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Exposure to (bi)sulfite (HSO3–) and sulfite (SO32–) has been shown to induce a wide range of adverse reactions in sensitive individuals. Studies have shown that peroxidase-catalyzed oxidation of (bi)sulfite leads to formation of several reactive free radicals, such as sulfur trioxide anion (.SO3–), peroxymonosulfate (–O3SOO.), and especially the sulfate (SO4. –) anion radicals. One such peroxidase in neutrophils is myeloperoxidase (MPO), which has been shown to form protein radicals. Although formation of (bi)sulfite-derived protein radicals is documented in isolated neutrophils, its involvement and role in in vivo inflammatory processes, has not been demonstrated. Therefore, we aimed to investigate (bi)sulfite-derived protein radical formation and its mechanism in LPS aerosol-challenged mice, a model of non-atopic asthma. Using immuno-spin trapping to detect protein radical formation, we show that, in the presence of (bi)sulfite, neutrophils present in bronchoalveolar lavage and in the lung parenchyma exhibit, MPO-catalyzed oxidation of MPO to a protein radical. The absence of radical formation in LPS-challenged MPO- or NADPH oxidase-knockout mice indicates that sulfite-derived radical formation is dependent on both MPO and NADPH oxidase activity. In addition to its oxidation by the MPO-catalyzed pathway, (bi)sulfite is efficiently detoxified to sulfate by the sulfite oxidase (SOX) pathway, which forms sulfate in a two-electron oxidation reaction. Since SOX activity in rodents is much higher than in humans, to better model sulfite toxicity in humans, we induced SOX deficiency in mice by feeding them a low molybdenum diet with tungstate. We found that mice treated with the SOX deficiency diet prior to exposure to (bi)sulfite had much higher protein radical formation than mice with normal SOX activity. Altogether, these results demonstrate the role of MPO and NADPH oxidase in (bi)sulfite-derived protein radical formation and show the involvement of protein radicals in a mouse model of human lung disease.

International audience; Background: Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom ofWalterinnesia aegyptia. Methods: Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionatedWalterinnesia aegyptiasnake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results: Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4-C6, C7-C8and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide fromBungarus candidusvenom. Conclusions: This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.

Background Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4-C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.

Free radicals were characterised by mass spectrometry (MS) and immuno-spin trapping on α-lactalbumin (α-lac) after a lactoperoxidase (LPO)-catalysed reaction. Although spin traps have historically been used for electron spin resonance (ESR) studies, here free radicals were detected by immuno-spin trapping using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), Western blotting, and MS; the latter yields information about the location of radical formation in the protein structure. Radical formation was highly specific towards Tyr residues; DMPO-trapped radicals were detected at Tyr18 and Tyr50, and the results also indicated a radical on Tyr36. These three oxidation sites on α-lac are all exposed on the surface of the protein, indicating that accessibility for LPO or DMPO was important. Applying immuno-spin trapping in combination with MS is a good approach to understand how different oxidation mechanisms affect the generation of radicals on proteins, and this oxidomics approach has future potential in food science.

Previous studies focused on catalyzed oxidation of (bi)sulfite, leading to the formation of reactive sulfur trioxide (•SO3−), peroxymonosulfate (−O3SOO•) and sulfate (SO4•−) anion radicals, which can damage target proteins and oxidize them to protein radicals. It is known that these very reactive sulfur- and oxygen-centered radicals can be formed by oxidation of (bi)sulfite by peroxidases. Myeloperoxidase (MPO), an abundant heme protein secreted from activated neutrophils that play a central role in host defense mechanisms, allergic reactions and asthma, is a likely candidate for initiating the respiratory damage caused by sulfur dioxide. The objective of the present study is to examine the oxidative damage caused by (bi)sulfite-derived free radicals in human neutrophils through formation of protein radicals. We used immuno-spin trapping and confocal microscopy to study the protein oxidations driven by sulfite-derived radicals. We found that the presence of sulfite can cause MPO-catalyzed oxidation of MPO to a protein radical in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. We trapped the MPO-derived radicals in situ using the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and detected them immunologically as nitrone adducts in cells. Our present study demonstrates that myeloperoxidase initiates (bi)sulfite oxidation leading to MPO radical damage possibly leading to (bi)sulfite-exacerbated allergic reactions.

