Identification of proteins in activated human neutrophils susceptible to tyrosyl radical attack. A proteomic study using a tyrosylating fluorophore

Tyrosyl radicals cross-linked to protein tyrosine residues (tyrosylated proteins) represent hallmarks of neutrophil-mediated injury at the inflammatory locus. Yet the proteins targeted by tyrosyl radicals in an intact cellular system remain to be elucidated. Here, we show that tyrosyl radicals generated by human neutrophils after activation by phorbol 12-myristate 13-acetate (PMA), interferon-gamma (IFN-gamma) or TNF-alpha could act in an autocrine manner by cross-linking to endogenous proteins. We have identified the tyrosylated proteins by using a membrane-impermeable tyrosine analogue, tyramine coupled to fluorescein (TyrFluo), in combination with proteomics techniques. Confocal microscopy images indicated that initially the tyrosylated proteins were localized in patches at the cell surface to become internalized subsequently. In the neutrophil membrane-associated proteome, lactoferrin was the prime target of tyrosylation. Out of three isoforms identified, an 80 kDa neutral isoform was tyrosylated more extensively than the 85 kD basic isoform, particularly after PMA activation. Although all three stimuli induced tyrosylation of the filamentous component vimentin, additional tyrosylated vimentin fragments were detected after IFN-gamma- and TNF-alpha-stimulation. Moreover, upon activation the bulk of vimentin behaved as a dimer (M(r) 120 kDa) being slightly tyrosylated, yet phosphorylated at Thr-425 possibly as a requirement for its externalization. Unexpectedly, bovine catalase added to end tyrosyl radicals formation was detected as a highly tyrosylated neutrophil-associated protein. A moderate stimulus-dependent tyrosylation of ATP synthase-beta, alpha-enolase, glyceraldehyde 3-phosphate dehydrogenase, cytokeratin-10, filamin-A, and annexin-I was also observed. When the membrane-permeable probe (acetylTyrFluo) was used, protein tyrosylation was not observed indicating that the intracellular proteins were well protected against oxidative attack. This study shows that human neutrophils can modulate their proteome via a tyrosine oxidation pathway induced by pro-inflammatory mediators.     

Probing the free radicals formed in the metmyoglobin-hydrogen peroxide reaction

The reaction between metmyoglobin and hydrogen peroxide results in the two-electron reduction of H2O2 by the protein, with concomitant formation of a ferryl-oxo heme and a protein-centered free radical. Sperm whale metmyoglobin, which contains three tyrosine residues (Tyr-103, Tyr-146, and Tyr-151) and two tryptophan residues (Trp-7 and Trp-14), forms a tryptophanyl radical at residue 14 that reacts with O2 to form a peroxyl radical and also forms distinct tyrosyl radicals at Tyr-103 and Tyr-151. Horse metmyoglobin, which lacks Tyr-151 of the sperm whale protein, forms an oxygen-reactive tryptophanyl radical and also a phenoxyl radical at Tyr-103. Human metmyoglobin, in addition to the tyrosine and tryptophan radicals formed on horse metmyoglobin, also forms a Cys-110-centered thiyl radical that can also form a peroxyl radical. The tryptophanyl radicals react both with molecular oxygen and with the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) traps the Tyr-103 radicals and the Cys-110 thiyl radical of human myoglobin, and 2-methyl-2-nitrosopropane (MNP) traps all of the tyrosyl radicals. When excess H2O2 is used, DBNBS traps only a tyrosyl radical on horse myoglobin, but the detection of peroxyl radicals and the loss of tryptophan fluorescence support tryptophan oxidation under those conditions. Kinetic analysis of the formation of the various free radicals suggests that tryptophanyl radical and tyrosyl radical formation are independent events, and that formation of the Cys-110 thiyl radical on human myoglobin occurs via oxidation of the thiol group by the Tyr-103 phenoxyl radical. Peptide mapping studies of the radical adducts and direct EPR studies at low temperature and room temperature support the conclusions of the EPR spin trapping studies.     

