1,812 research outputs found

    Assembly in vitro of rhodococcus jostii RHA1 encapsulin and peroxidase DypB to form a nano-compartment

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    Rhodococcus jostii RHA1 peroxidase DypB has been recently identified as a bacterial lignin peroxidase. The dypB gene is co-transcribed with a gene encoding an encapsulin protein, shown in Thermotoga maritima to assemble to form a 60-subunit nano-compartment, and DypB contains a C-terminal sequence motif thought to target the protein to the encapsulin nanocompartment. R. jostii RHA1 encapsulin protein has been overexpressed in R. jostii RHA1, and purified as a high molecular weight assembly (Mr >106). The purified nanocompartment can be denatured to form a low molecular weight species by treatment at pH 3.0, and can be re-assembled to form the nanocompartment at pH 7.0. Recombinant DypB can be assembled in vitro with monomeric encapsulin to form an assembly of similar size and shape to the encapsulin-only nanocompartment, assessed by dynamic light scattering. The assembled complex shows enhanced lignin degradation activity per mg DypB present, compared with native DypB, using a nitrated lignin UV-vis assay method. The measured stoichiometry of 8.6 µmoles encapsulin/µmol DypB in the complex is comparable to the value of 10 predicted from the crystal structure

    Clinical relevance of biomarkers of oxidative stress

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    SIGNIFICANCE Oxidative stress is considered to be an important component of various diseases. A vast number of methods have been developed and used in virtually all diseases to measure the extent and nature of oxidative stress, ranging from oxidation of DNA to proteins, lipids, and free amino acids. Recent Advances: An increased understanding of the biology behind diseases and redox biology has led to more specific and sensitive tools to measure oxidative stress markers, which are very diverse and sometimes very low in abundance. CRITICAL ISSUES The literature is very heterogeneous. It is often difficult to draw general conclusions on the significance of oxidative stress biomarkers, as only in a limited proportion of diseases have a range of different biomarkers been used, and different biomarkers have been used to study different diseases. In addition, biomarkers are often measured using nonspecific methods, while specific methodologies are often too sophisticated or laborious for routine clinical use. FUTURE DIRECTIONS Several markers of oxidative stress still represent a viable biomarker opportunity for clinical use. However, positive findings with currently used biomarkers still need to be validated in larger sample sizes and compared with current clinical standards to establish them as clinical diagnostics. It is important to realize that oxidative stress is a nuanced phenomenon that is difficult to characterize, and one biomarker is not necessarily better than others. The vast diversity in oxidative stress between diseases and conditions has to be taken into account when selecting the most appropriate biomarker. Antioxid. Redox Signal. 00, 000-000

    Quantification of nitrotyrosine in nitrated proteins

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    For kinetic studies of protein nitration reactions, we have developed a method for the quantification of nitrotyrosine residues in protein molecules by liquid chromatography coupled to a diode array detector of ultraviolet-visible absorption. Nitrated bovine serum albumin (BSA) and nitrated ovalbumin (OVA) were synthesized and used as standards for the determination of the protein nitration degree (ND), which is defined as the average number of nitrotyrosine residues divided by the total number of tyrosine residues in a protein molecule. The obtained calibration curves of the ratio of chromatographic peak areas of absorbance at 357 and at 280 nm vs. nitration degree are nearly the same for BSA and OVA (relative deviations <5%). They are near-linear at low ND (< 0.1) and can be described by a second-order polynomial fit up to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}ND=0.5(R2>0.99) {\hbox{ND}} = 0.5\left( {{R^2} > 0.99} \right) \end{document}. A change of chromatographic column led to changes in absolute peak areas but not in the peak area ratios and related calibration functions, which confirms the robustness of the analytical method. First results of laboratory experiments confirm that the method is applicable for the investigation of the reaction kinetics of protein nitration. The main advantage over alternative methods is that nitration degrees can be efficiently determined without hydrolysis or digestion of the investigated protein molecules

    Water-soluble organic compounds in air particulate matter analyzed by HPLC-DAD-MS and HPLC-MS/MS: abundance, sources and transformation of carboxylic acids, nitrophenols and nitrated proteins