The photosensitizer, methylene blue (MB), generates singlet oxygen ((1)O2) that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark MB inhibits reversibly, binding being accompanied by a bathochromic shift that can be used to show its displacement by other reversible inhibitors binding to the catalytic 'anionic' subsite (CAS), the peripheral 'anionic' subsite (PAS), or bridging them. Data concerning both reversible and irreversible inhibition are here reviewed. MB protects TcAChE from thermal denaturation, and differential scanning calorimetry reveals a ~8 °C increase in the denaturation temperature. The crystal structure of the MB/TcAChE complex reveals a single MB stacked against W279 in the PAS, pointing down the gorge towards the CAS. The intrinsic fluorescence of the irreversibly inhibited enzyme displays new emission bands that can be ascribed to N'-formylkynurenine (NFK); this was indeed confirmed using anti-NFK antibodies. Mass spectroscopy revealed that two Trp residues, Trp84 in the CAS, and Trp279 in the PAS, were the only Trp residues, out of a total of 14, significantly modified by photo-oxidation, both being converted to NFK. In the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Thus, photo-oxidative damage caused by MB involves targeted release of (1)O2 by the bound photosensitizer within the aqueous milieu of the active-site gorge.

4-Hydroxynonenal (4-HNE) is a major product of the oxidation of ω-6-polyunstaturated lipids and an effector of radical-mediated oxidative damage, whose analytical determination requires chemical derivatization. In this work, its reactivity with fluorinated phenylhydrazines was explored both under preparative and analytical settings. A five-step synthesis of 4-HNE on gram-scale with an overall yield of 30 % is described. Reaction of 4-HNE with ortho-, meta-, or para-CF3-phenylhydrazine, as well as with the 3,5-di-CF3, 2,4-di-CF3, or pentafluoro analogues, in MeCN with 0.5 mM TFA yields the corresponding hydrazones with rate constants kf of 2.8 ± 0.4, 1.7 ± 0.1, 3.0 ± 0.2, 0.6 ± 0.1, 0.5 ± 0.1, and 3.5 ± 0.5 M–1 s–1, respectively at 298 K. At higher temperatures, the hydrazones undergo intramolecular cyclization to form 1,6-dihydropyridazines that, depending on the solvent and temperature, may further react with the hydrazine to yield tetrahydropyridazine adducts and their oxidation products. Other reaction products were isolated, depending on the reaction conditions, and the complex reactivity of 4-HNE with the above nucleophiles is discussed. Due to the good yield and rate of formation of the hydrazone adducts, their stability and favorable UV absorbance, 2-(trifluoromethyl)phenylhydrazine and 2,3,4,5,6-pentafluorophenylhydrazine are the most interesting candidates for the development of rapid and efficient analytical derivatizations of 4-HNE.

Free radicals and other oxidation products were characterized on α-lactalbumin with electron spin resonance (ESR), immuno-spin trapping, and mass spectrometry (MS) after riboflavin-mediated oxidation. Radicals were detected using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in immuno-spin trapping with both enzyme-linked immunosorbent assay (ELISA) and Western blotting and further characterized with mass spectrometry. A DMPO-trapped radical was identified at His68 and another at one of the tyrosine residues, Tyr50 or Tyr36, respectively, generated by a type II or I mechanism. Not all tyrosyl radicals were trapped, as the secondary oxidation product, 3,4-dihydroxyphenylalanine (DOPA), was detected by mass spectrometry at Tyr18 and Tyr50. A further oxidation of DOPA resulted in the DOPA o-semiquinone radical, which was characterized by ESR. Both surface exposure and the neighboring residues in the local environment of the tertiary structure of α-lactalbumin seem to play a role in the generation of DMPO trapped radicals and secondary oxidation products.

