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

Mesquita, Cristina S.; Oliveira, Raquel; Bento, M. Fátima; Geraldo, M. Dulce; Rodrigues, João V.; Marcos, João Carlos

doi:10.1016/j.ab.2014.04.034

Cited by 355 publications

(175 citation statements)

References 25 publications

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“…Given that early stage ovarian cancer has a greater five year survival status than advanced presentation, the difficulty lies in the detection of early malignant or premalignant changes in the ovaries [1, 3]. ROS are short lived oxygen-containing derivatives developed as by-products of normal cellular respiration that help to maintain cellular homeostasis along with the body’s innate antioxidant system [1,16]. Oxidative damage is a major factor inducing the adaptive expression of antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and enzymes of the glutathione system which could aid in our understanding of signaling cascades involved in promoting normal ovarian cells to malignant cells [9].…”

Section: Discussionmentioning

confidence: 99%

“…Many studies show continuous release of ROS induce oxidative reactions which damage nucleic acids, proteins, and lipids [19–21]. Further, oxidative stress has been associated with the promotion of many human diseases including various cancers [1, 2, 16, 17, 18]. Specifically, ovarian tumors have known to secrete excessive amounts of ROS leading to increase tumor size [3, 11].…”

Section: Discussionmentioning

confidence: 99%

“…The dinitrophenylhydrazine (DNPH) spectrophotometric assay was used to quantify oxidized proteins within the samples as previously described by Mesquita et al [16] with some modifications. The assay permits detection of carbonyl proteins modified by ROS as an indication of elevated oxidative damage.…”

Section: Methodsmentioning

confidence: 99%

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Reactive Oxygen Species and Serous Epithelial Ovarian Adenocarcinoma

Cohen

Mehrabi

Yao

et al. 2016

CRJ

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Serous ovarian cancer (SOC) is usually diagnosed at late stage and stage-adjusted five year survival rate is low. Mortality is relatively heavy on African-Americans/Black (AA) affected with SOC compared to their Caucasian counterparts, though the cause for the disparity remains unclear. DNA damage induced by oxidative stress has been linked to ovarian cancer, but the role of oxidative stress in distinguishing differences in aggressive SOC tumors among patients is yet to be determined. This study aims to determine the levels of reactive oxygen species (ROS), malondialdehyde (MDA), reactive carbonyl groups and antioxidants in primary SOC normal, precancerous (cystadenoma, borderline) and invasive (III/IV) tissue samples obtained from AA and Caucasian subgroups. Additionally, the study seeks to investigate significant changes in the level of ROS between AA and Caucasian SOC samples. A fluorogenic probe, dichlorodihydrofluorescein (DCFH-DiOxyQ), was used to scavenge reactive oxygen species in SOC normal, precancerous and malignant stages III/IV tissue samples. Malondialdehyde (MDA), a lipid peroxidation marker, and reactive carbonyl groups were measured as indicators of oxidative injury. Moreover, antioxidant status was assessed by estimating glutathione peroxidase 3 (GPX3) enzyme levels. Results indicate ROS concentration was approximately 96% higher in the malignant tissues in comparative to the normal non-diseased controls. In addition, ROS concentration among AA women was approximately 9% higher than Caucasian women. MDA levels increased exponentially from non-disease control and precancerous tissues relative to malignant tissues. Furthermore, malignant serous ovarian samples showed significantly higher reactive carbonyl content compared to the non-disease controls (p=0.009), while GPX3 levels decreased considerably in serous cystadenoma and malignant tissue samples, and non-diseased control compared to borderline disease. The results suggest accumulation of ROS and MDA levels may be a causative factor for SOC. Elevated levels of MDA and reactive carbonyl proteins could override the GPX3 enzyme capacity therefore, initiating serous ovarian neoplasm.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Reactive Oxygen Species and Serous Epithelial Ovarian Adenocarcinoma

Cohen

Mehrabi

Yao

et al. 2016

CRJ

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“…DNPH reacted with ketones or aldehydes to form a derivative with an absorbance peak around 370 nm, similar to the absorbance of free DNPH. The maximum wavelength of the hydrazine derivative was shifted by the addition of 1.2 M NaOH and subsequent 10 minute incubation, as described by Mesquita et al [40]. Carbonyl concentrations were determined using a molar extinction coefficient of 22,308 M −1 cm −1 [40].…”