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    Wasserlösliche organische Verbindungen (WSOCs) sind Hauptbestandteile atmosphärischer Aerosole, die bis zu ~ 50% und mehr der organischen Aerosolfraktion ausmachen. Sie können die optischen Eigenschaften sowie die Hygroskopizität von Aerosolpartikeln und damit deren Auswirkungen auf das Klima beeinflussen. Darüber hinaus können sie zur Toxizität und Allergenität atmosphärischer Aerosole beitragen.In dieser Studie wurde Hochleistungsflüssigchromatographie gekoppelt mit optischen Diodenarraydetektion und Massenspektrometrie ( HPLC-DAD-MS und HPLC-MS/MS) angewandt, um WSOCs zu analysieren, die für verschiedene Aerosolquellen und -prozesse charakteristisch sind. Niedermolekulare Carbonsäuren und Nitrophenole wurden als Indikatoren für die Verbrennung fossiler Brennstoffe und die Entstehung sowie Alterung sekundärer organischer Aerosole (SOA) aus biogenen Vorläufern untersucht. Protein-Makromoleküle wurden mit Blick auf den Einfluss von Luftverschmutzung und Nitrierungsreaktionen auf die Allergenität primärer biologischer Aerosolpartikel – wie Pollen und Pilzsporen – untersucht.rnFilterproben von Grob- und Feinstaubwurden über ein Jahr hinweg gesammelt und auf folgende WSOCs untersucht: die Pinen-Oxidationsprodukte Pinsäure, Pinonsäure und 3-Methyl-1,2,3-Butantricarbonsäure (3-MBTCA) sowie eine Vielzahl anderer Dicarbonsäuren und Nitrophenole. Saisonale Schwankungen und andere charakteristische Merkmale werden mit Blick auf Aerosolquellen und -senken im Vergleich zu Daten anderen Studien und Regionen diskutiert. Die Verhätlnisse von Adipinsäure und Phthalsäure zu Azelainsäure deuten darauf hin, dass die untersuchten Aerosolproben hauptsächlich durch biogene Quellen beeinflusst werden. Eine ausgeprägte Arrhenius-artige Korrelation wurde zwischen der 3-MBTCA-¬Konzentration und der inversen Temperatur beobachtet (R2 = 0.79, Ea = 126±10 kJ mol-1, Temperaturbereich 275–300 K). Modellrechnungen zeigen, dass die Temperaturabhängigkeit auf eine Steigerung der photochemischen Produktionsraten von 3-MBTCA durch erhöhte OH-Radikal-Konzentrationen bei erhöhten Temperaturen zurückgeführt werden kann. Im Vergleich zur chemischen Reaktionskinetik scheint der Einfluss von Gas-Partikel-Partitionierungseffekten nur eine untergeordnete Rolle zu spielen. Die Ergebnisse zeigen, dass die OH-initiierte Oxidation von Pinosäure der geschwindigkeitsbestimmende Schritt der Bildung von 3-MBTCA ist. 3-MBTCA erscheint somit als Indikator für die chemische Alterung von biogener sekundärer organischer Aerosole (SOA) durch OH-Radikale geeignet. Eine Arrhenius-artige Temperaturabhängigkeit wurde auch für Pinäure beobachtet und kann durch die Temperaturabhängigkeit der biogenen Pinen-Emissionen als geschwindigkeitsbestimmender Schritt der Pinsäure-Bildung erklärt werden (R2 = 0.60, Ea = 84±9 kJ mol-1).rn rnFür die Untersuchung von Proteinnitrierungreaktionen wurde nitrierte Protein¬standards durch Flüssigphasenreaktion von Rinderserumalbumin (BSA) und Ovalbumin (OVA) mit Tetranitromethan (TNM) synthetisiert.Proteinnitrierung erfolgt vorrangig an den Resten der aromatischen Aminosäure Tyrosin auf, und mittels UV-Vis-Photometrie wurde der Proteinnnitrierungsgrad (ND) bestimmt. Dieser ist definiert als Verhältnis der mittleren Anzahl von Nitrotyrosinresten zur Tyrosinrest-Gesamtzahl in den Proteinmolekülen. BSA und OVA zeigten verschiedene Relationen zwischen ND und TNM/Tyrosin-Verhältnis im Reaktionsgemisch, was vermutlich auf Unterschiede in den Löslichkeiten und den molekularen Strukturen der beiden Proteine zurück zu führen ist.rnDie Nitrierung von BSA und OVA durch Exposition mit einem Gasgemisch aus Stickstoffdioxid (NO2) und Ozon (O3) wurde mit einer neu entwickelten HPLC-DAD-¬Analysemethode untersucht. Diese einfache und robuste Methode erlaubt die Bestimmung des ND ohne Hydrolyse oder Verdau der untersuchten Proteine und ernöglicht somit eine effiziente Untersuchung der Kinetik von Protein¬nitrierungs-Reaktionen. Für eine detaillierte Produktstudien wurden die nitrierten Proteine enzymatisch verdaut, und die erhaltenen Oligopeptide wurden mittels HPLC-MS/MS und Datenbankabgleich mit hoher Sequenzübereinstimmung analysiert. Die Nitrierungsgrade individueller Nitrotyrosin-Reste (NDY) korrelierten gut mit dem Gesamt-Proteinnitrierungsgrad (ND), und unterschiedliche Verhältnisse von NDY zu ND geben Aufschluss über die Regioselektivität der Reaktion. Die Nitrierungmuster von BSA und OVA nach Beahndlung mit TNM deuten darauf hin, dass die Nachbarschaft eines negativ geladenen Aminosäurerestes die Tyrosinnitrierung fördert. Die Behandlung von BSA durch NO2 und O3 führte zu anderend Nitrierungemustern als die Behandlung mit TNM, was darauf hindeutet, dass die Regioselektivität der Nitrierung vom Nitrierungsmittel abhängt. Es zeigt sich jedoch, dass Tyrosinreste in Loop-Strukturen bevorzugt und unabhängig vom Reagens nitriert werden.Die Methoden und Ergebnisse dieser Studie bilden eine Grundlage für weitere, detaillierte Untersuchungen der Reaktionskinetik sowie der Produkte und Mechanismen von Proteinnitrierungreaktionen. Sie sollen helfen, die