Sialic acid attenuates the cytotoxicity of the lipid hydroperoxides HpODE and HpETE

Reduction of peroxide molecular species is an essential function in living organisms. In previous studies, we proposed a new function for the sialic acid N-acetylneuraminic acid (Neu5Ac)—that of antioxidant/hydrogen peroxide scavenging agent. On the basis of the reaction scheme, Neu5Ac is thought to act as a general antioxidant of all hydroperoxide-type species (R-OOHs). The concentration of tert-butyl hydroperoxide (t-BuOOH) decreased after co-incubation with N-acetylneuraminic acid. Neu5Ac also decreased the R-OOH concentration in solutions of peroxylinolenic acid (13(S)-hydroperoxy-(9Z,11E)-octadecadienoic acid, HpODE) and peroxyarachidonic acid (15(S)-hydroperoxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid, HpETE)—two lipid hydroperoxides that participate in many physiological events. Moreover, the cytotoxicity of both these lipid hydroperoxides was attenuated by reaction with Neu5Ac acid. Our results suggest that N-acetylneuraminic acid is a potential antioxidant of most hydroperoxides that accumulate in organisms.     

Highly Sensitive Flow Injection Analysis of Lipid Hydroperoxides in Foodstuffs

Lipid hydroperoxides in oils and foods were measured by a flow injection analysis system with high sensitivity and selectivity. After sample injection, lipid hydroperoxides were reacted with diphenyl-1-pyrenylphosphine (DPPP) in a stainless steel coil, then the fluorescence intensity of DPPP oxide, that was produced by the reaction, was monitored. By this method, trilinolein hydroperoxide showed good linearity between 0.4 and 79pmol and their detection limits were 0.2pmol (signal-to-noise ratio = 3). The method made it possible to inject samples at 2-min intervals. There was a good agreement of the amounts of lipid hydroperoxides in oils and foods between by the batch method with DPPP and by the proposed method (coefficient of correlation: r = 0.999; n = 21; peroxide value = 0.09–167 meq/g). With this method, the calibration graph of trilinolein hydroperoxide was useful for all samples tested.     

Ohr Protects Corynebacterium glutamicum against Organic Hydroperoxide Induced Oxidative Stress

Ohr, a bacterial protein encoded by the Organic Hydroperoxide Resistance (ohr) gene, plays a critical role in resistance to organic hydroperoxides. In the present study, we show that the Cys-based thiol-dependent Ohr of Corynebacterium glutamicum decomposes organic hydroperoxides more efficiently than hydrogen peroxide. Replacement of either of the two Cys residues of Ohr by a Ser residue resulted in drastic loss of activity. The electron donors supporting regeneration of the peroxidase activity of the oxidized Ohr of C. glutamicum were principally lipoylated proteins (LpdA and Lpd/SucB). A Δohr mutant exhibited significantly decreased resistance to organic hydroperoxides and marked accumulation of reactive oxygen species (ROS) in vivo; protein carbonylation was also enhanced notably. The resistance to hydrogen peroxide also decreased, but protein carbonylation did not rise to any great extent. Together, the results unequivocally show that Ohr is essential for mediation of organic hydroperoxide resistance by C. glutamicum.     

Oxidizing substrate specificity of Mycobacterium tuberculosis alkyl hydroperoxide reductase E: kinetics and mechanisms of oxidation and overoxidation

Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~ 10³ times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pK_a reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~ 10⁸ and 10⁵ M^− 1 s^− 1, respectively, at pH 7.4 and 25 °C) were much faster than expected according to the Brønsted relationship with leaving-group pK_a. A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity.     

Enhanced stereoselectivity of α-mannosylation under thermodynamic control using trichloroacetimidates

O-Specific polysaccharides of Vibrio cholerae O1, serotypes Inaba and Ogawa, consist of α-(1→2)-linked N-(3-deoxy-l-glycero-tetronyl)perosamine (4-amino-4,6-dideoxy-d-mannose). The blockwise synthesis of larger fragments of such O-PSs involves oligosaccharide glycosyl donors that contain a nonparticipating 2-O-glycosyl group at the position vicinal to the anomeric center where the new glycosidic linkage is formed. Such glycosyl donors may bear at C-4 either a latent acylamino (e.g., azido) or the 3-deoxy-l-glycero-tetronamido group. While monosaccharide glycosyl donors, even those bearing a nonparticipating group at O-2 (e.g., methyl), and the 4-N-(3-deoxy-l-glycero-tetronyl) side chain form α-linked oligosaccharides with excellent stereoselectivity, α-mannosylation with analogous oligosaccharide donors in this series is adversely affected by the presence of the side chain. Consequently, the unwanted β-product is formed in a considerable amount. Conducting the reaction at elevated temperature under thermodynamic control substantially enhances formation of the α-linked oligosaccharide. This effect is much more pronounced when glycosyl trichloroacetimidates, rather than thioglycosides or glycosyl chlorides, are used as glycosyl donors.     

