Simultaneous determination of nitrated and oligomerized proteins by size exclusion high-performance liquid chromatography coupled to photodiode array detection.

Chemical modifications such as nitration and cross-linking may enhance the allergenic potential of proteins. The kinetics and mechanisms of the underlying chemical processes, however, are not yet well understood. Here, we present a size-exclusion chromatography/spectrophotometry method (SEC-HPLC-DAD) that allows a simultaneous detection of mono-, di-, tri-, and higher protein oligomers, as well as their individual nitration degrees (NDs). The ND results of proteins from this new method agree well with the results from an alternative well-established method, for the analysis of tetranitromethane (TNM)- and nitrogen dioxide and ozone (NO2/O3)-nitrated protein samples. Importantly, the NDs for individual oligomer fractions can be obtained from the new method, and also, we provide a proof of principle for the calculation of the concentrations for individual protein oligomer fractions by their determined NDs, which will facilitate the investigation of the kinetics and mechanism for protein tyrosine nitration and cross-linking.

Nitration of the birch pollen allergen Bet v 1.0101: efficiency and site-selectivity of liquid and gaseous nitrating agents.

Nitration of the major birch pollen allergen Bet v 1 alters the immune responses toward this protein, but the underlying chemical mechanisms are not yet understood. Here we address the efficiency and site-selectivity of the nitration reaction of recombinant protein samples of Bet v 1.0101 with different nitrating agents relevant for laboratory investigations (tetranitromethane, TNM), for physiological processes (peroxynitrite, ONOO(-)), and for the health effects of environmental pollutants (nitrogen dioxide and ozone, O3/NO2). We determined the total tyrosine nitration degrees (ND) and the NDs of individual tyrosine residues (NDY). High-performance liquid chromatography coupled to diode array detection and HPLC coupled to high-resolution mass spectrometry analysis of intact proteins, HPLC coupled to tandem mass spectrometry analysis of tryptic peptides, and amino acid analysis of hydrolyzed samples were performed. The preferred reaction sites were tyrosine residues at the following positions in the polypeptide chain: Y83 and Y81 for TNM, Y150 for ONOO(-), and Y83 and Y158 for O3/NO2. The tyrosine residues Y83 and Y81 are located in a hydrophobic cavity, while Y150 and Y158 are located in solvent-accessible and flexible structures of the C-terminal region. The heterogeneous reaction with O3/NO2 was found to be strongly dependent on the phase state of the protein. Nitration rates were about one order of magnitude higher for aqueous protein solutions (∼20% per day) than for protein filter samples (∼2% per day). Overall, our findings show that the kinetics and site-selectivity of nitration strongly depend on the nitrating agent and reaction conditions, which may also affect the biological function and adverse health effects of the nitrated protein.

Glutamine synthetase is a molecular target of nitric oxide in root nodules of Medicago truncatula and is regulated by tyrosine nitration.

Nitric oxide (NO) is emerging as an important regulatory player in the Rhizobium-legume symbiosis, but its biological role in nodule functioning is still far from being understood. To unravel the signal transduction cascade and ultimately NO function, it is necessary to identify its molecular targets. This study provides evidence that glutamine synthetase (GS), a key enzyme for root nodule metabolism, is a molecular target of NO in root nodules of Medicago truncatula, being regulated by tyrosine (Tyr) nitration in relation to active nitrogen fixation. In vitro studies, using purified recombinant enzymes produced in Escherichia coli, demonstrated that the M. truncatula nodule GS isoenzyme (MtGS1a) is subjected to NO-mediated inactivation through Tyr nitration and identified Tyr-167 as the regulatory nitration site crucial for enzyme inactivation. Using a sandwich enzyme-linked immunosorbent assay, it is shown that GS is nitrated in planta and that its nitration status changes in relation to active nitrogen fixation. In ineffective nodules and in nodules fed with nitrate, two conditions in which nitrogen fixation is impaired and GS activity is reduced, a significant increase in nodule GS nitration levels was observed. Furthermore, treatment of root nodules with the NO donor sodium nitroprusside resulted in increased in vivo GS nitration accompanied by a reduction in GS activity. Our results support a role of NO in the regulation of nitrogen metabolism in root nodules and places GS as an important player in the process. We propose that the NO-mediated GS posttranslational inactivation is related to metabolite channeling to boost the nodule antioxidant defenses in response to NO.