Zusammenhänge zwischen verkehrsbedingten Luftschadstoffen wie Stickoxiden und Ozon und der Allergenität von Luftstaub aufzuklären.rnWater-soluble organic compounds (WSOCs) are major components of atmospheric aerosols, accounting for ~50% or more of the organic fraction of particulate matter. They can influence the optical properties and hygroscopicity of aerosol particles and their effects on climate. Moreover, they can contribute to the toxicity and allergenicity of air particulate matter.rnIn this study, high performance liquid chromatography coupled with optical diode array detection and mass spectrometry (HPLC-DAD-MS and HPLC-MS/MS) has been applied to measure WSOCs with a wide range of molecular masses and structures that are characteristic for different aerosol types, sources, and processes. Carboxylic acids and nitrophenols with low molecular mass were investigated as tracers for fossil fuel combustion and the formation and aging of secondary organic aerosols (SOA) from biogenic precursors. Protein macromolecules were investigated with regard to the influence of air pollution and nitration on the allergenicity of primary biological particles like pollen and fungal spores. rnFilter samples of fine and coarse aerosol particles collected over a period of one year in central Europe (Mainz, Germany) were analyzed for WSOCs, including the α- and β-pinene oxidation products (pinic acid, pinonic acid and 3-methyl¬-1,2,3-¬butane¬tricarboxylic acid (3-MBTCA) ) as well as a variety of dicarboxylic acids and nitrophenols. Seasonal variations and other characteristic features are discussed with regard to aerosol sources and sinks in comparison to data from other studies and regions. The ratios of adipic acid and phthalic acid to azelaic acid indicate that the investigated aerosol samples were mainly influenced by biogenic sources. A strong Arrhenius-type correlation was found between the 3-MBTCA concentration and inverse temperature (R2 = 0.79, Ea = 126±10 kJ mol-1, temperature range: 275–300 K). Model calculations suggest that the temperature dependence observed for 3-MBTCA can be explained by enhanced photochemical production due to an increase of OH radical concentration with increasing temperature, whereas the influence of gas-particle partitioning appears to play a minor role. The results suggest that the OH-initiated oxidation of pinonic acid is the rate-limiting step in the formation of 3-MBTCA, and that 3-MBTCA may be a suitable tracer for the chemical aging of biogenic SOA by OH radicals. An Arrhenius-type temperature dependence was also observed for the concentration of pinic acid (R2 = 0.60, Ea = 84±9 kJ mol-1); it can be tentatively explained by the temperature dependence of biogenic pinene emission as the rate-limiting step of pinic acid formation.rn rnFor the investigation of protein nitration, nitrated protein standards were synthesized by liquid-phase reaction of bovine serum albumin (BSA) and ovalbumin (OVA) with tetranitromethane (TNM). Protein nitration occurs primarily on residues of the aromatic amino acid tyrosine, and UV-Vis photometry was used to determine the protein nitration degree (ND), which is defined as the average number of nitrotyrosine residues divided by the total number of tyrosine residues in a protein molecule. BSA and OVA exhibited different relations between ND and TNM/tyrosine ratios in the reaction mixture, which is likely due to the different solubilities and molecular structures of the two proteins. rnThe nitration of BSA and OVA upon exposure to gas mixtures of nitrogen dioxide (NO2) and (O3) was investigated with a newly developed analytical method using HPLC-DAD. This simple and robust method enables the determination of ND without hydrolysis or digestion of the investigated protein macromolecules, and thus efficient investigation of the reaction kinetics of protein nitration. For detailed product studies, however, nitrated BSA and OVA were enzymatically digested. The obtained oligopeptides were analyzed using advanced HPLC-MS/MS techniques and database search functions, and high sequence coverage was achieved. The nitration degrees of individual nitrotyrosine residues (NDY) were well correlated with the overall protein ND, and different ratios of NDY to ND provided insight into the regioselectivity of the reaction. The nitration patterns observed in BSA and OVA nitrated with TNM indicate that the vicinity of negatively charge amino acids promotes the nitration of tyrosine residues. The nitration patterns observed in BSA nitrated by NO2 and O3 differed from those obtained with TNM, which indicates that the regioselectivity of protein nitration depends on the nitrating agent. However, tyrosine residues located in loop structures were preferentially nitrated regardless of the nitrating agent.rnThe methods and results of this study provide a basis for further detailed investigations of the reaction kinetics, products and mechanisms of protein nitration. They shall help to elucidate the relations between traffic-related air pollutants like nitrogen oxides and O3 and the allergenicity of air particulate matter.rn116 S

