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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 (NO 2 /O 3 )-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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.

Amino acid residue-specific reactivity in proteins is of great current interest in structural biology as it provides information about solvent accessibility and reactivity of the residue and, consequently, about protein structure and possible interactions. In the work presented tyrosine residues of three model proteins with known spatial structure are modified with two tyrosine-specific reagents: tetranitromethane and iodine. Modified proteins were specifically digested by proteases and the mass of resulting peptide fragments was determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Our results show that there are only small differences in the extent of tyrosine residues modification by tetranitromethane and iodine. However, data dealing with accessibility of reactive residues obtained by chemical modifications are not completely identical with those obtained by nuclear magnetic resonance and X-ray crystallography. These interesting discrepancies can be caused by local molecular dynamics and/or by specific chemical structure of the residues surrounding.

A screening method is described for determining whether a drug or small solute has significant interactions at the two major binding sites on human serum albumin (HSA). This method uses affinity capillary electrophoresis (ACE) to perform a mobility shift assay, where the solute of interest is injected in both the presence of pH 7.4, 0.067 M phosphate buffer, and the same buffer containing a known concentration of HSA. Dextran is also used in the running buffer to adjust the mobility of HSA. Two types of modified HSA are used in this assay. The first is modified with 2-hydroxy-5-nitrobenzyl bromide (HNB), which selectively blocks HSA's warfarin-azapropazone site. The second type of HSA is modified with tetranitromethane (TNM), which decreases binding at the indole-benzodiazepine site. By comparing the mobility of a solute in the presence of these two modified forms of HSA vs normal HSA, it is possible to detect solute interactions at these binding sites. This approach is illustrated using warfarin and ibuprofen as examples of test solutes.

Reversal of the superoxide dismutase (SOD) reaction was measured in terms of the reduction of tetranitromethane (TNM) by O2-. Cu,ZnSOD caused a biphasic reduction of TNM by H2O2. The rapid initial phase was stoichiometric with the enzyme and was followed by a slower catalytic phase that was oxygen dependent and was augmented by HCO3-. The reaction scheme explaining this behavior is presented and a rate constant for the reduction of O2 by the cuprous enzyme is estimated. This rate constant is so low that it precludes significant O2- production by the reduced enzyme under the conditions explored.

A technique based on affinity capillary electrophoresis (ACE) and chemically modified proteins was used to screen the binding sites of various drugs on human serum albumin (HSA). This involved using HSA as a buffer additive, following the site-selective modification of this protein at two residues (tryptophan 214 or tyrosine 411) located in its major binding regions. The migration times of four compounds (warfarin, ibuprofen, suprofen and flurbiprofen) were measured in the presence of normal or modified HSA. These times were then compared and the mobility shifts observed with the modified proteins were used to identify the binding regions of each injected solute on HSA. Items considered in optimizing this assay included the concentration of protein placed into the running buffer, the reagents used to modify HSA, and the use of dextran as a secondary additive to adjust protein mobility. The results of this method showed good agreement with those of previous reports. The advantages and disadvantages of this approach are examined, as well as its possible extension to other solutes.

The inactivation of horseradish peroxidase A2 (HRP-A2) with H2O2 as the sole substrate has been studied. In incubation experiments it was found that the fall in HRP-A2 activity was non-linearly dependent on H2O2 concentrations and that a maximum level of inactivation of approximately 80% (i.e. approximately 20% residual activity) was obtained with 2,000 or more equivalents of H2O2. Further inactivation was only induced at much higher H2O2 concentrations. Spectral changes during incubations of up to 5 days showed the presence of a compound III-like species whose abundance was correlated to the level of resistance observed. Inactivation was pH dependent, the enzyme being much more sensitive under acid conditions. A partition ratio (r1 approximately equals 1,140 at pH 6.5) between inactivation and catalysis was calculated from the data. The kinetics of inactivation followed single exponential time curves and were H2O2 concentration dependent. The apparent maximum rate constant of inactivation was lambdamax=3.56+/-0.07x10(-4)s(-1) and the H2O2 concentration required to give lambdamax/2 was K2=9.94+/-0.52 mM. The relationship lambdamax

The amine buffer Tris slowly reduces tetranitromethane (TNM) to the nitroform anion in a non-accelerating reaction. The amine buffers HEPES and MOPS also (slowly) react with TNM but the dialkylaminoalkyl radicals formed from these two buffers undergo further reactions resulting in a rapid, accelerating, free radical chain process whereby the amine is oxidized and TNM reduced. The chemical functionality in any reaction component, not necessarily the buffer, required for this radical chain mechanism is >N-CH<. In the presence of such groups, the quantification of superoxide by TNM is impossible.

