oxyR showed throughout growth an 8-to 10-fold-higher frequency of spontaneous mutation than was seen for wild-type bacteria. The ahp⌬5 allele also had a mutator effect half of that of ⌬oxyR in exponential and stationary phases and equal to that of ⌬oxyR in lag phase, perhaps by affecting organic peroxide levels. These results show that oxyR-regulated catalase expression is not solely an emergency response of E. coli to environmental oxidative stress, but also that it mediates a homeostatic regulation of the H 2 O 2 produced by normal aerobic metabolism. The activation of the oxyR regulon in this process occurs at much lower levels of H 2 O 2 (ϳ10 ؊7 M) than those reported for oxyR activation by exogenous H 2 O 2 (ϳ10 ؊5 M).

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Micro method for determination of reactive carbonyl groups in proteins and peptides, using 2,4-dinitrophenylhydrazine

Cited by 69 publications

References 8 publications

An alkyl etherase in rat liver.

An alkyl etherase in rat liver.

Resveratrol accelerates erythroid maturation by activation of FoxO3 and ameliorates anemia in beta-thalassemic mice

Homeostatic regulation of intracellular hydrogen peroxide concentration in aerobically growing Escherichia coli

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