Activation of the iron-containing B2 protein of ribonucleotide reductase by hydrogen peroxide

The active form of protein B2, the small subunit of ribonucleotide reductase, contains two dinuclear Fe(III) centers and a tyrosyl radical. The inactive metB2 form also contains the same diferric complexes but lacks the tyrosyl radical. We now demonstrate that incubation of metB2 with hydrogen peroxide generates the tyrosyl radical. The reaction is optimal at 5.5 nM hydrogen peroxide, with a maximum of 25-30% tyrosyl radical being formed after approximately 1.5 hr of incubation. The activation reaction is counteracted by a hydrogen peroxide-dependent reduction of the tyrosyl radical. It is likely that the generation of the radical proceeds via a ferryl intermediate, as in the proposed mechanisms for cytochrome P-450 and the peroxidases.     

Oxidation pathways underlying the pro-oxidant effects of apigenin

Apigenin, a natural flavone, is emerging as a promising compound for the treatment of several diseases. One of the hallmarks of apigenin is the generation of intracellular reactive oxygen species (ROS), as judged by the oxidation of reduced dichlorofluorescein derivatives seen in many cell types. This study aimed to reveal some mechanisms by which apigenin can be oxidized and how apigenin-derived radicals affect the oxidation of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (H₂DCF), a probe usually employed to detect intracellular ROS. Apigenin induced a rapid oxidation of H₂DCF in two different immortalized cell lines derived from rat and human hepatic stellate cells. However, apigenin did not generate ROS in these cells, as judged by dihydroethidium oxidation and extracellular hydrogen peroxide production. In cell-free experiments we found that oxidation of apigenin leads to the generation of a phenoxyl radical, which directly oxidizes H₂DCF with catalytic amounts of hydrogen peroxide. The net balance of the reaction was the oxidation of the probe by molecular oxygen due to redox cycling of apigenin. This flavonoid was also able to deplete NADH and glutathione by a similar mechanism. Interestingly, H₂DCF oxidation was significantly accelerated by apigenin in the presence of horseradish peroxidase and xanthine oxidase, but not with other enzymes showing peroxidase-like activity, such as cytochrome c or catalase. We conclude that in cells treated with apigenin oxidation of reduced dichlorofluorescein derivatives does not measure intracellular ROS and that pro- and antioxidant effects of flavonoids deduced from these experiments are inconclusive and must be confirmed by other techniques.     

Tyrosinase scavenges tyrosyl radical

Melanosomes scavenged tyrosyl radical that was generated by ultraviolet irradiation of tyrosine. Purified mushroom tyrosinase also removed tyrosyl radical in a dose-dependent manner. To elucidate the underlying mechanism, we analyzed the reaction of mushroom tyrosinase with tyrosyl radical generated by horseradish peroxidase and hydrogen peroxide. Resting tyrosinase, which contained a small amount of oxytyrosinase, did not oxidize tyrosine to DOPAchrome until horseradish peroxidase exhausted H(2)O(2) and thereafter the enzyme recovered its full activity. During the inhibition period most tyrosine was converted to dityrosine, suggesting that only a small amount of tyrosyl radical was enough to interact with a fraction of tyrosinase which was in the active oxy-form. When horseradish peroxidase and H(2)O(2) were added to oxytyrosinase, which was prepared by allowing it to turn over beforehand, DOPAchrome production was abolished with an accelerated consumption of H(2)O(2). Dityrosine formation was totally suppressed and tyrosine concentration stayed constant during the inhibition period with a concomitant production of O(2). The results are accounted for by a mechanism in which tyrosyl radical is reduced to tyrosine by oxytyrosinase and the resulting met-form reacts with H(2)O(2) to return to the oxy-form.     