    Development of an Analytical Assay for Electrochemical Detection and Quantification of Protein-Bound 3-Nitrotyrosine in Biological Samples and Comparison with Classical, Antibody-Based Methods.

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    Reactive oxygen and nitrogen species (RONS) cause oxidative damage, which is associated with endothelial dysfunction and cardiovascular disease, but may also contribute to redox signaling. Therefore, their precise detection is important for the evaluation of disease mechanisms. Here, we compared three different methods for the detection of 3-nitrotyrosine (3-NT), a marker of nitro-oxidative stress, in biological samples. Nitrated proteins were generated by incubation with peroxynitrite or 3-morpholino sydnonimine (Sin-1) and subjected to total hydrolysis using pronase, a mixture of different proteases. The 3-NT was then separated by high performance liquid chromatography (HPLC) and quantified by electrochemical detection (ECD, CoulArray) and compared to classical methods, namely enzyme-linked immunosorbent assay (ELISA) and dot blot analysis using specific 3-NT antibodies. Calibration curves for authentic 3-NT (detection limit 10 nM) and a concentration-response pattern for 3-NT obtained from digested nitrated bovine serum albumin (BSA) were highly linear over a wide 3-NT concentration range. Also, ex vivo nitration of protein from heart, isolated mitochondria, and serum/plasma could be quantified using the HPLC/ECD method and was confirmed by LC-MS/MS. Of note, nitro-oxidative damage of mitochondria results in increased superoxide (O2•-) formation rates (measured by dihydroethidium-based HPLC assay), pointing to a self-amplification mechanism of oxidative stress. Based on our ex vivo data, the CoulArray quantification method for 3-NT seems to have some advantages regarding sensitivity and selectivity. Establishing a reliable automated HPLC assay for the routine quantification of 3-NT in biological samples of cell culture, of animal and human origin seems to be more sophisticated than expected

    Characterization and quantifi cation of endogenous fatty acid nitroalkene metabolites in human urine