Determination of a catalytic tyrosine in Trametes cervina lignin peroxidase with chemical modification techniques.

Trametes cervina lignin peroxidase (LiP) lacks a catalytic tryptophan strictly conserved in other LiP and versatile peroxidases. It contains tyrosine(181) at the potential catalytic site. This protein and the well-characterized Phanerochaete chrysosporium LiP with the catalytic tryptophan(171) have been chemically modified: the tryptophan-specific modification with N-bromosuccinimide sufficiently disrupted oxidation of veratryl alcohol by P. chrysosporium LiP, whereas the activity of T. cervina LiP was not affected, suggesting no catalytic tryptophan in T. cervina LiP. On the other hand, the tyrosine-specific modification with tetranitromethane did not affect the activities of P. chrysosporium LiP lacking tyrosine but inactivated T. cervina LiP due to the nitration of tyrosine(181). These results strongly suggest that tyrosine(181) is at the catalytic site in T. cervina LiP.

Synthesis of unusual oxime ethers by reaction of tetranitromethane with B-alkylcatecholboranes.

The reaction of tetranitromethane with B-alkylcatecholboranes leads to the formation of unusual dinitrooxime ethers. A tentative mechanism is provided, which suggests the involvement of extremely fast addition of alkyl radicals to tetranitromethane. The substitution of one of the nitro groups in the oxime ethers by nucleophiles (such as secondary amines, halogens and styrene) and by radicals generated from B-alkylcatecholboranes is reported.

Unexpected heterocyclization of electrophilic alkenes by tetranitromethane in the presence of triethylamine. Synthesis of 3-nitroisoxazoles.

Novel reaction of tetranitromethane (TNM) with electrophilic alkenes in the presence of triethylamine yielding substituted 3-nitroisoxazoles was found and studied. Triethylamine increases the reactivity of TNM toward electrophilic alkenes promoting their heterocyclization, and the reactions proceed in an unusual way. A variety of alpha,beta-unsaturated aldehydes, ketones, esters, amides, phosphonates, and nitro and sulfur compounds was involved in the heterocyclization reaction, and a wide range of functionalized 3-nitroisoxazoles was obtained in good to high yields. The scope and limitations of the reaction and the mechanistic aspects are discussed.

In vitro and in vivo protein-bound tyrosine nitration characterized by diagonal chromatography.

A new proteomics technique for analyzing 3-nitrotyrosine-containing peptides is presented here. This technique is based on the combined fractional diagonal chromatography peptide isolation procedures by which specific classes of peptides are isolated following a series of identical reverse-phase HPLC separation steps. Here dithionite is used to reduce 3-nitrotyrosine to 3-aminotyrosine peptides, which thereby become more hydrophilic. Our combined fractional diagonal chromatography technique was first applied to characterize tyrosine nitration in tetranitromethane-modified BSA and further led to a high quality list of 335 tyrosine nitration sites in 267 proteins in a peroxynitrite-treated lysate of human Jurkat cells. We then analyzed a serum sample of a C57BL6/J mouse in which septic shock was induced by intravenous Salmonella infection and identified six in vivo nitration events in four serum proteins, thereby illustrating that our technique is sufficiently sensitive to identify rare in vivo tyrosine nitration sites in a very complex background.

Protein tyrosine nitration: selectivity, physicochemical and biological consequences, denitration, and proteomics methods for the identification of tyrosine-nitrated proteins.