Two alpha-neurotoxins, Oh-4 and Oh-7, from king cobra (Ophiophagus hannah) venom were subjected to Tyr modification with tetranitromethane. Selective nitration of Tyr4 in Oh-4 caused a slight decrease in lethal toxicity of 11% and a decrease in nicotinic acetylcholine receptor (nAchR)-binding activity of 28%, whereas nitration of Tyr4 in Oh-7 resulted in an approximately equal 60% decrease in lethality and nAchR-binding activity. When the Tyr23 in Oh-4 or Tyr22 in Oh-7 appears to be 'buried' in the toxin following further modification, the toxins lost their biological activity and conformational change concurrently. Nevertheless, the dinitrated Oh-4 retained a beta-sheet structure as revealed by CD spectra and exhibited a precipitin reaction with anti-Oh-4 sera. These results indicate that both Tyr4 and Tyr22 play a crucial role in the neurotoxicity of Oh-7, whereas intact Tyr23 is involved in the manifestation of the toxicity of Oh-4 to a greater extent. In contrast to Oh-4, the conformational stability of Oh-7 depends heavily upon the integrity of Tyr22.

The flavoprotein nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with production of nitrite and hydrogen peroxide. The enzyme is irreversibly inactivated by incubation with tetranitromethane, a tyrosine-directed reagent, at pH 7.3. The inactivation is time-dependent and shows first-order kinetics for two half-lives of inactivation. Further inactivation can be achieved upon a second addition of tetranitromethane. A saturation kinetic pattern is observed when the rate of inactivation is determined versus the concentration of tetranitromethane, indicating that a reversible enzyme-inhibitor complex is formed before irreversible inactivation occurs. Values of 0.096 +/- 0.013 min(-1) and 12.9 +/- 3.8 mM were determined for the first-order rate constant for inactivation and the dissociation constant for the reversibly formed complex, respectively. The competitive inhibitor valerate protects the enzyme from inactivation by tetranitromethane, suggesting an active-site-directed inactivation. The UV-visible absorbance spectrum of the inactivated enzyme is perturbed with respect to that of the native enzyme, suggesting that treatment with tetranitromethane resulted in nitration of the enzyme. Comparison of tryptic maps of nitroalkane oxidase treated with tetranitromethane in the presence and absence of valerate shows a single peptide differentially labeled in the inactivated enzyme. The spectral properties of the modified peptide are consistent with nitration of a tyrosine residue. The amino acid sequence of the nitrated peptide is L-L-N-E-V-M-C-(NO(2)-Y)-P-L-F-D-G-G-N-I-G-L-R. The possible role of this tyrosine in substrate binding is discussed.

Chemical modification of ovomucoid third domain (DIII) has been conducted to characterize the binding site residues that determine antigenecity and allergenecity of DIII. Nitration of Tyr, ethoxyformylation of His and succinylation of Lys residues led to a decrease of alpha-helix content of DIII. Modification of His, Tyr, Glu, Asp and Lys residues on DIII resulted in a reduction of human IgG binding activity, but little effect on IgE binding activity. These results suggest that hydrophilic residues appear to be more critical for human IgG binding site, whereas hydrophobic residues may be more important for IgG binding site., (Copyright 1999 Academic Press.)

The endo-beta-1,4-xylanase (EC 3.2.1.8) from Trichosporon cutaneum was chemically modified using amino acid-specific reagents. The enzyme does not bear arginines essential for activity, since 1,2-cyclohexanedione and 2,3-butanedione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Reaction of the enzyme with tetranitromethane and N-acetylimidazole did not result in a significant activity loss as a result of modification of tyrosine residues. The water-soluble carbodiimide 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide inactivated the xylanase rapidly and completely in a pseudo-first-order process, and kinetic analysis indicated that at least one molecule of carbodiimide binds to the enzyme for inactivation. A mixture of neutral xylooligomers provided significant protection of the enzyme against this carbodiimide inactivation. Reaction of the xylanase with 2,4,6-trinitrobenzene sulfonic acid did not result in a significant activity loss as a result of modification of lysine residues. Titration of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetamide and p-chloromercuribenzoate indicated the presence of a free/active thiol group. Xylan completely protected the enzyme from inactivation by p-hydroxymercuribenzoate, suggesting the presence of cysteine at the substrate-binding site. Inactivation of xylanase by p-hydroxymercuribenzoate could be restored by cysteine.