The effect of neighboring amino acid residues and solution environment on the oxidative stability of tyrosine in small peptides

The effects of neighboring residues and formulation variables on tyrosine oxidation were investigated in model dipeptides (glysyl tyrosine, N-acetyl tyrosine, glutamyl tyrosine, and tyrosyl arginine) and tripeptide (lysyl tyrosyl lysine). The tyrosyl peptides were oxidized by light under alkaline conditions by a zero-order reaction. The rate of the photoreaction was dependent on tyrosyl pK(a), which was perturbed by the presence of neighboring charged amino acid residues. The strength of light exposure, oxygen headspace, and the presence of cationic surfactant, cetyltrimethylammonia chloride had a significant effect on the kinetics of tyrosyl photo-oxidation. Tyrosine and model tyrosyl peptides were also oxidized by hydrogen peroxide/metal ions at neutral pH. Metal-catalyzed oxidation followed first-order kinetics. Adjacent negatively charged amino acids accelerated tyrosine oxidation owing to affinity of the negative charges to metal-ions, whereas positively charged amino acid residues disfavored the reaction. The oxidation of tyrosine in peptides was greatly affected by the presence of adjacent charged residues, and the extent of the effect depended on the solution environment.     

Nitration and oxidation of a hydrophobic tyrosine probe by peroxynitrite in membranes: comparison with nitration and oxidation of tyrosine by peroxynitrite in aqueous solution.

It has been reported that peroxynitrite will initiate both oxidation and nitration of tyrosine, forming dityrosine and nitrotyrosine, respectively. We compared peroxynitrite-dependent oxidation and nitration of a hydrophobic tyrosine analogue in membranes and tyrosine in aqueous solution. Reactions were carried out in the presence of either bolus addition or slow infusion of peroxynitrite, and also using the simultaneous generation of superoxide and nitric oxide. Results indicate that the level of nitration of the hydrophobic tyrosyl probe located in a lipid bilayer was significantly greater than its level of oxidation to the corresponding dimer. During slow infusion of peroxynitrite, the level of nitration of the membrane-incorporated tyrosyl probe was greater than that of tyrosine in aqueous solution. Evidence for hydroxyl radical formation from decomposition of peroxynitrite in a dimethylformamide/water mixture was obtained by electron spin resonance spin trapping. Mechanisms for nitration of the tyrosyl probe in the membrane are discussed. We conclude that nitration but not oxidation of a tyrosyl probe by peroxynitrite is a predominant reaction in the membrane. Thus, the local environment of target tyrosine residues is an important factor governing its propensity to undergo nitration in the presence of peroxynitrite. This work provides a new perspective on selective nitration of membrane-incorporated tyrosine analogues.     

Transmembrane nitration of hydrophobic tyrosyl peptides. Localization,characterization, mechanism of nitration,and biological implications

We have shown previously that peroxynitrite-induced nitration of a hydrophobic tyrosyl probe is greater than that of tyrosine in the aqueous phase (Zhang, H., Joseph, J., Feix, J., Hogg, N., and Kalyanaraman, B. (2001) Biochemistry 40, 7675-7686). In this study, we have tested the hypothesis that the extent of tyrosine nitration depends on the intramembrane location of tyrosyl probes and on the nitrating species. To this end, we have synthesized membrane spanning 23-mer containing a single tyrosyl residue at positions 4, 8, and 12. The location of the tyrosine residues in the phospholipid membrane was determined by fluorescence and electron spin resonance techniques. Nitration was initiated by slow infusion of peroxynitrite, co-generated superoxide and nitric oxide ((.)NO), or a myeloperoxidase/hydrogen peroxide/nitrite anion (MPO/H(2)O(2)/NO(2)(-)) system. Results indicate that with slow infusion of peroxynitrite, nitration of transmembrane tyrosyl peptides was much higher (10-fold or more) than tyrosine nitration in aqueous phase. Peroxynitrite-dependent nitration of tyrosyl-containing peptides increased with increasing depth of the tyrosyl residue in the bilayer. In contrast, MPO/H(2)O(2)/ NO(2)(-)-induced tyrosyl nitration decreased with increasing depth of tyrosyl residues in the membrane. Transmembrane nitrations of tyrosyl-containing peptides induced by both peroxynitrite and MPO/H(2)O(2)/NO(2)(-) were totally inhibited by (.)NO that was slowly released from spermine NONOate. Nitration of peptides in both systems was concentration-dependently inhibited by unsaturated fatty acid. Concomitantly, an increase in lipid oxidation was detected. A mechanism involving (.)NO(2) radical is proposed for peroxynitrite and MPO/H(2)O(2)/NO(2)(-)-dependent transmembrane nitration reactions.     

