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Metals, Metalloproteins and Redox Reactions
METALS, METALLOPROTEINS AND REDOX REACTIONS
01: Metals, Metalloproteins and Redox Reactions 1 Mn Porphyrin-Based SOD Mimic, MnTe-2-PyP5+ (AEOL-10113), Targets Mouse Heart Mitochondria After Single 10 Mg/Kg IP Administration Ivan Spasojevic1, Ines Batinic-Haberle1, Yiqun Yu1, Yumin Chen2, Teresa J Noel2, Marsha P Cole3, Yunfeng Zhao4, and Daret St. Clair2 1 Duke University Medical Center, 2University of Kentucky, Lexington, 3 University of Pittsburgh, 4Louisiana State University The Mn(III) tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-25+ PyP (AEOL 10113) has been proven effective in treating oxidative stress-induced diseases including cancer, radiation, diabetes, and injuries of the central nervous system. in addition to 5+ elimination of O2•− , MnTE-2-PyP is equally or more effective in − reducing levels of ONOO and CO3− , respectively. Thus decreasing levels of these reactive species is considered its mode of action and may be involved in redox-regulation of signaling pathways as well. Recently, Ferrer-Sueta at al (Free Radic Biol. Med. 2006) have shown with submitochondrial 5+ particles that MnTE-2-PyP was able to protect components of the mitochondrial electron transport chain from peroxynitritemediated damage at μM levels. Our study complements their data in showing, for the first time, that μM mitochondrial concentrations 5+ of MnTE-2-PyP are obtainable in vivo. for this study we have 5+ developed a new and sensitive method for in vivo MnTE-2-PyP 2+ detection based on the exchange of porphyrin Mn metal site 2+ with Zn , followed by the HPLC/fluorescence detection of ZnTE4+ 2-PyP . at 3 and 6 hours after a single 10 mg/kg IP 5+ administration of MnTE-2-PyP , the mice (11 in total) were sacrificed and mitochondria isolated using Mela and Seitz method 5+ (Methods Enzymol. 1979). We found that MnTE-2-PyP localizes in heart mitochondria, on average, at 3.0 ng/mg protein levels. Assuming a mitochondrial volume of 0.2 μL/mg protein, the 5+ concentration of MnTE-2-PyP in mitochondriawas found to be 15.5 μM which is sufficient to protect mitochondria from oxidative damage. at that concentration Mn porphyrin can compete in equality for ONOO− with at least 3 mM CO2 and can additionally intercept a substantial amount of CO3•− (Ferrer-Sueta et al, Chem. Res Toxicol. 1999; Ferrer-Sueta et al, J. Biol. Chem. 2003).
2 Radical Production from Hydrogen Peroxide/ Bicarbonate:Modulation by Cu(II) Complexes Giselle Cerchiaro1, and Ohara Augusto1 1 Departamento de Bioquímica, Universidade de São Paulo, Brazil The bicarbonate/carbon dioxide pair has been long known to accelerate transition metal ion-catalyzed peroxidation of several biotargets by mechanisms that remain under scrutiny. on the other hand, our previous studies showed that stable Cu(II) II 2complexes of different imine ligands ([Cu L) ] that cycle between Cu(I) and Cu(II) display diverse apoptotic effects on cell cultures II (1). Also, it has been reported that Cu(II)-tetraglicine ([Cu (H-2G4)] − ) forms a stable Cu(III) complex. Thus, it became interesting to compare radical generation from H2O2 and H2O2/HCO3− in the presence of these different complexes in phosphate buffer pH 7.4. Hydroxyl and CO3•− formation was monitored by spin trapping experiments in the presence of DMPO and in the presence and absence of DMSO ( 2); also ABTS oxidation was used to probe CO3•− formation. in parallel, O2•− formation and oxygen evolution was monitored by reduction of tetranitromethane and oxymetry, respectively. Taken together, the results showed that Cu(II) imine complexes produce predominantly O2•− whereas Cu(II) tetraglicine
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produces mainly •OH. in the presence of H2O2/HCO3− , the Cu(II) tetraglicine produces mainly the CO3•− . in contrast, the Cu(II) imine complexes increased DMPO/•OH production by a Fentonand carbonate radical-independent mechanism. the produced oxidant remains to be identified but it is likely to be important in vivo because its formation correlates with apoptotic effects. (1) Cerchiaro, G., Aquilano, K., Filomeni, G., Rotilio, G., Ciriolo, M.R., Ferreira, A.M.D. J. Inorg. Biochem. (2005) 99, 1433. (2) Zhang, H., Joseph, J., Felix, C., Kalyanaraman, B. J. Biol. Chem. (2000) 275, 14038.