Internal fatty acylation of proteins is a recognized means of modifying biological behavior. Escherichia coli hemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of two lysine residue epsilon-amino groups; HlyC catalyzes the acyl transfer from acyl-acyl carrier protein (ACP), the obligate acyl donor. Conserved residues among the respective homologous C proteins that activate 13 different RTX (repeats in toxin) toxins of which HlyA is the prototype likely include some residues that are important in catalysis. Possible roles of two conserved tyrosines and two conserved arginines were investigated by noting the effects of chemical modifiers and site-directed mutagenesis. TNM modification of HlyC at pH 8.0 led to extensive inhibition that was prevented by the presence of the substrate myristoyl-ACP but not by the product, ACPSH. NAI had no effect. Y70G and Y150G greatly diminished enzyme activity, whereas mutations Y70F and Y150F exhibited wild-type activity. Modification of arginine residues with PG markedly lowered acyltransferase activity with moderate protection by both myristoyl-ACP and ACPSH. Under optimum conditions, four separate mutations of the two conserved arginine residues (R24A, R24K, R87A, and R87K) had little effect on acyltransferase activity.

The direct energy transfer technique was modified and applied to probe the relative localization of apomyoglobin A-, G- and H-helixes, which are partly protected from deuterium exchange in the equilibrium molten globule state and in the molten globule-like kinetic intermediate. The non-radiative transfer of tryptophan electronic energy to 3-nitrotyrosine was studied in different conformational states of apomyoglobin (native, molten globule, unfolded) and interpreted in terms of average distances between groups of the protein chain. The experimental data show that the distance between the middle of A-helix and the N-terminus of G-helix as well as the distance between the middle of the A-helix and the C-terminus of the H-helix in the molten globule state are close to those in the native state. This is a strong argument in favor of similarity of the overall architecture of the molten globule and native states.

Several lines of evidence have been accumulated for occurrence of nitration in vivo. In this brief review, we summarized nitration studies on functional changes of proteins, hormones and neurotransmitters, before as well as after the discovery of peroxynitrite. Most of nitrated molecules exhibit less active properties than the parental compounds. It is still unknown whether nitration is merely a footprint of oxidative stress, an important pathway of nitric oxide metabolisms or a part of integral processes for maintaining cellular homeostasis.

Properties of active site of Zn2+-glycerophosphocholine cholinephosphodiesterase from ox brain were examined using substrates and inhibitors of the phosphodiesterase. The anionic binding site expressed a selectivity for a positively-charged group. Meanwhile, the glyceryl moiety-binding site appeared to be a narrow crevice of a limited size, excluding the entry of acylglycerophospholipids containing long acyl chains. While endogenous quaternary ammonium compounds such as phosphocholine, choline or carnitine inhibited the enzyme, divalent metal ions such as Co2+, Mn2+ or Zn2+ enhanced the activity by 1.5 to 2-folds. The pH dependence for the inhibition by phosphocholine or the hydrolysis of substrate implies the involvement of a basic amino acid residue with a pK value of 9.6-9.7, probably lysine, in the binding of phosphoryl group. In further support, the lysine modifiers such as trinitrobenzene sulfonic acid or diethylpyrocarbonate expressed some inactivation. The pH-rate profile indicates that an amino acid residue with a pK value of 10.2, presumably tyrosine, may participate as a nucleophile in the catalysis. This might be further supported by the inactivation of the enzyme by tyrosine modifiers such as tetranitromethane or HOI-generating system. Separately, the phosphodiesterase was observed to be susceptible to the action of hydrogen peroxide or peroxynitrite-generating system. From these results, it is implied that the phosphodiesterase may be affected by endogenous sources.

Nitration of ovine placental lactogen (oPL) with a 10-fold molar excess of tetranitromethane over protein content resulted in the modification of 0.8 tyrosine residue. No conformational changes were observed by either fourth-derivative spectral analysis or circular dichroism. Nitration significantly decreased the binding capacity of the hormone to lactogenic and somatogenic rat liver receptors. This binding capacity was not restored by reduction of the nitro groups, thus indicating that the decrease was not due to the difference in pK between tyrosine and nitrotyrosine. The nitrotyrosine-containing peptide was isolated from a tryptic digest by HPLC and its modification extent was of 67%, which is consistent with the decrease in binding capacities (65% and 70%). Its amino acid sequence was determined and the modified tyrosine residue was identified as Tyr-46. These results provide the first evidence of the involvement of a tyrosine residue in the binding of oPL to both lactogenic and somatogenic receptors. This tyrosine appears to be a shared binding epitope between oPL and the prolactins.