Oxidation of thiamine on reaction with nitrogen dioxide generated by ferric myoglobin and hemoglobin in the presence of nitrite and hydrogen peroxide

It is shown that nitrogen dioxide oxidizes thiamine to thiamine disulfide, thiochrome, and oxodihydrothiochrome (ODTch). The latter is formed during oxidation of thiochrome by nitrogen dioxide. Nitrogen dioxide was produced by incubation of nitrite with horse ferric myoglobin and human hemoglobin in the presence of hydrogen peroxide. After addition of tyrosine or phenol to aqueous solutions containing oxoferryl forms of the hemoproteins, thiamine, and nitrite, the yield of thiochrome greatly increased, whereas the yield of ODTch decreased. In the presence of high concentrations of tyrosine or phenol compounds ODTch was not formed at all. The neutral form of thiamine with the closed thiazole cycle and minor tricyclic form of thiamine do not enter the heme pocket of the protein and do not interact with the oxoferryl heme complex Fe(IV=O) or porphyrin radical. The tricyclic form of thiamine is oxidized to thiochrome by tyrosyl radicals located on the surface of the hemoprotein. The thiol form of thiamine is oxidized to thiamine disulfide by both hemoprotein tyrosyl radicals and oxoferryl heme complexes. Nitrite and also tyrosine, tyramine, and phenol readily penetrate into the heme pocket of the protein and reduce the oxyferryl complex to ferric cation. These reactions yield nitrogen dioxide as well as tyrosyl and phenoxyl radicals of tyrosine molecules and phenol compounds, respectively. Tyrosyl and phenoxyl radicals of low molecular weight compounds oxidize thiamine only to thiochrome and thiamine disulfide. The effect of oxoferryl forms of myoglobin and hemoglobin, nitrogen dioxide, and phenol on thiamine oxidative transformation as well as antioxidant properties of the hydrophobic thiamine metabolites thiochrome and ODTch are discussed.     

Immunochemical detection of nitric oxide and nitrogen dioxide trapping of the tyrosyl radical and the resulting nitrotyrosine in sperm whale myoglobin

We demonstrate herein that nitric oxide (*NO) and nitrogen dioxide (*NO2) both react with the tyrosyl radical formed in sperm whale myoglobin (swMb) by reaction with hydrogen peroxide. The tyrosyl radical was detected by Western blotting using a novel anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes protein radical-derived DMPO nitrone adducts. In the presence of DMPO, hydrogen peroxide reacts with swMb to form the DMPO tyrosyl radical as is known from both electron spin resonance and immuno-spin trapping investigations. Both *NO and NO2- significantly suppressed DMPO-Mb formation under the physiological oxygen tension of 30 mm Hg. If this inhibition of DMPO trapping of the tyrosyl radical is due, at least in part, to the reaction of the tyrosyl radical with *NO and *NO2, then nitrotyrosine should be formed. In line with this expectation, swMb treated with low concentrations of *NO or NO2- formed nitrotyrosine when hydrogen peroxide was added under 30 mm Hg oxygen tension as detected by Western blotting. The amount of nitrotyrosine generated with *NO was higher than with NO2-, implying that there are two different peroxynitrite-independent nitrotyrosine formation mechanisms and that *NO is not just a source of *NO2.     

Effect of phenols on the oxidation of estradiol by uterine peroxidase.

The effect of various phenols on the conversion of [4 -14C]estradiol to water-soluble products by estrogen-induced uterine peroxidase (EC 1.11.1.7) has been investigated. Evidence was provided that those phenols which enhanced the oxidation of estradiol exerted their effect by activating peroxidase or protecting the enzyme from inactivation by the products of the reaction rather than by inhibiting the breakdown of hydrogen peroxide by catalase (EC 1.11.1.6). It has also been shown that tyrosine acted both as an activator of uterine peroxidase and as a water-soluble acceptor for the metabolites of estradiol. The ability of tyrosyl peptides to form conjugates with estradiol was influenced by the other amino acids and decreased with the number of adjacent tyrosyl residues.     