The principle biological role of acetylcholinesterase (AChE) is the termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine (ACh) [1]. In keeping with its biological role, AChE possesses very high specific activity, functioning at a rate approaching that of a diffusion-limited reaction [2]. The toxicity of organophosphate nerve agents and insecticides is due to their potent inhibition of AChE [3]. The first generation of drugs for the treatment of Alzheimer’s disease are AChE inhibitors that act to overcome the cholinergic insufficiency resulting from degeneration of cholinergic nerve endings [4,5]. The electric organs of Torpedo californica and Electrophorus electricus are rich sources of AChE that is structurally homologous to that found in mammalian nerve and muscle [6]. Unexpectedly for such a rapid enzyme, the crystal structure of Torpedo californica AChE (TcAChE) revealed that its active site is near the bottom of a deep and narrow gorge lined by the rings of 14 conserved aromatic residues [7]. It contains two subsites: a catalytic triad very similar to that found in other serine hydrolases, and a so-called catalytic ‘anionic’ site (CAS), which recognizes the quaternary group of ACh. A peripheral ‘anionic’ site (PAS) at the entrance to the active site gorge mediates substrate trapping [8]. At both sites, recognition of ACh is primarily via π-cation interactions with conserved aromatic residues [9,10]. Recently, Weiner et al. [11] investigated the interaction of methylene blue (MB) with TcAChE. Methylene blue is a singlet-oxygen (1O2)-generating photosensitizer that is also a strong reversible inhibitor of TcAChE in the dark [12,13]. The mechanism of generation of 1O2 by light irradiation of MB is shown in Fig. 1a. The principal amino acid residues in proteins oxidized by 1O2 are His, Cys, Met, Tyr and Trp. Oxidation of Trp residues results in the formation principally of N-formylkynurenine (NFK) and kynurenine (Fig. 1b). The MB/TcAChE complex was used as a model system for studying the chemical and structural consequences of photo-oxidation of residues within the active-site gorge (it is assumed, a priori, that the photochemical properties of MB in its complex with TcAChE, viz., the life time of MB* and the efficacy of energy transfer to oxygen are similar to its properties in its uncomplexed state). Figure 1 Singlet oxygen formation from methylene blue and its reaction with Trp. a) Formation of singlet oxygen generated by the irradiation of methylene blue and b) oxidation of a tryptophan residue to NFK and kynurenine by 1O2. (T1: triplet state; ISC: intersystem ... Indeed, illumination of TcAChE in the presence of MB produces time- and concentration-dependent inactivation of the enzyme, attributable to 1O2. Addition of reversible inhibitors of TcAChE, such as edrophonium (ED), which interacts at the CAS [10], prior to sample illumination in the presence of MB protects the enzyme from inactivation, suggesting that the 1O2 produced by MB is localized to the active-site gorge. As mentioned, TcAChE contains 14 highly conserved aromatic amino acid residues that line ~40% of its surface [7]. Both the CAS and the PAS contain a tryptophan residue, Trp 84 and Trp 279, respectively (TcAChE numbering) [10]. The crystal structure of the MB/TcAChE complex [14,15] (Protein Data Bank entry 2w9i) reveals a single MB molecule stacked against Trp 279, at the top or the gorge, and oriented along the gorge axis towards the active site (Fig. 2). Comparison of the fluorescence spectra of photo-inactivated TcAChE and of the native protein showed changes characteristic of Trp modification that are also correlated with loss of activity [11]. Figure 2 Cross-section through the active-site gorge of TcAChE in the MB/TcAChE complex. The MB molecule is seen stacked against Trp 279 in the peripheral anionic site (PAS). Also shown are Trp 84, in the catalytic anionic subsite of the active site (CAS), and ... This study was undertaken to characterize and identify the specific structural modifications produced in the Trp residues of TcAChE by MB photo-inactivation. We used a recently developed antiserum to N-formyl-kynurenine [16,17], an oxidation product of Trp and of Trp residues (Fig. 2), in Western analysis of TcAChE samples that had been photo-oxidized in the presence of MB. A correlation was revealed between photo-inactivation of TcAChE and Trp oxidation to NFK. Mass spectrometry was then used to precisely identify which of the 14 Trps of TcAChE were oxidized to NFK in the photo-inactivated samples, as well as to identify other potential structural Trp modifications. It was revealed that MB-mediated photo-oxidation involves primarily Trp 84, within the CAS, and Trp 279, within the PAS.

The objective of this study was to determine the effect of (bi)sulfite (hydrated sulfur dioxide) on human neutrophils and the ability of these immune cells to produce reactive free radicals due to (bi)sulfite oxidation. Myeloperoxidase (MPO) is an abundant heme protein in neutrophils that catalyzes the formation of cytotoxic oxidants implicated in asthma and inflammatory disorders. In this study sulfite ((•)SO(3)(-)) and sulfate (SO(4)(•-)) anion radicals are characterized with the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the reaction of (bi)sulfite oxidation by human MPO and human neutrophils via sulfite radical chain reaction chemistry. After treatment with (bi)sulfite, phorbol 12-myristate 13-acetate-stimulated neutrophils produced DMPO-sulfite anion radical, -superoxide, and -hydroxyl radical adducts. The last adduct probably resulted, in part, from the conversion of DMPO-sulfate to DMPO-hydroxyl radical adduct via a nucleophilic substitution reaction of the radical adduct. This anion radical (SO(4)(•-)) is highly reactive and, presumably, can oxidize target proteins to protein radicals, thereby initiating protein oxidation. Therefore, we propose that the potential toxicity of (bi)sulfite during pulmonary inflammation or lung-associated diseases such as asthma may be related to free radical formation.