The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid anchored receptor (uPAR) is essential for the confinement of plasminogen activation to cell surfaces where it is thought to play an important role in cancer cell invasion and metastasis. The receptor binding site of uPA is retained within its isolated growth factor-like module (GFD; residues 4-43). The NH2-terminal domain of uPAR has a primary role in uPA binding, although maintenance of its multidomain structure has been shown to be necessary for the high affinity of this interaction [Ploug, M., Ellis, V., & Danø, K. (1994) Biochemistry 33, 8991-8997]. To identify residues engaged in the uPAR-uPA interaction, we have performed a "protein-protein footprinting" study on preformed uPAR-GFD complexes by chemical modification with tetranitromethane. All six tyrosine residues in uPAR and the single tyrosine residue in GFD (Tyr24) were susceptible to nitration in the native uncomplexed proteins, whereas in the receptor-ligand complexes both Tyr57 of uPAR and Tyr24 of GFD were protected from modification. Modification of uPAR alone led to a parallel reduction in the potential to bind pro-uPA and 8-anilino-1-naphthalenesulfonate, an extrinsic fluorophore reporting on the accessibility of a hydrophobic site involved in uPA binding. These data clearly demonstrate that Tyr57 in the NH2-terminal domain of uPAR and Tyr24 in uPA are intimately engaged in the receptor-ligand interaction, whereas Tyr87 positioned in the linker region between the first two domains of uPAR does not appear to be shielded by the resulting intermolecular interface.

Lysozyme and ribonuclease were used as model proteins to explore the feasibility of detecting protein-bound nitrotyrosines by 15N-NMR spectroscopy. The reporter group was introduced via synthesized [15N]tetranitromethane. Several experiments for detection of the 15N resonance in the model [3-15N]nitrotyrosine demonstrated a substantial pH-dependence of the chemical shift. When lysozyme was nitrated, either two or three 15N resonances were detected, depending on the extent of nitration. The pH-dependence of the detected resonances clearly described an apparent microscopic pK in accord with reported values, while addition of Gd(III) gave selective line broadening, indicating that the 15N reporter group could also monitor relative distances from paramagnetic sources. Nitration of ribonuclease showed five 15N resonances, of which three persisted in the purified monomer. The pH-dependence of these resonances also described apparent microscopic pK values. The [3-15N]nitrotyrosine model was reduced to the [3-15N]aminotyrosine and its 15N resonance was easily monitored by several methods, including selective population inversion. When the protein-bound nitrotyrosines were similarly reduced, much sample decomposition resulted, a possible result of photooxidation, and/or reduction of disulfide bond(s), thereby making interpretation difficult.

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with two thiol groups of the dimeric horse erythrocyte glutathione transferase at pH 5.0, with strong inactivation reversible on dithiothreitol treatment. The inactivation kinetic follows a biphasic pattern, similar to that caused by other thiol reagents as recently reported. Both S-methylglutathione and 1-chloro-2,4-dinitrobenzene protect the enzyme from inactivation. Analysis of the reactive SH group-containing peptide gives the sequence Ala-Ser-Cys-Leu-Tyr, identical with that of the peptide that contains the reactive cysteine 47 of the human placental transferase. In the presence of glutathione, the enzyme is not inactivated by this reagent, but it catalyzes its conjugation to glutathione. At higher pH values, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with 2 tyrosines/dimer and lysines, as well as with cysteines. Reaction with lysine seems essentially without effect on activity; whether the reactive tyrosines are important for activity could not be determined using this reagent only. However, 2 tyrosines among the 4 that are nitrated by tetranitro-methane are important for activity.

We found that the reaction of hen egg-white lysozyme with an equimolar amount of tetranitromethane (TNM) at pH 8.0 and room temperature yielded derivatives in which the N-C bond of Gly104 is oxidatively cleaved, and a mono-nitrotyrosine lysozyme in which Tyr23 is nitrated. This bond cleavage occurred more predominantly with a decrease in the nitration of Tyr23, when the reaction was carried out under more dilute conditions. A possible mechanism in which a phenoxyl radical of Tyr 23 (an intermediate of nitration) is involved was proposed for this oxidative bond cleavage. When lysozyme was reacted with a 10 times molar excess of TNM, in addition to a mono-nitrotyrosine lysozyme in which only Try23 is nitrated, a di-nitrotyrosine lysozyme in which Tyr20 and Tyr23 are both nitrated and a tri-nitrotyrosine lysozyme in which Tyr20, Tyr23, and Tyr53 are all nitrated were obtained. However, no other possible mono- or di-nitrotyrosine lysozymes could be isolated. Thus, it is concluded that the three tyrosine residues in lysozyme are essentially nitrated sequentially with TNM in the order of Tyr23, Tyr20, and Tyr53. Since the derivatives obtained here were all active, none of the three tyrosine residues or the residues around Gly104 are considered to be very important for the lysozyme activity.