[49] Determination of carbonyl content in oxidatively modified proteins
Publisher Summary
This chapter discusses methods to determine carbonyl content in oxidatively modified proteins. The methods described are (1) reduction of the carbonyl group to an alcohol with tritiated borohydride; (2) reaction of the carbonyl group with 2,4-dinitrophenylhydrazine to form the 2,4-dinitrophenylhydrazone; (3) reaction of the carbonyl with fluorescein thiosemicarbazide to form the thiosemicarbazone; and (4) reaction of the carbonyl group with fluorescein amine to form a Schiff base followed by reduction to the secondary amine with cyanoborohydride. Van Poelje and Snell have also quantitated protein-bound pyruvoyl groups through formation of a Schiff base with p-aminobenzoic acid followed by reduction with cyanoborohydride. Although a systematic investigation has not appeared, this method should also be useful in detecting other protein-bound carbonyl groups. Carbonyl content of proteins is expressed as moles carbonyl/mole subunit for purified proteins of known molecular weight. For extracts, the results may be given as nanomoles carbonyl/milligram protein. For a protein having a molecular weight of 50,000, a carbonyl content of 1 mol carbonyl/mol protein corresponds to 20 nmol carbonyl/mg proteins.
References (38)
- E.R. Stadtman
Trends Biol. Sci.
(1986) - K.J.A. Davies
J. Free Radicals Biol. Med.
(1986) - R.L. Levine
J. Biol. Chem.
(1983) - N. Mori et al.
Anal. Biochem.
(1986) - A. Benedetti et al.
Biochim. Biophys. Acta
(1982) - C.N. Oliver
Arch. Biochem. Biophys.
(1987) - C.N. Oliver et al.
J. Biol. Chem.
(1987) - A. Amici et al.
J. Biol. Chem.
(1989) - R.L. Levine
J. Biol. Chem.
(1983) - P.D. van Poelje et al.
Anal. Biochem.
(1987)
Anal. Biochem.
Biochim. Biophys. Acta
Anal. Biochem.
Anal. Biochem.
Arch. Biochem. Biophys.
Biochem. Biophys. Acta
Anal. Biochem.
J. Am. Geriatr. Soc.
Cited by (5223)
Arsenic exposure and oxidative damage to lipid, DNA, and protein among general Chinese adults: A repeated-measures cross-sectional and longitudinal study
2025, Journal of Environmental Sciences (China)Arsenic-related oxidative stress and resultant diseases have attracted global concern, while longitudinal studies are scarce. To assess the relationship between arsenic exposure and systemic oxidative damage, we performed two repeated measures among 5236 observations (4067 participants) in the Wuhan-Zhuhai cohort at the baseline and follow-up after 3 years. Urinary total arsenic, biomarkers of DNA oxidative damage (8-hydroxy-2′-deoxyguanosine (8-OHdG)), lipid peroxidation (8-isoprostaglandin F2alpha (8-isoPGF2α)), and protein oxidative damage (protein carbonyls (PCO)) were detected for all observations. Here we used linear mixed models to estimate the cross-sectional and longitudinal associations between arsenic exposure and oxidative damage. Exposure-response curves were constructed by utilizing the generalized additive mixed models with thin plate regressions. After adjusting for potential confounders, arsenic level was significantly and positively related to the levels of global oxidative damage and their annual increased rates in dose-response manners. In cross-sectional analyses, each 1% increase in arsenic level was associated with a 0.406% (95% confidence interval (CI): 0.379% to 0.433%), 0.360% (0.301% to 0.420%), and 0.079% (0.055% to 0.103%) increase in 8-isoPGF2α, 8-OHdG, and PCO, respectively. More importantly, arsenic was further found to be associated with increased annual change rates of 8-isoPGF2α (β: 0.147; 95% CI: 0.130 to 0.164), 8-OHdG (0.155; 0.118 to 0.192), and PCO (0.050; 0.035 to 0.064) in the longitudinal analyses. Our study suggested that arsenic exposure was not only positively related with global oxidative damage to lipid, DNA, and protein in cross-sectional analyses, but also associated with annual increased rates of these biomarkers in dose-dependent manners.