3 Role of Lipoic Acid in the Redox Regulation of Mitochondrial Aldehyde Dehydrogenase (ALDH2) Activity: Implications for Mitochondrial Oxidative Stress and Nitrate Tolerance Andreas Daiber1, Philip Wenzel1, Matthias Oelze1, Ulrich Hink1, Karin Schäuble2, Stefan Schildknecht3, Markus Bachschmid3, and Thomas Münzel1 1 University Hospital Mainz, 2University Konstanz, 3Boston University School of Medicine Background: the chronic therapy with nitroglycerin (GTN) results in a rapid development of nitrate tolerance which is associated with an increased production of reactive oxygen species (ROS). We have recently shown that mitochondria are an important source of GTN-induced ROS and that the GTN-bioactivating mitochondrial aldehyde dehydrogenase (ALDH-2) is oxidatively inactivated in the setting of tolerance. Here we investigated the effect of various oxidants on ALDH-2 activity and restoration of dehydrogenase activity by dihydrolipoic acid. Methods: in vivo tolerance in Wistar rats was induced by infusion of GTN (6.6 µg/kg/min for 4 d). Vascular reactivity was measured by isometric tension studies of isolated aortic rings in response to GTN. Aldehyde dehydrogenase (ALDH-2) activity in isolated mitochondria was determined by an HPLC-based assay and the activity of yeast ALDH was measured spectrophotometrically. Results: Chronic GTN infusion lead to impaired vascular responses to GTN and decreased ALDH-2 activity which was corrected by dihydrolipoic acid coincubation. Superoxide, peroxynitrite and GTN itself were highly efficient in inhibiting ALDH-2 and yeast ALDH activity which was restored by dithiol compounds such as dihydrolipoic acid and dithiothreitol. Hydrogen peroxide and nitric oxide were rather insensitive inhibitors. Conclusions: Our observations indicate that mitochondrial oxidative stress (especially superoxide and peroxynitrite) in response to organic nitrate treatment may inactivate ALDH-2 thereby leading to nitrate tolerance. Furthermore, the present data demonstrate that the mitochondrial dithiol compound dihydrolipoic acid restores ALDH-2 dehydrogenase activity via reduction of a disulfide at the active site and thereby improves nitrate tolerance.
4 Nitroxyl Formation from NH2OH and NOHA Oxidation Sonia Donzelli1, Christopher H Switzer1, Michael G Espey1, Douglas D Thomas1, Lisa a Ridnour1, Katrina M Miranda2, Carlo G Tocchetti3, Giuseppe Lazzarino4, Nazareno Paolocci5, and David a Wink1 1 NIH/NCI, 2University of Arizona, 3Johns-Hopkins University, 4 University of Catania, 5Johns Hopkins University School of Medicine The interactions of nitric oxide (NO) with heme proteins such as soluble guanylate cyclase and oxyhemoglobin are well established. in contrast, the chemistry between other nitrogen oxides and heme/metal proteins is still under appreciated while mechanisms for such reactions are not fully elicited yet. as such, products of nitric oxide syntase like the decoupled intermediate
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N -hydroxy-L-arginine (NOHA) and hydroxylamine (NH2OH) can be oxidized by hypervalent heme species through a two-electron oxidation resulting nitroxyl (HNO) production. Here we sought to determine whether the formation of free diffusible HNO combined with GSH ultimately forms sulfinamide [GS(O)NH2]. If so, then the detection of GS(O)NH2 may serve as useful marker for tracing, and perhaps quantifying, free HNO production in biological settings. to this purpose, GSH was incubated with NOHA/NH2OH, different heme proteins (metmyoglobin (metMb), methemoglobin) and H2O2 in phosphate buffer. to confirm that this reaction occurs also with peroxidases we tested GSH reaction towards horseradish peroxidase, catalase and myeloperoxidase. in our model, the highest yield of GS(O)NH2 was observed with GSH reaction with NH2OH and metMb. We tested next how the yield of GS(O)NH2 formation varies as a function of the concentrations of mtMb, NH2OH and H2O2. in general, the formation of GS(O)NH2 was dose-dependence with all these reagents. Present evidence shows that under our experimental conditions measurable levels of GS(O)NH2 are formed, strongly suggesting that significant HNO amount could be generated and can freely diffuse from its generation site (i.e. the heme metMb pocket) to reach the medium (or likely cellular cytoplasm) where it can directly interact with biological targets, such as free thiols. Our data provides first, critical ground of knowledge to explore the relevance of GSH and NH2OH reaction with heme-proteins to generate and trace HNO formation in intact cells or in vivo systems.