Formation of nitrotyrosine by methylene blue photosensitized oxidation of tyrosine in the presence of nitrite.

Methylene blue photosensitized oxidation of tyrosine in the presence of nitrite produces 3-nitrotyrosine, with maximum yield at pH 6. The formation of 3-nitrotyrosine requires oxygen and increases using deuterium oxide as solvent, suggesting the involvement of singlet oxygen in the reaction. The detection of dityrosine as an additional reaction product suggests that the first step in the interaction of tyrosine with singlet oxygen generates tyrosyl radicals which can dimerize to form dityrosine or react with a nitrite-derived species to produce 3-nitrotyrosine. Although the chemical identity of the nitrating species has not been established, the possible generation of nitrogen dioxide (*NO(2)) by indirect oxidation of nitrite by intermediately produced tyrosyl radical, via electron transfer, is proposed. One important implication of the results of this study is that the oxidation of tyrosine by singlet oxygen in the presence of nitrite may represent an alternative or additional pathway of 3-nitrotyrosine formation of potential importance in oxidative injures such as during inflammatory processes.     

Dps proteins prevent Fenton-mediated oxidative damage by trapping hydroxyl radicals within the protein shell

Dps (DNA-binding proteins from starved cells) proteins belong to a widespread bacterial family of proteins expressed under nutritional and oxidative stress conditions. In particular, Dps proteins protect DNA against Fenton-mediated oxidative stress, as they catalyze iron oxidation by hydrogen peroxide at highly conserved ferroxidase centers and thus reduce significantly hydroxyl radical production. This work investigates the possible generation of intraprotein radicals during the ferroxidation reaction by Escherichia coli and Listeria innocua Dps, two representative members of the family. Stopped-flow analyses show that the conserved tryptophan and tyrosine residues located near the metal binding/oxidation center are in a radical form after iron oxidation by hydrogen peroxide. DNA protection assays indicate that the presence of both residues is necessary to limit release of hydroxyl radicals in solution and the consequent oxidative damage to DNA. In general terms, the demonstration that conserved protein residues act as a trap that dissipates free electrons generated during the oxidative process brings out a novel role for the Dps protein cage.     

Submolecular adventures of brain tyrosine: what are we searching for now?

This overview summarizes recent findings on the role of tyrosyl radical (TyrO(*)) in the multitudinous neurochemical systems of brain, and theorizes on the putative role of TyrO(*) in neurological disorders [Parkinson's disease (PD), Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS)]. TyrO(*) and tyrosine per se can interact with reactive oxygen species (ROS) and reactive nitrogen species (RNS) via radical mechanisms and chain propagating reactions. The concentration of TyrO(*), ROS and RNS can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive as well, and this can induce damage directly (by lipid peroxidation) or indirectly (by proteins oxidation and/or nitration), potentially causing apoptotic neuronal cell death or autoschizis.Evidence of lesion-induced neuronal oxidative stress includes the presence of protein peroxides (TyrOOH), DT (o,o'-dityrosine) and 3-NT (3-nitrotyrosine). Mechanistic details of protein- and enzymatic oxidation/nitration in vivo remain unresolved, although recent in vitro data strongly implicate free radical pathways via TyrO(*). Nitration/denitration processes can be pathological, but they also may play: 1). a signal transduction role, because nitration of tyrosine residues through TyrO(*) formation can modulate, as well the phosphorylation (tyrosine kinases activity) and/or tyrosine hydroxylation (tyrosine hydroxylase inactivation), leading to consequent dopamine synthesis failure and increased degradation of target proteins, respectively; 2). a role of "blocker" for radical-radical reactions (scavenging of NO(*), NO(*)(2) and CO(3)(*-) by TyrO(*)); 3). a role of limiting factors for peroxynitrite formation, by lowering O(2)(*-) formation, which is strongly linked to the pathogenesis of neural diseases.It is still not known if tyrosine oxidation/nitration via TyrO(*) formation is 1). a footprint of generalized stress and neuronal disorders, or 2). an important part of O(2)(*-) and NO(*) metabolism, or 3). merely a part of integral processes for maintaining of neuronal homeostasis. The full answer to these questions should be of top research priority, as the problem of increased free radical formation in brain and/or imbalance of the ratios ROS/RNS/TyrO(*) may be all important in defining whether oxidative stress is the critical determinant of tissue and neural cell injury that leads to pathological end-points.     