Antioxidative and anti-inflammatory effects of taxifolin in H<inf>2</inf>O<inf>2</inf>-induced oxidative stress in HTR-8/SVneo trophoblast cell line
2024, Reproductive ToxicologyOxidative stress has been implicated in numerous pregnancy-related disorders. Biologically active plant secondary metabolites, which are present in everyday diet, could prove effective therapeutic agents in preventing these disorders. This study evaluated effects of taxifolin (dihydroquercetin) on ROS production, markers of oxidative damage to lipids and proteins, activity of antioxidant enzymes and production of pro-inflammatory cytokines in H2O2-induced oxidative stress in trophoblast HTR-8/SVneo cells. Taxifolin in 10 µM and 100 µM concentrations attenuated oxidative damage to lipids and proteins, as evidenced by a decrease in MDA content, extracellular LDH activity, carbonyl groups and nitrite contents. A reduction in the activity of antioxidant enzymes SOD, CAT and GPx in cells pre-treated with taxifolin, prior to H2O2 exposure, was also observed, along with a reduction in intracellular ROS production. Both evaluated concentrations of taxifolin showed anti-inflammatory activity in trophoblast cells, by reducing production of pro-inflammatory cytokines IL-1β and IL-6. In this model of H2O2-induced oxidative stress, taxifolin showed marked antioxidative and anti-inflammatory activities in trophoblast cells, adding further evidence of its protective effects and showing potential as a therapeutic agent in preventing adverse pregnancy outcomes.
Transcriptomic insights into vibrio-induced mortality in the clam Meretrix petechialis under high temperature
2024, Comparative Biochemistry and Physiology - Part D: Genomics and ProteomicsIn this study, we investigate the mortality of the clam Meretrix petechialis facing a vibrio challenge under different temperatures and the underlying molecular mechanisms. Our experiment distinctly revealed that clam mortality was predominantly observed under high temperature, highlighting the critical impact of thermal stress on clam susceptibility to infection. Using RNA-seq, we further compared the global transcriptional response to vibrio in clam gills between high and low temperatures. Compared to other groups, the differentially expressed genes in vibrio-challenged group at high temperature associated with immunity, oxidative stress, and membrane transport. Key results show a weakened immune response in clams at high temperature, especially in the TNF signaling pathway, and a decrease in membrane transport efficiency, notably in SLC proteins. Additionally, high temperature enhanced pro-inflammatory related unsaturated fatty acid metabolism, leading to increased oxidative damage. This was further evidenced by our biochemical assays, which showed significantly higher levels of lipid peroxidation and protein carbonylation in clams at high temperature, indicating heightened oxidative damage. RT-PCR validation of selected DEGs corroborated the RNA-seq findings. Our findings contribute to the understanding of more frequent shellfish mortality in summer, emphasizing the role of temperature in pathogen response, elucidating the molecular mechanisms underlying the synergistic effect of pathogen and high temperature stresses. The key genes identified provide potential targets for resistance-assisted breeding. This research has significant implications for bivalve aquaculture and their physiology, particularly in light of global climate changes affecting marine ecosystems.
Effect of levan polysaccharide on chronological aging in the yeast Saccharomyces cerevisiae
2024, International Journal of Biological MacromoleculesLevan is a fructose-based biopolymer with diverse applications in the medicinal, pharmaceutical, and food industries. However, despite its extensive biological and pharmacological actions, including antioxidant, anti-inflammatory, and antidiabetic properties, research on its anti-aging potential is limited. This study explored levan's impact on the chronological lifespan (CLS) of yeast Saccharomyces cerevisiae for the first time. The results show that levan treatment significantly extended the CLS of wild-type (WT) yeast by preventing the accumulation of oxidative stress markers (reactive oxygen species, malondialdehyde, and protein carbonyl content) and ameliorating apoptotic features such as reduced mitochondrial membrane potential, loss of plasma membrane integrity, and externalization of phosphatidylserine. By day 40 of the CLS, a significant increase in yeast viability of 6.8 % (p < 0.01), 11.9 % (p < 0.01), and 20.8 % (p < 0.01) was observed at 0.25, 0.5, and 1 mg/mL of levan concentrations, respectively, compared to control (0 %). This study's results indicate that levan treatment substantially modulates the expression of genes involved in the TORC1/Sch9 pathway. Moreover, levan treatment significantly extended the CLS of yeast antioxidant-deficient mutant sod2Δ and antiapoptotic gene-deficient mutant pep4Δ. Levan also extended the CLS of signaling pathway gene-deficient mutants such as pkh2Δ, rim15Δ, atg1, and ras2Δ, while not affecting the CLS of tor1Δ and sch9Δ.