5 Novel Copper(II) Macrocyclic Complexes with Superoxide Scavenging Activity Ana Sofia Fernandes1, M. Fátima Cabral1, José Rueff2, Cátia Caneiras1, Jorge Gaspar2, Matilde Castro1, Judite Costa1, and Nuno G. Oliveira1 1 Fac. Pharmacy - Univ. of Lisbon, 2Fac. Medical Sciences - New Univ. of Lisbon Several human pathologies involve the overproduction of reactive oxygen species, namely superoxide anion radical. Preclinical studies have revealed that superoxide dismutase (SOD) enzymes play a protective role in animal models of several diseases. Synthetic SOD mimetics have thus emerged as a potential novel class of drugs. Among these agents, copper(II) complexes with macrocyclic ligands may constitute an important group. in this work we synthesized the copper(II) complexes of five macrocyclic ligands: [9]aneN2O, ac2[9]aneN2O, [15]aneN4O, [15]aneN3O2 and ac4[15]aneN4O. the ability of these complexes to scavenge the superoxide anion generated by the xanthine-xanthine oxidase system was evaluated using two different methods: the classical nitroblue tetrazolium (NBT) reduction assay (colorimetric) and the dihydroethidium (DHE) oxidation assay (fluorimetric). All the macrocyclic complexes tested, with the exception of [15]aneN3O2, revealed an effective superoxide anion scavenging activity, showing IC50 in the low micromolar range. a very good correlation 2 between the two methods was observed (r =0.985), being the IC50 values obtained with the DHE method about 3-fold lower than those from the NBT method. Cytotoxicity was assessed in V79 Chinese hamster cells using the MTT method. the results revealed that the copper complexes were not considerably toxic in the range of concentrations tested in the SOD assays. in summary, these novel macrocyclic copper(II) complexes, specially those with a high stability constant and low IC50 appear to be highly promising superoxide scavenger agents. Acknowledgements: the authors acknowledge FCT (Project POCTI/49114/QUI/2002) for the financial support.