Tyrosine iminoxyl radical formation from tyrosyl radical/nitric oxide and nitrosotyrosine

The quenching of the Y(D)(.) tyrosyl radical in photosystem II by nitric oxide was reported to result from the formation of a weak tyrosyl radical-nitric oxide complex (Petrouleas, V., and Diner, B. A. (1990) Biochim. Biophys. Acta 1015, 131-140). This radical/radical reaction is expected to generate an electron spin resonance (ESR)-silent 3-nitrosocyclohexadienone species that can reversibly regenerate the tyrosyl radical and nitric oxide or undergo rearrangement to form 3-nitrosotyrosine. It has been proposed that 3-nitrosotyrosine can be oxidized by one electron to form the tyrosine iminoxyl radical (>C=N-O*). This proposal was put forth as a result of ESR detection of the iminoxyl radical intermediate when photosystem II was exposed to nitric oxide (Sanakis, Y., Goussias, C., Mason, R. P., and Petrouleas, V. (1997) Biochemistry 36, 1411-1417). A similar iminoxyl radical was detected in prostaglandin H synthase-2 (Gunther, M. R., Hsi, L. C., Curtis, J. F., Gierse, J. K., Marnett, L. J., Eling, T. E., and Mason, R. P. (1997) J. Biol. Chem., 272, 17086-17090). Although the iminoxyl radicals detected in the photosystem II and prostaglandin H synthase-2 systems strongly suggest a mechanism involving 3-nitrosotyrosine, the iminoxyl radical ESR spectrum was not unequivocally identified as originating from tyrosine. We report here the detection of the non-protein L-tyrosine iminoxyl radical generated by two methods: 1) peroxidase oxidation of synthetic 3-nitroso-N-acetyl-L-tyrosine and 2) peroxidase oxidation of free L-tyrosine in the presence of nitric oxide. A newly developed ESR technique that uses immobilized enzyme was used to perform the ESR experiments. Analysis of the high resolution ESR spectrum of the tyrosine iminoxyl radical generated from free tyrosine and nitric oxide reveals a 28.4-G isotropic nitrogen hyperfine coupling and a 2.2-G proton hyperfine coupling assigned to the proton originally ortho to the phenoxyl oxygen.     

Involvement of reactive oxygen species produced via NADPH oxidase in tyrosine phosphorylation in human B- and T-lineage lymphoid cells

In several human B- and T-lymphoid cell lines, reactive oxygen species (ROS) were produced in a time- and dose-dependent manner in response to menadione (vitamin K3) and anti-Fas (CD95/APO-1) mAb when ROS formation was determined by a chemiluminescence-based method. The ROS evoked by menadione and anti-Fas could be first observed as rapidly as within 20 seconds after the stimulation, reaching a maximum within 5-10 min, and declining slowly thereafter. Both menadione and anti-Fas also induced increased tyrosine phosphorylation of multiple cellular proteins whose pattern was similar to that observed upon hydrogen peroxide treatment. For each agent, the kinetics of the increased tyrosine phosphorylation was similar to that of ROS production, and an NADPH oxidase inhibitor, diphenyleneiodonium, prevented both of these two events. Our results suggest a close link between ROS production and tyrosine phosphorylation induced by divergent extracellular stimuli and the possible role of NADPH oxidase or its related enzyme.     