Protein tyrosine nitration (PTN) is a post-translational modification occurring under the action of a nitrating agent. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group (NO2). In the present article, we review the main nitration reactions and elucidate why nitration is not a random chemical process. The particular physical and chemical properties of 3-nitrotyrosine (e.g., pKa, spectrophotometric properties, reduction to aminotyrosine) will be discussed, and the biological consequences of PTN (e.g., modification of enzymatic activity, sensitivity to proteolytic degradation, impact on protein phosphorylation, immunogenicity and implication in disease) will be reviewed. Recent data indicate the possibility of an in vivo denitration process, which will be discussed with respect to the different reaction mechanisms that have been proposed. The second part of this review article focuses on analytical methods to determine this post-translational modification in complex proteomes, which remains a major challenge.

Charge-transfer complexations of 1,n-di(9-ethylcarbazol-3-yl)alkanes with tetracyanoethylene and tetranitromethane.

1,n-Di(9-ethylcarbazol-3-yl)alkanes, where n=1-5, as the dichromophoric model compounds of poly-3-vinylcarbazoles were synthesized to examine their complexation behaviors with the electron acceptors tetracyanoethylene (TCNE) and tetranitromethane (TNM). 9,9'-Diethyl-3,3'-dicarbazolyl, di(3-ethylcarbazol-9-yl)methane, and three monomeric analogues were also included for comparison. In dichloromethane solution, the dicarbazoles formed stable 1:1 electron donor-acceptor complexes with TCNE having formation enthalpies around -3.5kcal/mol. With TNM they formed more weakly bound complexes that showed little dependence on concentration and almost zero dependence on temperature changes having nearly 0kcal/mol enthalpies of formation. The smaller gap between the two carbazole groups in 1,n-di(9-ethylcarbazol-3-yl)alkanes with nor=3.

Identification of the tyrosine nitration sites in human endothelial nitric oxide synthase by liquid chromatography-mass spectrometry.

The formation of nitric oxide (NO) in biological systems has led to the discovery of a number of post- translational protein modifications that can affect biological conditions such as vasodilation. Studies both from our laboratory and others have shown that beside its effect on cGMP generation from soluble guanylate cylcase, NO can produce protein modifications through both S-nitrosylation of cysteine residues. Previously, we have identified the potential S-nitrosylation sites on endothelial NO synthase (eNOS). Thus, the goal of this study was to further increase our understanding of reactive nitrogen protein modifications of eNOS by identifing tyrosine residues within eNOS that are susceptible to nitration in vitro. To accomplish this, nitration was carried out using tetranitromethane followed by tryptic digest of the protein. The resulting tryptic peptides were analyzed by liquid chromatography/mass spectrometry (LC/MS) and the position of nitrated tyrosines in eNOS were identified. The eNOS sequence contains 30 tyrosine residues and our data indicate that multiple tyrosine residues are capable of being nitrated. We could identify 25 of the 30 residues in our tryptic digests and 19 of these were susceptible to nitration. Interstingly, our data identified four tyrosine residues that can be modified by nitration that are located in the region of eNOS responsible for the binding to heat shock protein 90 (Hsp90), which is responsible for ensuring efficient coupling of eNOS.

Mass spectrometry analysis of in vitro nitration of a recombinant human IgG1 monoclonal antibody.

Nitration of a recombinant human monoclonal antibody was carried out in vitro by incubating the antibody with the nitrating reagent tetranitromethane (TNM). The susceptible sites of nitration were identified using high-performance liquid chromatography/mass spectrometry (HPLC/MS). In general, tyrosine residues in the variable domains of the antibody are more susceptible to nitration, while tyrosine residues in the constant domains are relatively resistant to nitration. However, one tyrosine residue in the CH1 domain and one tyrosine residue in the CH2 domain are highly susceptible to nitration. Interestingly, the susceptible tyrosine residue in the CH2 domain is followed by the conserved asparagine residue that is glycosylated.