6 Ferritin Heavy Chain, an Antioxidant/Stress Response/ Iron Storage Protein, Induces a Phenotypic Cure for Sickle Cell Disease in Eythroid Precursor Cells from Sickle Cell Patients Robert H. Broyles1, Visar Belegu2, Austin C. Roth1, Emily J. Clarkson1, Kelly S. Williamson1, Charles A. Stewart1, Quentin N. Pyr1, Robert A. Floyd1, Klodiana Jani3, Marie Trudel3, Paolo Santambrogio4, Sonia Levi4, Paolo Arosio5, and Joan Parkhurst Cain1 1
Oklahoma Medical Research Foundation, & University of Oklahoma Health Sciences Center, Oklahoma City, 2Kennedy Krieger Institute, Johns Hopkins University School of Medicine, Baltimore, 3Institut de Reserches Clinques de Montreal, 4Instituto di Ricera Biologica e Tecnologica, H. San Raffaele, Milano, 5University of Brescia Ferritin heavy chain (FtH), an antioxidant/stress response/ironstorage protein, localizes to the nucleus in K562 cells and represses the human adult beta-globin promoter (Broyles et al., PNAS 98: 9145, 2001). Since other work indicates FtH is an activator of fetal-globin genes, we hypothesize that FtH is a developmental hemoglobin (Hb) switching factor and that delivery of FtH to human adult erythroid cell precursors will reverse the phenotype to HbF, offering a phenotypic cure for sickle cell disease (SCD). Chromatin immunoprecipitation (ChIP) assays, antisense treatments, and an FtH transgenic mouse have confirmed that FtH is a globin gene regulatory protein in vivo. with erythroid precursor cells isolated from blood of pediatric SCD patients, under an IRB-approved protocol, we have used a twophase system for in vitro maturation of erythroid cells in the presence of FtH, delivered as pure protein, as an expression plasmid, or as a priority inducer compound that activates the endogenous FtH gene. in each mode of delivery, FtH stimulates a complete switch from HbS to HbF in five experiments using erythroid precursor cells from three different SCD donors. Fluorescently-labeled FtH protein is taken into red cell precursors in culture, suggesting that the purified protein can be directly delivered without gene therapy. This method of producing a phenotypic cure in SCD patients should be easy and inexpensive to deliver in vivo.
7 Redox Reactions of Nitrite with Oxy- and DeoxyHemoglobin and Myoglobin Along the Physiological Oxygen Gradient Rozalina Grubina1, Zhi Huang1, Sruti Shiva1, and Mark T. Gladwin1 1
National Institutes of Health The complex autocatalytic reaction of nitrite with oxyhemoglobin has been studied for a century; this reaction ultimately oxidizes nitrite to nitrate and oxyhemoglobin to methemoglobin. in contrast, the anaerobic reaction of nitrite with deoxyhemoglobin has recently been characterized as a unique allosteric autocatalytic reaction with the potential to generate bioavailable NO. Interestingly, the physiologically relevant reaction of nitrite simultaneously with both oxy- and deoxyhemoglobin at various hemoglobin oxygen saturations has never been characterized. Using least squares deconvolution absorption spectroscopy and electron paramagnetic resonance spectrometry of experiments evaluating the reaction of nitrite with both hemoglobin and myoglobin at various oxygen partial pressures, we find that the reaction of nitrite with both deoxyhemoglobin and deoxymyoglobin inhibits the autocatalytic phase of the nitrite oxy-hemoglobin and oxymyoglobin reactions. the inhibition of the oxy-reactions occurs at oxymyoglobin saturations below approximately 70% and oxyhemoglobin saturations below 60%. from a mechanistic standpoint, we consider that the NO produced from the reduction of nitrite by deoxyhemoglobin and deoxymyoglobin reacts with
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and inactivates nitrogen dioxide and ferryl-hemoglobin, both necessary intermediates in the catalytic phase of the nitriteoxyglobin reactions. the nitrite reductase activity of deoxyglobins is further potentiated by intra-cellular reductants, while the autcatalytic phase of the nitrite-oxyglobin reactions is inhibited in this reductive milieu. These chemistries determine the oxygendependent disposition of nitrite in red cells and tissue by limiting the oxidative inactivation of nitrite by oxyhemoglobin and oxymyoglobin and promoting nitrite reduction to NO during globin deoxygenation.