Induction of apoptosis in a human leukemic cell line via reactive oxygen species modulation by antioxidants

In the human acute myeloid leukemia cell line M07e, the growth factor interleukin-3 (IL-3) induces ROS formation, positively affecting Glut1-mediated glucose uptake and cell survival. The effect of IL-3 and exogenous hydrogen peroxide on cell viability seems to be mediated through inhibition of the cell death commitment, as shown by apoptotic markers such as caspase activities, apoptotic nuclei, and changes in the amount of proteins belonging to the Bcl-2 family. The pivotal role of ROS is confirmed using various antioxidants, such as EUK-134, ebselen, TEMPO, and hydroxylamine probe. In fact, these antioxidants, acting through different mechanisms, decrease glucose transport activity and cell proliferation activated by IL-3 or by low concentrations of hydrogen peroxide. Moreover, antioxidants foster programmed cell death commitment, as shown by the cited apoptotic parameters. EUK-134, a combined superoxide dismutase/catalase mimetic, opposes the effects of IL-3 and H₂O₂, decreasing phosphorylation levels of signaling enzymes such as Akt, Src tyrosine kinase, and ERK. Results show that ROS production induced by IL-3 can protect leukemic cells from apoptosis, the effect being counteracted by antioxidants. This mechanism may play an important role in supporting acute myeloid leukemia treatment, thus representing a novel therapeutic strategy.     

The main components of St John's Wort inhibit low-density lipoprotein atherogenic modification: A beneficial “side effect” of an OTC antidepressant drug?

Hypericin and pseudohypericin are polycyclic–phenolic structurally related compounds found in Hypericum perforatum L. (St John's wort). As hypericin has been found to bind to LDL one may assume that it can act as antioxidant of LDL lipid oxidation, a property which is of prophylactic/therapeutic interest regarding atherogenesis as LDL oxidation may play a pivotal role in the onset of atherosclerosis. Therefore, in the present paper hypericin, pseudohypericin and hyperforin, an other structurally unrelated constituent in St John's wort were tested in their ability to inhibit LDL oxidation. LDL was isolated by ultracentrifugation and oxidation was initiated either by transition metal ions (copper), tyrosyl radical (myeloperoxidase/hydrogen peroxide/tyrosine) or by endothelial cells (HUVEC). LDL modification was monitored by conjugated diene and malondialdehyde formation. The data show that all compounds (hypericin, pseudohypericin and hyperforin) at doses as low as 2.5 μmol/l are potent antioxidants in the LDL oxidation systems used. The results indicate that the derivatives found in Hypericum perforatum have possible antiatherogenic potential.     

Nitrite as a substrate and inhibitor of myeloperoxidase. Implications for nitration and hypochlorous acid production at sites of inflammation

Myeloperoxidase is a heme enzyme of neutrophils that uses hydrogen peroxide to oxidize chloride to hypochlorous acid. Recently, it has been shown to catalyze nitration of tyrosine. In this study we have investigated the mechanism by which it oxidizes nitrite and promotes nitration of tyrosyl residues. Nitrite was found to be a poor substrate for myeloperoxidase but an excellent inhibitor of its chlorination activity. Nitrite slowed chlorination by univalently reducing the enzyme to an inactive form and as a consequence was oxidized to nitrogen dioxide. In the presence of physiological concentrations of nitrite and chloride, myeloperoxidase catalyzed little nitration of tyrosyl residues in a heptapeptide. However, the efficiency of nitration was enhanced at least 4-fold by free tyrosine. Our data are consistent with a mechanism in which myeloperoxidase oxidizes free tyrosine to tyrosyl radicals that exchange with tyrosyl residues in peptides. These peptide radicals then couple with nitrogen dioxide to form 3-nitrotyrosyl residues. With neutrophils, myeloperoxidase-dependent nitration required a high concentration of nitrite (1 mM), was doubled by tyrosine, and increased 4-fold by superoxide dismutase. Superoxide is likely to inhibit nitration by reacting with nitrogen dioxide and/or tyrosyl radicals. We propose that at sites of inflammation myeloperoxidase will nitrate proteins, even though nitrite is a poor substrate, because the co-substrate tyrosine will be available to facilitate the reaction. Also, production of 3-nitrotyrosine will be most favorable when the concentration of superoxide is low.