International audience; Continuous wave X-band EPR spectroscopy was used as an analytical tool to follow the formation of nitroxide radicals after the reaction between various nucleophiles and 2-methyl-2-nitrosopropane (MNP). The addition of amines or of trihalogeno-acetic acids onto the nitroso moiety was found to depend on experimental parameters such as exposure to white light or the nature of the solvent, while no nitroxide adducts were ever detected when alcohols or weak carboxylic acids were used. In contrast to results previously obtained with cysteine, the reaction of ten standard amino acids (Gly, Lys, Arg, His, Trp, Ser, Thr, Tyr, Asp and Glu) on MNP never led to an EPR observable adduct, further confirming the particular reactivity of this nitroso compound towards thiols.

International audience; The combination of electron paramagnetic resonance (EPR) and mass spectrometry (MS) was used as an efficient tool to elucidate the mechanism of an ene-reaction between a dienol compound and 2-methyl-2-nitrosopropane. (C) 2010 Elsevier Ltd. All rights reserved.

International audience; An approach combining EPR spectroscopy and electrospray ionisation mass spectrometry was employed to study the reactivity of two alkylthiols and of cysteine towards 2-methyl-2-narosopropane (MNP) in the presence of H(2)O(2) Depending on the experimental conditions, various nitroxides could be EPR-detected, while their diamagnetic derivatives were structurally characterised after tandem mass spectrometry experiments Sample dilution in methanol before electrospray ionisation was also found to generate methyl-hydroxylamine derivatives and could thus constitute a simple derivatisation process to stabilise hardly MS-detectable radical species Upon white light irradiation, the three thiols tested led to alkoxynitroxides according to an inverted spin trapping process On the other hand, only the reduced form of cysteine was found to react with MNP in the dark, yielding an alkylthionitroxide adduct via a Forrester-Hepburn mechanism Besides a better understanding of the reactivity of thiols towards nitroso compounds, the results obtained underlined the particular behaviour of cysteine, suggesting that the use of a molecule as simple as MNP could allow a nitroxide moiety to be linked to a cysteinyl residue

International audience; The spontaneous addition of air oxygen to a dienolic compound, yielding a cyclic peroxide, was followed by spin trapping (ST) combined with EPR spectroscopy and mass spectrometry (MS). Using two different nitrones, the ST/EPR study allowed the detection of the spin adduct of a radical intermediate, and the radical centre in the addend was identified after similar experiments performed with two different (13)C-labelled analogues of the substrate. The media were also submitted to electrospray ionisation, in both positive and negative modes, for structural characterisation of the spin adducts by tandem mass spectrometry. This allowed the structure of the hydroxylamine derivatives of the nitroxides formed to be identified. Following these results, a mechanism pathway was proposed for this autoxidation.

International audience; The title compound behaves as an efficient cheletropic trap for both NO and NO(2) radicals in the presence of oxygen, yielding EPR observable nitroxide and alkoxynitroxide, respectively.

Ants use venom for predation, defence and communication, however, the molecular diversity, function and potential applications of ant venom remains understudied compared to other venomous lineages such as arachnids, snakes and cone snails. In this work, we used a multidisciplinary approach that encompassed field work, proteomics, sequencing, chemical synthesis, structural analysis, molecular modelling, stability studies, and a series ofin vitroandin vivobioassays to investigate the molecular diversity of the venom of the AmazonianPseudomyrmex penetratorants. We isolated a potent insecticidal heterodimeric peptide Δ-pseudomyrmecitoxin-Pp1a (Δ-PSDTX-Pp1a) composed of a 27-residue long A-chain and a 33-residue long B-chain crosslinked by two disulfide bonds in an antiparallel orientation. We chemically synthesised Δ-PSDTX-Pp1a, its corresponding parallel AA and BB homodimers, and its monomeric chains and demonstrated that Δ-PSDTX-Pp1a had the most potent insecticidal effects in blow fly assays (LD50= 3 nM). Molecular modelling and circular dichroism studies revealed strong alpha-helical features, indicating its cytotoxic effects could derive from membrane disruption, which was further supported by insect cell calcium assays. The native heterodimer was also substantially more stable against proteolytic degradation (t1/2=13 h) than its homodimers or monomers (t1/2Pseudomyrmex penetratorvenom and in-depth characterisation of Δ-PSDTX-Pp1a provide novel insights in the structural complexity of ant venom, and further exemplifies how nature exploits disulfide-bond formation and dimerization to gain an evolutionary advantageviaimproved stability; a concept that is also highly relevant for the design and development of peptide therapeutics, molecular probes and bioinsecticides.