Inverse correlation between cyclic GMP and tyrosine aminotransferase degradation in rat hepatoma tissue culture cells

[1,2-³H]Cholesterol was epoxidized to radioactive cholesterol α- and β-epoxides (5,6α-epoxy-5α- and 5,6β-epoxy-5β-cholestan-3β-ols) in the ratio 1:4 by hepatic microsomal lipid hydroperoxides (MsOOH, 1 mM as active oxygen) in the presence of ferrous ion. MsOOH could be replaced by methyl linoleate hydroperoxides (MOOH) under the same conditions although the latter was less effective than the former. None of cumene hydroperoxide, t-butyl hydroperoxide, and hydrogen peroxide was an effective oxidant even at 10 mM. Neither ADP nor EDTA had an effect on the epoxidation of cholesterol by MsOOH as well as by MOOH. Ferrous ion could not be replaced by ferric ion in the hydroperoxide-mediated epoxidation. Cyanide anion potentially inhibited the reaction.     

Detection of Picomole Levels in Lipid Hydroperoxides by a Chemiluminescence Assay

A highly sensitive and simple chemiluminescent method for the quantitation of lipid hydroperoxides at the picomole level is described. The method is based on detecting the chemiluminescence generated during the oxidation of luminol by the reaction with hydroperoxide and cytochrome c under mild conditions. A semilogarithmic relationship was observed between the hydroperoxide added and the chemiluminescence produced. For lipid hydroperoxides, cytochrome c was a most favorable catalyst for generating the chemiluminescence, rather than cytochrome c heme peptide and horseradish peroxidase. This method had high sensitivity to methyl linoleate hydroperoxide, arachidonic acid hydroperoxide and cholesterol hydroperoxide, but low to /-butyl hydroperoxide, J-butyl perbenzoate, diacyl peroxides (lauroyl peroxode and benzoyl peroxide) and dialkyl peroxides (di-/-butyl peroxide and dicumyl peroxide).     

Radical intermediates in peroxide-dependent reactions catalyzed by cytochrome P-450

Highly purified liver microsomal cytochrome P-450 acts as a peroxygenase in catalyzing the reaction, RH+ XOOH→ROH+XOH, Where RH represents any of a large variety of foreign or physiological substrates and ROH the corresponding product, and XOOH represents any of a series of peroxy compounds such as hydroperoxides or peracids serving as the oxygen donor and XOH the resulting alcohol or acid. Several experimental approaches in this and other laboratories have yielded results compatible with a homolytic mechanism of oxygen-oxygen bond cleavage but not with the heterolytic formation of a common iron-oxo intermediate from the various peroxides. Recently, we have found a new reaction, catalyzed by the reconstituted system containing the phenobarbital-inducible form of P-450, which catalyzes the reductive cleavage of hydroperoxides: XRR’C-OOH+ NADPH+H⁺→ XR’CO + R’H+H₂O + NADP⁺ Thus, cumyl hydroperoxide yields acetophenone and methane, and 13-hydroperoxyoctadeca-9, 11-dienoic acid yields pentane and an as yet unidentified additional product. Since hydroperoxide reduction does not produce the corresponding alcohol, it is concluded that homolytic cleavage of the oxygen-oxygen bond occurs with rearrangement of the resulting alkoxy radical. Studies are in progress to determine how broad a role the new hydroperoxide cleavage reaction plays in the biological peroxidation of lipids.     