Nitroavidin as a ligand for the surface capture and release of biotinylated proteins.

Discussed here is the preparation, detailed purification, and evaluation of nitroavidin as a ligand for surface capture and release of biotinylated proteins. Avidin from chicken egg white was nitrated using dilute tetranitromethane solutions. UV-vis spectroscopy was used to show decreased binding of the biotin analogue, 2-(4'-hydroxyazobenzene)benzoic acid, HABA, to nitroavidin compared to binding of HABA to native avidin. From enzyme-linked immunosorbent assay (ELISA)-based assays of the modified avidin, it was found that there are approximately three tyrosine residues converted to nitrotyrosine out of the total four tyrosine residues in the protein tetramer. For the first time, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to demonstrate the point of nitration in nitroavidin as that of the tyrosine associated with the binding of biotin (Y33). From surface plasmon resonance spectroscopy (SPR) experiments, it is shown that biotin has less binding propensity to immobilized nitroavidin (K(D) = 4.4 +/- 1.9 x 10(-6) M) than immobilized avidin (K(D) < or = 10(-11) M). Importantly, the use of pH 10 carbonate buffer as eluent resulted in facile release of bound biotin from the nitroavidin-functionalized surfaces, allowing for readily regenerated biotin capture surfaces (reversible binding surfaces). These outcomes are important for the development of protein concentration methods directed at isolation of select proteins from a large population using gentle target protein isolation/release conditions.

Probing structural differences in prion protein isoforms by tyrosine nitration.

Two conformational isomers of recombinant hamster prion protein (residues 90-232) have been probed by reaction with two tyrosine nitration reagents, peroxynitrite and tetranitromethane. Two conserved tyrosine residues (tyrosines 149 and 150) are not labeled by either reagent in the normal cellular form of the prion protein. These residues become reactive after the protein has been converted to the beta-oligomeric isoform, which is used as a model of the fibrillar form that causes disease. After conversion, a decrease in reactivity is noted for two other conserved residues, tyrosine 225 and tyrosine 226, whereas little to no effect was observed for other tyrosines. Thus, tyrosine nitration has identified two specific regions of the normal prion protein isoform that undergo a change in chemical environment upon conversion to a structure that is enriched in beta-sheet.

Tyrosine-dependent oxidative DNA damage induced by carcinogenic tetranitromethane.

Tetranitromethane (TNM) is used as an oxidizer in rocket propellants and explosives and as an additive to increase the cetane number of diesel fuel. TNM was reported to induce pulmonary adenocarcinomas and squamous cell carcinomas in mice and rats. However, the mechanisms underlying carcinogenesis induced by TNM has not yet been clarified. We previously revealed that nitroTyr and nitroTyr-containing peptides caused Cu(II)-dependent DNA damage in the presence of P450 reductase, which is considered to yield nitroreduction. Since TNM is a reagent for nitration of Tyr in proteins and peptides, we have hypothesized that TNM-treated Tyr and Tyr-containing peptides induce DNA damage by the modification of Tyr. We examined DNA damage induced by TNM-treated amino acids or peptides using (32)P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. TNM-treated Tyr and Lys-Tyr-Lys induced DNA damage including the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the presence of Cu(II) and NADH. DNA damage was inhibited by catalase and bathocuproine, indicating the involvement of H(2)O(2) and Cu(I). The cytosine residue of the ACG sequence complementary to codon 273, well-known hotspots of the p53 gene, was cleaved with piperidine and Fpg treatments. On the other hand, nitroTyr and Lys-nitroTyr-Lys did not induce DNA damage in the presence of Cu(II) and NADH. Time-of-flight mass spectrometry confirmed that reactions between Lys-Tyr-Lys and TNM yielded not only Lys-nitroTyr-Lys but also Lys-nitrosoTyr-Lys. Therefore, it is speculated that the nitrosotyrosine residue can induce oxidative DNA damage in the presence of Cu(II) and NADH. It is concluded that Tyr-dependent DNA damage may play an important role in the carcinogenicity of TNM. TNM is a new type of carcinogen that induces DNA damage not by itself but via Tyr modification.