8 Oxidative Potential and Sources of Endogenous “Free” Iron in Rats Subjected to Endotoxic Shock Johanna Catharina Duvigneau1, Christina Piskernik2, Susanne Haindl2, Romana Hartl1, Ingeborg Kehrer2, Thomas Ebel1, Manfred Gemainer1, Heinz Redl2, and Andrey V. Kozlov2 1 Institute for Medical Chemistry, University of Veterinary Medicine, Vienna, Austria, 2Ludwig Boltzmann Institute for Experimental and Clinical Traumatology Iron ions multiply damaging potential of reactive oxygen species catalyzing Fenton reaction. the aim of this study was to clarify whether bacterial endotoxin (lipopolysaccharide) influence iron metabolism in a manner which increases the intracellular levels of potentially dangerous “free” iron in liver. “Free” iron levels in liver (EPR-spectroscopy) were increased at 4h after endotoxin challenge. Expression of transferrin receptor (reverse transcription polymerase chain reaction) was decreased, suggesting that the observed increase in “free” iron levels is not a consequence of increased iron uptake. the expression of heme oxygenase-1 (HO-1) was increased, while that of ferritin was decreased. HO-1 degrades heme thereby liberating iron, and can thus increase intracellular iron levels. Cytochrome p450, a potential substrate for HO-1, is known to degrade in endotoxic shock. We have found a positive correlation between HO-1 expression and “free” iron levels and a negative correlation between HO-1 expression and respiratory function of mitochondria. In-vitro we have shown that within all products of HO-1 only free iron reduced the performance of mitochondria. Our data suggest that endotoxin induces up-regulation of HO-1, leading to degradation of P450 and subsequent increase in intracellular free iron levels. the latter possibly contributes to liver damage.
9 Identification of Protein Radicals Formed in the Human Neuroglobin-H2O2 Reaction Olivier M Lardinois1, Leesa J Deterding1, Kenneth B Tomer1, and Ronald P Mason1 1 National Institute of Environmental Health Sciences Neuroglobin (Ngb) is a newly discovered protein that belongs to the globin family of proteins. Ngb is predominantly expressed in the brain, the retina and other nerve tissues. Although the functional role of Ngb remains unclear, Ngb contains a hemebinding domain and may participate in diverse processes such as oxygen transport, oxygen storage, nitric oxide detoxification, or modulation of terminal oxidase activity. We have utilized the immuno-spin trapping technique to investigate whether a protein radical intermediate is formed in the Ngb-H2O2 reaction. Immuno-spin trapping uses polyclonal antiserum raised against the nitrone spin trap 5,5-dimethyl-1pyrroline-N-oxide (DMPO) to detect the presence of DMPO covalently attached to proteins, an attachment which occurs only subsequent to DMPO trapping of a radical species. When DMPO spin trapping in the presence of H2O2 was combined with ELISA, Ngb/DMPO adducts were detected. the absence of H2O2 or its elimination by catalase results in nearly complete loss of trapping.
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To determine the actual site of DMPO covalent attachment to the protein moiety, the spin-trapped protein adducts were digested with trypsin, the peptides from the enzymatic digest were separated by HPLC, and the fractions eluted from the column were analyzed by ELISA. Only one fraction showed a strong positive ELISA signal and was further characterized by tandem mass spectrometry. An abundant ion that corresponds in mass to tryptic peptide T8 (amino acids residues 67-93) plus a DMPO molecule was observed. Sequencing by collision-induced dissociation revealed Tyr 87 to be the site of modification by DMPO. Liquid chromatography/peptide mapping in combination with immuno-spin trapping is an extremely powerful technique for determining the location of radical formation in proteins; it makes MS analysis substantially easier allowing greater certainty in assignment.