Two pathways of photoproduction of organic hydroperoxides on the donor side of photosystem 2 in subchloroplast membrane fragments

Earlier the catalase-insensitive formation of organic hydroperoxides (via the interaction of organic radicals produced due to redox activity of P₆₈₀ ^+· (or TyrZ^·) with molecular oxygen) has been found in Mn-depleted PS2 preparations (apo-WOC-PS2) by Khorobrykh et al. (Biochemistry 50:10658–10665, 2011). The present work describes a second pathway of the photoproduction of organic peroxides on the donor side of PS2. It was shown that illumination of CaCl₂-treated PS2 membranes (deprived of the PS2 extrinsic proteins without removal of the Mn-containing water-oxidizing complex) (CaCl₂-PS2) led to the photoproduction of highly lipophilic organic hydroperoxides (LP-OOH) (in amount corresponding to 1.5 LP-OOH per one reaction center of PS2) which significantly increased upon the addition of exogenous electron acceptor potassium ferricyanide (to 4.2 LP-OOH per one reaction center). Addition of catalase (200 U/ml) before illumination inhibited ferricyanide-induced photoproduction of hydroperoxides while no effect was obtained by adding catalase after illumination or by adding inactivated catalase before illumination. The hydroperoxide photoproduction was inhibited by the addition of exogenous electron donor for PS2, diphenylcarbazide or diuron (inhibitor of the electron transfer in PS2). The addition of exogenous hydrogen peroxide to the CaCl₂-PS2 led to the production of highly lipophilic organic hydroperoxides in the dark (3.2 LP-OOH per one reaction center). We suggest that the photoproduction of highly lipophilic organic hydroperoxides in CaCl₂-PS2 preparations occurs via redox activity of H₂O₂ produced on the donor side of PS2.     

Synthesis of glycosyl halides and glycosides via 1-O-sulfonyl derivatives

The reaction of an aldose derivative containing a free anomeric hydroxyl group with trifluoromethanesulfonic anhydride or methanesulfonic anhydride, in the presence of halide ion and s-collidine, furnishes a glycosyl halide; if an alcohol is then introduced, glycoside synthesis is effected in an overall, “one-pot” reaction. Several α-d-glucopyranosides, including disaccharides, have been prepared in high yield by using 2,3,4,6-tetra-O-benzyl-d-glucose as the aldose, and generating the corresponding glycosyl bromide(s) in situ. As a halide-exchange step is incorporated in the reaction sequence, orthoacetate formation was favored in reactions of 2,3,4,6-tetra-O-acetyl-d-glucose, such as occurs with per-O-acetylglycosyl halides. Methanesulfonic anhydride promotes glycosidation or orthoester formation in the absence of halide ion, as well as in its presence, whereas formation of an intermediate glycosyl halide appears to be necessary in order to moderate the more vigorous reactions of the trifluoro derivative. The analogous reaction of methanesulfonyl chloride with an aldose provides a ready route to glycosyl chlorides. Under the conditions employed for these various syntheses, acid-sensitive protecting groups may be used, including cyclic and acyclic acetals and O-trityl substituents.     

Theoretical analysis of a site-specific chemiluminescence reaction and its application to quantitation of lipid hydroperoxides

A system was designed for chemiluminescent measurement of lipid hydroperoxides by their site-specific reaction in sodium dodecylsulfate micelles. Ferrous ion-induced decomposition of lipid hydroperoxides in the sodium dodecylsulfate micelles resulted in strong chemiluminescence of the Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (CLA). After addition of ferrous sulfate to the micelles containing lipid hydroperoxide and luciferin, the chemiluminescence intensity reached a maximum rapidly and then decreased. The sequence of this reaction was elucidated by theoretical analysis, which demonstrated that the maximum chemiluminescence intensity is proportional to the initial concentration of hydroperoxide. Good linear relationships were observed between the maximum counts of chemiluminescence and the amounts of hydroperoxides of linoleic acid, phosphatidylcholine, choresterol (5 alpha), cumene and tert-butyl and hydrogen peroxide. This chemiluminescence method was simple and sensitive enough to detect picomole levels of linoleic acid and phosphatidylcholine hydroperoxides.     

Use of ionically tagged glycosyl donors in the synthesis of oligosaccharide libraries

We have developed a new procedure based on the random glycosyl reaction of a partially benzoylated glycosyl acceptor with a glycosyl donor containing a 4,6-O-(4-methoxycarbonylbenzylidene) protecting group as a masked/caged ion-tag. Glycosylated products are ionically tagged by saponification of the methyl ester and the use of this anion-tag greatly simplifies the separation of the desired oligosaccharides from unreacted or excess glycosyl acceptors as well as from over-glycosylated oligosaccharides. In addition, the use of partially benzoylated acceptors greatly improves their solubility in dichloromethane increasing the yield of product formation and, also, of altering the distribution of positional isomers in favor of products derived by reaction of the donors at hydroxyl groups which otherwise would be considerably less reactive. Using this new approach in random glycosyl reactions, several oligosaccharide libraries were readily prepared in overall yields of 60–70% and the individual positional isomers present in the libraries were identified using the ‘reductive-cleavage’ method.     