Role of tyrosine and tryptophan in chemically modified serum albumin on its tissue distribution.

To investigate the effect of functional groups in bovine serum albumin (BSA) on its tissue distribution characteristics, tyrosine (Tyr) or tryptophan (Trp) residues of BSA were chemically modified by tetranitromethane (TNM) and 2-hydroxy-5-nitrobenzyl bromide (HNB), respectively. BSA was successfully modified with each reagent depending on the amount of the reagent added to the reaction mixture, and TNM- and HNB-modified BSA derivatives with different degrees of modification were obtained. Circular dichroism measurements showed that slight secondary and large tertiary changes were detectable as the degree of modification increased. After intravenous injection into mice, all synthetic BSA derivatives were eliminated very slowly from the systemic circulation. However, (111)In-TNM(6.6)- and (111)In-HNB(2.0)-BSA, derivatives with a high degree of modification, showed a slightly faster disappearance from the systemic circulation and slightly higher accumulation in the liver than (111)In-unmodified BSA. Pharmacokinetic analyses also demonstrated that the modification of Tyr or Trp residues on BSA had only marginal effects on tissue distribution. These results indicate that the Tyr and Trp residues have little effect on the tissue distribution characteristics of serum albumins, and that the specific modification of these residues may be a promising approach to designing sustained drug delivery systems using serum albumins.

Nitration enhances the allergenic potential of proteins.

BACKGROUND: Recent investigations have shown that proteins, including Bet v 1a, are nitrated by exposure to polluted urban air. We have investigated immunogenic and allergenic properties of in vitro nitrated allergens in in vivo models. METHODS: Untreated and nitrated samples of ovalbumin or Bet v 1a were compared for their ability to stimulate proliferation and cytokine secretion in splenocytes from DO11.10 or from sensitized BALB/c mice, and for their ability to induce specific immunoglobulin (Ig)G1, IgG2a and IgE in sensitized mice. Additionally, sera from birch pollen-allergic individuals were analysed for IgE and IgG specific for nitrated Bet v 1a. RESULTS: Upon splenocyte stimulation with nitrated as compared with unmodified allergens, proliferation as well as interleukin 5 and interferon-gamma production were enhanced. Sera of mice sensitized with nitrated allergens showed elevated levels of specific IgE, IgG1 and IgG2a, compared with sera from mice sensitized with unmodified allergens. Moreover, cross-reactivity of antibodies against unrelated, nitrated allergens was observed in mice. We also found higher amounts of functional, specific IgE against nitrated than against untreated Bet v 1a in sera from birch pollen-allergic patients. CONCLUSIONS: Our findings suggest that nitration enhances allergic responses, which may contribute to an increased prevalence of allergic diseases in polluted urban environments.

Correlation between induction of DNA fragmentation in lung cells from rats and humans and carcinogenic activity.

Six chemicals, known to induce lung tumors in rats, were examined for their ability to induce DNA fragmentation in primary cultures of rat and human lung cells, and in the lung of intact rats. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the single-cell gel electrophoresis (Comet) assay, were obtained in primary lung cells from male rats with the following, minimally toxic, concentrations of the six test compounds: N-nitrosodimethylamine (NDMA; 2.5-10 mM), hydrazine (HZ; 0.5-4 mM), cadmium sulfate (CD; 31.2 and 62.5 µM), 4,4'-methylene bis (2-chloroaniline) (MOCA; 31.2-125 µM), isobutyl nitrite (IBN; 7.8-31.2 µM) and tetranitromethane (TNM; 1.9-15.6 µM). Similar degrees of DNA fragmentation were obtained in primary human lung cells; however, due to inter-donor differences, the minimum effective concentrations were in some donors lower and in others higher than in rats, and IBN induced DNA damage only in one of three donors. The DNA-damaging potency of HZ was higher in rats than in humans, and the opposite was true for MOCA. In agreement with these findings, statistically significant increases in the average frequency of DNA breaks were obtained in the lung of rats given a single oral dose (1/2 LD50) of the six test compounds. These findings give evidence that genotoxic lung carcinogens may be identified by use of the DNA fragmentation/Comet assay on rat lung cells as targets cells, and show that the six compounds tested produce in primary cultures of lung cells from human donors DNA-damaging effects substantially similar to those observed in rats.