Section: Methodsmentioning

confidence: 99%

Exposure of fibrinogen and thrombin to nitric oxide donor ProliNONOate affects fibrin clot properties

Helms

Kapadia

Gilmore

et al. 2017

Blood Coagulation &Amp; Fibrinolysis

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Background Fibrin fibers form the structural backbone of blood clots. The structural properties of fibrin clots are highly dependent on formation kinetics. Environmental factors such as protein concentration, pH, salt and protein modification, to name a few, can affect fiber kinetics through altered fibrinopeptide release, monomer association and/or lateral aggregation. Objective To determine the effect of thrombin and fibrinogen exposed to nitric oxide on fibrin clot properties. Methods ProliNONOate (5 μM) was added to fibrinogen and thrombin before clot initiation and immediately following the addition of thrombin to the fibrinogen solution. Resulting fibrin fibers were probed with an atomic force microscope to determine their diameter and extensibility and fibrin clots were analyzed for clot density using confocal microscopy. Fiber diameters were also determined by confocal microscopy and the rate of clot formation was recorded using UV-vis spectrophotometry. Protein oxidation and S-nitrosation was determined by UV-vis, ELISA and chemiluminescence. Results The addition of ProliNONOate to fibrinogen or thrombin resulted a change in clot structure. ProliNONOate exposure produced clots with lower fiber density, thicker fibers and increased time to maximum turbidity. The effect of the exposure of nitric oxide to thrombin and fibrinogen were measured independently and indicated that each plays a role in altering clot properties. We detected thrombin S-nitrosation and protein carbonyl formation after nitric oxide exposure. Conclusions Our study reveals a regulation of fibrin clot properties by nitric oxide exposure and suggests a role of peroxynitrite in oxidative modifications of the proteins. These results relate NO bioavailability and oxidative stress to altered clot properties.

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“…Laccase was first submitted to undergo periodate oxidation and, after the elimination of the remaining periodate through a dialysis step, the oxidized glycoprotein is directly incubated with chitosan macro particles. The buffer favorable conditions and the optimal laccase concentration for the immobilization were investigated, as well as the aldehyde groups formed after the oxidation were qualitatively and quantitatively determined through 2,4‐dinitrophenylhydrazine spectrophotometric assay …”

Section: Introductionmentioning

confidence: 99%

Design of a heterogeneous enzymatic catalyst on chitosan: investigation of the role of conjugation chemistry in the catalytic activity of a Laccase from Trametes versicolor

Apriceno

Girelli

Scuto

2018

J of Chemical Tech & Biotech

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BACKGROUND Three protocols are presented in order to immobilise a laccase (EC 1.10.3.2) from Trametes versicolor on chitosan. In particular, chitosan is functionalized with glutaraldehyde and epichlorohydrin to explore to what extent the conjugation of the enzyme is affected by a carbonyl‐ or epoxy‐modified surface, respectively. In addition, an oxidation procedure is tested to modify, for the first time, the carbohydrate moiety of the enzyme and exploit it for linking with the amine group of chitosan. RESULTS The system in which laccase is directly conjugated to chitosan seems to be the best‐performing since it is able to maintain 100% of initial activity over a 30 day period and at least 50% of the starting activity after 3 catalytic cycles. CONCLUSION The methods show the capability to develop three biocatalysts suitable for the immobilization of laccase. All of them provide covalent bonds that don't inactivate the enzyme through any conformational change and any distortion of the active pocket. Specifically, the oxidation procedure proves to be feasible for the preparation of stabilized glycoproteins without the introduction of foreign molecules, suggesting that the carbohydrate moiety of laccase does not appear to be closely related to the catalytic site of the protein. © 2017 Society of Chemical Industry

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

Cited by 355 publications

References 25 publications

Reactive Oxygen Species and Serous Epithelial Ovarian Adenocarcinoma

Reactive Oxygen Species and Serous Epithelial Ovarian Adenocarcinoma

Exposure of fibrinogen and thrombin to nitric oxide donor ProliNONOate affects fibrin clot properties

Design of a heterogeneous enzymatic catalyst on chitosan: investigation of the role of conjugation chemistry in the catalytic activity of a Laccase from Trametes versicolor

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