Evidence for glutathione peroxidase activities in cultured plant cells

Extracts from cultured plant cells of spinach, maize and sycamore and from Lemna plants contain detectable glutathione peroxidase activity, using either hydrogen peroxide or t-butyl hydroperoxide as substrates. Using extracts from cultured maize cells, two peaks of glutathione peroxidase activity could be resolved by a combination of gel filtration and ion exchange chromatography. One peak was eluted along with glutathione transferase activity; the second was distinct from both glutathione transferase and ascorbic acid peroxidase, and was active with both hydrogen peroxide and organic hydroperoxides. It seems likely that at least two enzymes with glutathione peroxidase activity exist in higher plant cells.     

Chromate-mediated free radical generation from cysteine,penicillamine, hydrogen peroxide,and lipid hydroperoxides

The Cr(VI)-mediated free radical generation from cystein, penicillamine, hydrogen peroxide, and model lipid hydroperoxides was investigated utilizing the electron spin resonance (ESR) spin trapping technique. Incubation of Cr(VI) with cysteine (Cys) generated cysteinyl radical. Radical yield depended on the relative concentrations of Cr(VI) and Cys. The radical generation became detectable at a cysteine: Cr(VI) ration of about 5, reached its highest level at a ratio of 30, and declined thereafter. Cr(VI) or Cys alone did not generate a detectable amount of free radicals. Similar results were obtained with penicillamine. Incubation of Cr(VI), Cys or penicillamine adn H₂O₂ led to hydroxyl (·OH) radical generation, which was verified by quantitative competition experiments utilizing ethanol. The mechanism for ·OH radical generation is considered to be a Cr(VI)-mediated Fenton-like reaction. When model lipid hydroperoxides such as t-butylhydroperoxide and cumene hydroperoxide were used in place of H₂O₂, hydroperoxide-derived free radicals were produced. Since thiols, such as Cys, exist in cellular systems at relatively high concentrations, Cr(VI)-mediated free radical generation in the presence of thiols may participate in the mechanisms of Cr(VI)-induced toxicity and carcinogenesis.     

The 1-Cys peroxiredoxin, a regulator of seed dormancy, functions as a molecular chaperone under oxidative stress conditions

Peroxiredoxins are antioxidative enzymes that catalyze the reduction of alkyl hydroperoxides to alcohols and hydrogen peroxide to water. 1-Cys peroxiredoxins (1-Cys Prxs) perform important roles during late seed development in plants. To characterize their biochemical functions in plants, a 1Cys-Prx gene was cloned from a Chinese cabbage cDNA library and designated as “C1C-Prx”. Glutamine synthetase (GS) protection and hydrogen peroxide reduction assays indicated that C1C-Prx was functionally active as a peroxidase. Also C1C-Prx prevented the thermal- or chemical-induced aggregation of malate dehydrogenase and insulin. Hydrogen peroxide treatment changed the mobility of C1C-Prx on a two-dimensional gel, which implies overoxidation of the conserved Cys residue. Furthermore, after overoxidation, the chaperone activity of C1C-Prx increased approximately two-fold, but its peroxidase activity decreased to the basal level of the reaction mixture without enzyme. However, according to the structural analysis using far-UV circular dichroism spectra, intrinsic tryptophan fluorescence spectra, and native-PAGE, overoxidation did not lead to a conformational change in C1C-Prx. Therefore, our results suggest that 1-Cys Prxs function not only to relieve mild oxidative stresses but also as molecular chaperones under severe conditions during seed germination and plant development, and that overoxidation controls the switch in function of 1-Cys-Prxs from peroxidases to molecular chaperones.     

Limitations of the hemoglobin method for detecting lipid hydroperoxides

The method using peroxidase activity of hemoglobin (Hb) for the determination of lipid peroxides, trilinoleoylglycerol hydroperoxides and phosphatidylcholine hydroperoxides as substrates and tetramethyl benzidine as electron donor for the peroxidase reaction of Hb. The reactivities of these substrates were different. Some electron donors were tested for peroxidase activity of Hb, but none showed a complete reduction of methyl linoleate hydroperoxides. Front these results, the Hb method needs to be carefully applied to biological materials that contain mixtures of different typos of lipid classes.     