The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding.

The starch-hydrolysing enzyme GA (glucoamylase) from Rhizopus oryzae is a commonly used glycoside hydrolase in industry. It consists of a C-terminal catalytic domain and an N-terminal starch-binding domain, which belong to the CBM21 (carbohydrate-binding module, family 21). In the present study, a molecular model of CBM21 from R. oryzae GA (RoGACBM21) was constructed according to PSSC (progressive secondary structure correlation), modified structure-based sequence alignment, and site-directed mutagenesis was used to identify and characterize potential ligand-binding sites. Our model suggests that RoGACBM21 contains two ligand-binding sites, with Tyr32 and Tyr67 grouped into site I, and Trp47, Tyr83 and Tyr93 grouped into site II. The involvement of these aromatic residues has been validated using chemical modification, UV difference spectroscopy studies, and both qualitative and quantitative binding assays on a series of RoGACBM21 mutants. Our results further reveal that binding sites I and II play distinct roles in ligand binding, the former not only is involved in binding insoluble starch, but also facilitates the binding of RoGACBM21 to long-chain soluble polysaccharides, whereas the latter serves as the major binding site mediating the binding of both soluble polysaccharide and insoluble ligands. In the present study we have for the first time demonstrated that the key ligand-binding residues of RoGACBM21 can be identified and characterized by a combination of novel bioinformatics methodologies in the absence of resolved three-dimensional structural information.

Binding residues and catalytic domain of soluble Saccharomyces cerevisiae processing alpha-glucosidase I.

Alpha-glucosidase I initiates the trimming of newly assembled N-linked glycoproteins in the lumen of the endoplasmic reticulum (ER). Site-specific chemical modification of the soluble alpha-glucosidase I from yeast using diethylpyrocarbonate (DEPC) and tetranitromethane (TNM) revealed that histidine and tyrosine are involved in the catalytic activity of the enzyme, as these residues could be protected from modification using the inhibitor deoxynojirimycin. Deoxynojirimycin could not prevent inactivation of enzyme treated with N-bromosuccinimide (NBS) used to modify tryptophan residues. Therefore, the binding mechanism of yeast enzyme contains different amino acid residues compared to its mammalian counterpart. Catalytically active polypeptides were isolated from endogenous proteolysis and controlled trypsin hydrolysis of the enzyme. A 37-kDa nonglycosylated polypeptide was isolated as the smallest active fragment from both digests, using affinity chromatography with inhibitor-based resins (N-methyl-N-59-carboxypentyl- and N-59-carboxypentyl-deoxynojirimycin). N-terminal sequencing confirmed that the catalytic domain of the enzyme is located at the C-terminus. The hydrolysis sites were between Arg(521) and Thr(522) for endogenous proteolysis and residues Lys(524) and Phe(525) for the trypsin-generated peptide. This 37-kDa polypeptide is 1.9 times more active than the 98-kDa protein when assayed with the synthetic trisaccharide, alpha-D-Glc1,2alpha-D-Glc1,3alpha-D-Glc-O(CH2)(8)COOCH(3), and is not glycosylated. Identification of this relatively small fragment with catalytic activity will allow mechanistic studies to focus on this critical region and raises interesting questions about the relationship between the catalytic region and the remaining polypeptide.