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    The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction. The formation of 9-NO2-octadeca-9,11-dienoic acid and 12-NO2-octadeca- 9,11-dienoic acid stems from peroxynitrite- and myeloperoxidase-derived nitrogen dioxide reactions as well as secondary to nitrite disproportionation under the acidic conditions of digestion. Broad anti-inflammatory and tissue-protective responses are mediated by nitro-fatty acids. It is now shown that electrophilic fatty acid nitroalkenes are present in the urine of healthy human volunteers (9.9 + 4.0 pmol/mg creatinine); along with electrophilic 16- and 14-carbon nitroalkenyl β-oxidation metabolites. High resolution mass determinations and co-elution with isotopically-labeled metabolites support renal excretion of cysteine-nitroalkene conjugates. These products of Michael addition are in equilibrium with the free nitroalkene pool in urine and are displaced by thiol reaction with HgCl2. This reaction increases the level of free nitroalkene fraction >10-fold and displays a KD of 7.5x10-6 M. In aggregate, the data indicates that formation of Michael adducts by electrophilic fatty acids is favored under biological conditions and that reversal of these addition reactions is critical for detecting both parent nitroalkenes and their metabolites. The measurement of this class of mediators can constitute a sensitive non-invasive index of metabolic and inflammatory status.Fil: Salvatore, Sonia Rosana. University of Pittsburgh; Estados UnidosFil: Vitturi, Dario A.. University of Pittsburgh; Estados UnidosFil: Baker, Paul R. S.. University of Pittsburgh; Estados UnidosFil: Bonacci, Gustavo Roberto. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Koenitzer, Jeffrey R.. University of Pittsburgh; Estados UnidosFil: Woodcock, Steven R.. University of Pittsburgh; Estados UnidosFil: Freeman, Bruce A.. University of Pittsburgh; Estados UnidosFil: Schopfer, Francisco J.. University of Pittsburgh; Estados Unido

    Fluorogenic and Affinity Derivatization Methods to Enable Proteomic Study of 3-Nitrotyrosine and 3,4-Dihydroxyphenylalanine as Markers of Oxidative Stress

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    Purpose: Oxidative post-translational modification of protein-bound tyrosine residues can have a significant impact on protein structure and function and thus may be important to physiological and pathological processes. Oxidative stress has been correlated with biological aging and many disease states, including diabetes, atherosclerosis, and neurodegeneration. Proteomic methods targeted to these modifications are important tools for determining which specific modifications may be significant in these conditions. Toward this end, a method designed to fluorogenically label the protein oxidation products 3-nitrotyrosine (3NY) and 3,4-dihydroxyphenylalanine (DOPA) using benzylamine-dependent chemistry is applied to model peptides and proteins, as well as cardiac tissue samples from a rat model for aging. Methods: Peptides or proteins are reacted with excess benzylamine (or a derivative thereof), in the presence of potassium ferricyanide, to fluorescently label DOPA residues by forming 2-phenylbenzoxazole derivatives. To label 3NY residues, the peptides or proteins are first reduced with sodium dithionite to give 3-aminotyrosine, which can undergo a similar reaction with benzylamine and oxidant to give the same products. Products are characterized by fluorescence spectroscopy, high-performance liquid chromatography (HPLC) with UV and fluorescence detection, mass spectrometry, and amino acid analysis. For enrichment by boronate-affinity HPLC, the benzylamine derivative (3R, 4S)-1-(4-(aminomethyl)phenylsulfonyl) pyrrolidine-3,4-diol (APPD) is used as the tagging reagent. Results: Cardiac proteins have been fluorescently labeled and separated, and some putative identifications have been made. A model protein, glycogen phosphorylase b (Ph-b), has been nitrated in vitro and labeled within a matrix of cardiac homogenate, and the products exhibit concentration-dependent fluorescence. The loss of 3NY from nitrated Ph-b upon mixing with cardiac homogenate has been observed and examined. Five model peptides have been labeled with APPD to determine the effect of primary structure on labeling efficiency, fluorescence quantum yield, and molar absorptivity. Conclusions: This method has great potential to aid identification of the protein oxidation products DOPA and 3NY in proteomic studies of tissue samples and can also be adapted for affinity enrichment and relative quantification of these low-abundance species

    Probing Isoforms of the Prion Protein through Tyrosine Nitration

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    The prion protein (PrP) has multiple stable isoforms. When PrP misfolds, it aggregates and causes neurological disease and death in mammals. The structure of the non-pathogenic isoform has been determined while the structures of the disease related isoforms are unknown. The nitration labeling patterns of three PrP isoforms with peroxynitrite and tetranitromethane, as detected by mass spectrometry, are reported. Two conserved tyrosine residues (tyrosines 149 and 150) are not labeled by either reagent in the normal cellular form of the prion protein but these residues become reactive after the protein has been converted to one of two aggregated isoforms. Another difference observed is that two other conserved tyrosine residues, 225 and 226, are much less reactive in both aggregated isoforms, while all other tyrosine residues show virtually no isoform specific-labeling. Thus, two regions been identified in which Tyr residues undergo a change in solvent accessibility, which may be due to a conformational change in that region or to inter-subunit packing
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