Helicobacter pylori secretary Proteins-Induced oxidative stress and its role in NLRP3 inflammasome activation
2024, Cellular ImmunologyHelicobacter pylori-associated stomach infection is a leading cause of gastric ulcer and related cancer. H. pylori modulates the functions of infiltrated immune cells to survive the killing by reactive oxygen and nitrogen species (ROS and RNS) produced by these cells. Uncontrolled immune responses further produce excess ROS and RNS which lead to mucosal damage. The persistent oxidative stress is a major cause of gastric cancer. H. pylori regulates nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), nitric oxide synthase 2 (NOS2), and polyamines to control ROS and RNS release through lesser-known mechanisms. ROS and RNS produced by these pathways differentiate macrophages and T cells from protective to inflammatory phenotype. Pathogens-associated molecular patterns (PAMPs) induced ROS activates nuclear oligomerization domain (NOD), leucine rich repeats (LRR) and pyrin domain-containing protein 3 (NLRP3) inflammasome for the release of pro-inflammatory cytokines. This study evaluates the role of H. pylori secreted concentrated proteins (HPSCP) related oxidative stress role in NLRP3 inflammasome activation and macrophage differentiation. To perceive the role of ROS/RNS, THP-1 and AGS cells were treated with 10 μM diphenyleneiodonium (DPI), 50 μM salicyl hydroxamic acid (SHX), 5 μM Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), which are specific inhibitors of NADPH oxidase (NOX), Myeloperoxidase (MPO), and mitochondrial oxidative phosphorylation respectively. Cells were also treated with 10 μM of NOS2 inhibitor l-NMMA and 10 μM of N-acetyl cysteine (NAC), a free radical scavenger·H2O2 (100 μM) treated and untreated cells were used as positive controls and negative control respectively. The expression of gp91phox (NOX2), NOS2, NLRP3, CD86 and CD163 was analyzed through fluorescent microscopy. THP-1 macrophages growth was unaffected whereas the gastric epithelial AGS cells proliferated in response to higher concentration of HPSCP. ROS and myeloperoxidase (MPO) level increased in THP-1 cells and nitric oxide (NO) and lipid peroxidation significantly decreased in AGS cells. gp91phox expression was unchanged, whereas NOS2 and NLRP3 downregulated in response to HPSCP, but increased after inhibition of NO, ROS and MPO in THP-1 cells. HPSCP upregulated the expression of M1 and M2 macrophage markers, CD86 and CD163 respectively, which was decreased after the inhibition of ROS.
This study concludes that there are multiple pathways which are generating ROS during H. pylori infection which further regulates other cellular processes. NO is closely associated with MPO and inhibition of NLRP3 inflammasome. The low levels of NO and MPO regulates gastrointestinal tract homeostasis and overcomes the inflammatory response of NLRP3. The ROS also plays crucial role in macrophage polarization hence alter the immune responses duing H. pylori pathogenesis.
Changes in the blood redox status of horses subjected to combat training
2024, Research in Veterinary ScienceCombat training of police horses, involving physical activity in the presence of environmental stressors, poses a risk of oxidative stress. This study compared the oxidative imbalance after combat training in horses in the regular police service and in horses that had just been schooled. Blood collection was performed immediately after training and after 16 h rest. The activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and total antioxidant status (TAS) were determined as the markers of enzymatic antioxidant defence. At the same time, lipid peroxidation (TBARS) and protein carbonylation (Carb) were assessed as oxidation biomarkers. Additionally, oxidative imbalance indexes such as SOD/CAT, SOD/GPx, TBARS/TAS and TBARS/GPx were calculated. Animals during schooling had significantly lower SOD activity in erythrocytes than those experienced. CAT activity in erythrocytes was insignificantly higher immediately after training than during recovery. The SOD/GPx ratio was higher in experienced animals, which may reflect the intra-erythrocyte imbalance between enzymes producing and degrading hydrogen peroxide towards the first one. The concentration of carbonyl groups was significantly higher after the combat training compared to the recovery period in all horses. In inexperienced animals slight increase in TBARS/TAS and TBARS/GPx indexes were observed during the recovery time after exercises, contrary to experienced horses, in which these markers decreased slightly. These results suggest that the oxidative imbalance in inexperienced horses, although less pronounced just after combat training, was more prolonged as compared to horses in regular service.