Characterization of the extended carbohydrate binding site of concanavalin A: Specificity for interaction with the nonreducing termini of α-(1→2)-linked disaccharides

p-Nitrophenyl 2-O-α-d-galactopyranosyl-α-d-mannopyranoside and p-nitrophenyl 2-O-α-d-glucopyranosyl-α-d-mannopyranoside were synthesized and the interactions of these disaccharides with concanavalin A (con A) were characterized. The kinetics of binding of the galactopyranosyl-containing disaccharide to con A were found to be similar to those observed with monosaccharides in that monophasic time dependencies for binding were observed. The glucopyranosyl-containing disaccharide, however, exhibited biphasic time dependencies which were similar to those previously observed for the binding of p-nitrophenyl 2-O-α-d-mannopyranosyl-α-d-mannopyranoside to con A. These results support a model wherein the α-(1→2)-linked disaccharides which exhibit biphasic binding kinetics must be able to bind to con A in two different and mutually exclusive orientations. The ability to bind to con A in two orientations is shared by α-(1→2)-linked disaccharides in which both glycosyl residues can interact separately with the primary glycosyl binding site of con A. According to the model, the initial fast phase of the biphasic reaction reflects binding of the ligand in two orientations so that two complexes are formed in amounts determined by the relative values of the rate constants for formation of each complex. The subsequent slow phase is proposed to reflect a slow equilibration of the less stable complex to the thermodynamically more stable one. In the more stable complex, the glycosyl residue at the reducing end of the disaccharide occupies the primary glycosyl binding site. The added stability of this complex is attributed to extended interactions between con A and groups on the second glycosyl residue. An axial orientation of OH-2 of the second glycopyranosyl residue appears to be the most important determinant for the extended interaction.     

Phosphorylase-catalyzed N-formyl-α-glucosaminylation of maltooligosaccharides

This paper describes the phosphorylase-catalyzed enzymatic N-formyl-α-glucosaminylation of maltooligosaccharides for direct incorporation of 2-deoxy-2-formamido-α-d-glucopyranose units into maltooligosaccharides. When the reaction of 2-deoxy-2-formamido-α-d-glucopyranose-1-phosphate (GlcNF-1-P) as the glycosyl donor and maltotetraose as a glycosyl acceptor was performed in the presence of phosphorylase, the N-formyl-α-d-glucosaminylated pentasaccharide was produced, as confirmed by MALDI-TOF MS. Furthermore, the glucoamylase-catalyzed reaction of the crude products supported that the 2-deoxy-2-formamido-α-d-glucopyranoside unit was positioned at the non-reducing end of the pentasaccharide. The pentasaccharide was isolated from the crude products and its structure was further determined by the ¹H NMR analysis. On the other hand, when the phosphorylase-catalyzed reactions of maltotriose and maltopentaose using GlcNF-1-P were conducted, no N-formyl-α-glucosaminylation took place in the former system, whereas the latter system gave N-formyl-α-d-glucosaminylated oligosaccharides with various degrees of polymerization. These results could be explained by the recognition behavior of phosphorylase toward maltooligosaccharides.     

The relative effectiveness of human plasma glutathione peroxidase as a catalyst for the reduction of hydroperoxides by glutathione

To reveal clues to the function of human plasma glutathione peroxidase (GPx), we investigated its catalytic effectiveness with a variety of hydroperoxides. Comparisons of hydroperoxides as substrates for plasma GPx based on the ratio ofV _max /K _m were blocked by the limited solubility of the organic hydroperoxides, which prevented kinetic saturation of the enzyme at the chosen glutathione concentration. Therefore, we compared the hydroperoxides by the fold increase in the apparent first-order rate constants of their reactions with glutathione owing to catalysis by plasma GPx. The reductions of aromatic and small hydrophobic hydroperoxides (cumene hydroperoxide,t-amyl hydroperoxide,t-butyl hydroperoxide, paramenthane hydroperoxide) were better catalyzed by plasma GPx than were reductions of the more “physiological” substrates (linoleic acid hydroperoxide, hydrogen peroxide, peroxidized plasma lipids, and oxidized cholesterol).     

Bildung von ‘desoxo-narchinol A” aus Nardosinon

In boiling methanol, the sesquiterpene peroxide nardosinone (1), isolated from Nardostachys chinensis Batalin (Valerianaceae), forms a C₁₂-compound (“desoxo-narchinol A”) (5) besides several C₁₅-compounds. 5 has the same carbon skeleton as narchinol A (6), occurring in the same plant.