10 The Inactivation of Cu, Zn Superoxide Dismutase by Hydrogen Peroxide:The Mechanism of Protection Stefan I Liochev1, Andrew B Goldstone2, and Irwin Fridovich1 1 Duke University Medical Center, 2Duke University The Cu,Zn SOD is known to be inactivated by HO2− and to be protected against that inactivation by a number of small molecules including formate, imidazole, and urate. This inactivation has been shown to be due to oxidation of a ligand field histidine residue by a bound oxidant formed by reaction of the active site Cu(II) with HO2− . We now report that formate protects more effectively than does NADH and that the protective action of both increase as the pH was raised in the range 8.0 – 9.5 . This is taken to indicate increased accessibility of the Cu site with rising pH. Formate appears to act as a sacrificial substrate that protects by competing with the endogenous histidine residue for reaction with the bound oxidant, or that repairs the damage by reducing the histidyl radical intermediate
11 Relative Free Radical Scavenging Ability, GPx Activity and In Vitro Radioprotection by Two Selenocysteine Derivatives Indira Priyadarsini1, Beena Mishra1, Amit Kunwar1, Santosh Kumar2, Leeladhar Kumbhar1, and Vimal K Jain1 1 Bhabha Atomic Research Centre, Mumbai-400085, India, 2Deccan Medical College, Hyderabad, India Selenium is an essential trace element. It is present in the active site of glutathione peroxidase (GPx) enzyme of eukaryotic cells in the form of selenocysteine Selenium compounds show antioxidant activity and are being explored as new class of radioprotectors. with this aim, two diselenides, selenocysteine(SeC), and bis-selenopropanoic acid (SeP) were examined for free radical reactions and in vitro radioprotection studies. SeC was purchased from Sigma, USA and SeP was synthesized and purified in our laboratory. Reactions of hydroxyl radicals and peroxyl radicals with the compounds were studied using nanosecond pulse radiolysis, which indicated formation of selenium centered radical cations, stabilized by the oxygen and nitrogen atoms at γ-position. SeP showed better reactivity with peroxyl radicals than SeC. Inhibition of γ-radiation induced lipid peroxidation was studied in phasphatidyl choline liposomes and at a constant dose of 210 Gy, IC50 value for inhibition of lipid peroxidation was found to be, 300 and 110 μM respectively for SeC and SeP. Further γ-radiation (50 Gy) induced pBR322 plasmid DNA strand breakage studies revealed SeP to be very effective in inhibiting the DNA damage. the compounds exhibit GPx like activity and this activity is being correlated with their free radical induced redox reactions and radioprotection.
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12 Chromium(VI) Reduction by Human Cytochrome B5 Generates Hydroxyl Radical and Superoxide Griselda R.. Borthiry1, William E. Antholine1, B. Kalyanaraman1, Judith M. Myers1, and Charles R. Myers1 1
Medical College of Wisconsin The reduction of hexavalent chromium, Cr(VI), can generate reactive Cr intermediates and oxidative stress. the potential role of human microsomal enzymes in free radical generation was examined using reconstituted proteoliposomes (PLs) containing purified cytochrome b5 and NADPH:P450 reductase. Under room air, the reduction of Cr(VI) to Cr(V) by the PLs was confirmed by 53 ESR using isotopically pure Cr(VI). When 5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) was included as a spin trap, a very prominent signal for the hydroxyl radical (HO•) adduct was observed as well as a smaller signal for the superoxide (O2•− ) adduct. These adducts were observed even at very low Cr(VI) concentrations (10 µM). NADPH, Cr(VI), O2 and the PLs were all required for significant HO• generation. Superoxide dismutase eliminated the O2•− adduct, but resulted in a 30% increase in the HO• adduct. Catalase largely diminished the HO• adduct signal indicating its dependence on H2O2. Some sources of catalase were found to have Cr(VI)-reducing contaminants which could confound results, but a source of catalase free of these contaminants was used for these studies. Exogenous H2O2 was not needed, indicating that the PLs generated sufficient H2O2. Adding exogenous H2O2, however, did increase the amount of DEPMPO/HO• adduct. the inclusion of formate yielded the carbon dioxide radical adduct of DEPMPO. Experiments with dimethylsulfoxide (DMSO) plus the spin trap αphenyl-N-tert-butylnitrone (PBN) yielded the methoxy and methyl radical adducts of PBN, confirming the generation of HO•. Quantification of the various species over time was consistent with a stoichiometric excess of HO• relative to the net amount of Cr(VI) reduced. This represents the first demonstration of a role for cytochrome b5 in the generation of HO•. Overall, the simultaneous generation of Cr(V) and H2O2 by the PLs and the resulting generation of HO• at low Cr(VI) concentrations could have important implications for human Cr(VI) toxicity.
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0.010 ± 0.009% at Time 0 and 0.011 ± 0.010% at 30 hr. Unlike previous studies with a polymeric iron dextran complex that destabilized these admixtures and produced very high PFAT5 values, the novel iron salt used in this study did not affect emulsion stability. Conclusions. Iron is often necessary in patients receiving long-term PN therapy, but poses significant safety issues if given as a separate infusion or as part of the PN regimen. the use of a novel iron compound results in stable lipid emulsion-containing PN admixtures during their 30-hr period of clinical use.
14 Modification of Cysteine 111 in Human Cu, ZnSuperoxide Dismutase Ayako Okado-Matsumoto1, and Irwin Fridovich2 1 Osaka University Graduate School of Medicine, 2Duke University Medical Center Human Cu,Zn-superoxide dismutase (hSOD1) has 4 cysteines per subunit. Cys57 and Cys148 are involved in an intrasubunit disulfide bond, while Cys6 and Cys111 are free. Cys6 is buried within the protein while Cys111 is on the surface, near the dimer interface. We examined by liquid chromatography-mass spectrometry the commercially purchased hSOD1 isolated from erythrocytes as well as hSOD1s isolated from human erythrocytes, brain, and hSOD1 expressed in Sf9, yeast, and E. coli. Our goal was to ascertain whether the Cys111 modification occurred naturally in vivo. Only the Sigma erythrocyte hSOD1 appeared to contain a trisulfide crosslink between the Cys111 residues. Thus it failed to react with N-ethylmaleimide, showed absorbtion at 325nm that was eliminated by 2-mercaptoethanol, and had a mass 30 units more than expected for the native dimer. We examined the possibility that different purification methods might cause this modification in erythrocyte hSOD1. None of the procedures examined for hSOD1 purification produced such a trisulfide. in disagreement with Liu et al. [Biochemistry, 2000, 39, 8125-8132], complete derivitization of both Cys111s of hSOD1 from Sf9 cells with N-ethylmaleimide, 4-vinylpyridine, and by 5,5’dithiobis(2-nitrobenzoic acid) were readily achieved; indicating that steric hindrance was not a problem.
15 Pro-Inflammatory Impact of Reduced Thioredoxin on Rat Airways: Modulation by Airway Mucus
Stability of Lipid Emulsion-Based Parenteral Nutrition (PN) Admixtures Containing a Novel Iron Salt Deanna J. Nelson1, David Driscoll2, and Ajay Gupta3 1 BioLink LIfe Sciences, Inc., 2BIDMC/Harvard Medical School, 3 Drew Univ Medical School Purpose. to assess the effects of a novel iron compound on the stability of PN admixtures by applying the measures and limits proposed in USP <729>. Methods. the USP proposes two criteria to verify the stability of lipid emulsions. the first criterion, the intensity-weighted mean droplet size (MDS), expressed in nm, was determined using dynamic light scattering. the second measure, the large-diameter tail of the globule size distribution (GSD), expressed as the volume-weighted percent of fat > 5 µm or PFAT5, was determined using light extinction employing a single-particle optical sensing technique. the upper USP limits for these measures are 500 nm and 0.05%, respectively. Twenty clinically relevant PNs containing variable amounts of soluble ferric pyrophosphate were prepared in triplicate (n=60) under aseptic conditions and studied over 30 hr at room temperature. the MDS values were determined at the beginning (Time 0) and end of study (Time 30), while PFAT5 levels were determined at Time 0, and at 6, 24 and 30 hr. Results. the MDS at Time 0 was 275.0 ± 2.9 nm and 276.8 ± 13.8 nm at Time 30 hr. PFAT5 was
Ray C Rancourt1, Rees L Lee2, Heidi C O'Neill1, Frank J Accurso3, and Carl W White1 1 National Jewish Medical & Research Center, Denver CO, 2Pediatric Pulmonary Medicine, Navy, 3The Mike McMorris Cystic Fibrosis Research and Treatment Center;The Childrens Hospital, Denver CO. Thioredoxin (Trx) has previously been shown to decrease the viscosity of cystic fibrosis (CF) sputum. Since Trx intended for use as a mucolytic would be administered into the lung, this study examined the effects of intratracheal instillation of reduced recombinant human thioredoxin (rhTrx) in naïve rat airways. Delivery of reduced rhTrx (1.2 mg in 100 μl of 0.9% NaCl) resulted in significant increases in the number of neutrophils in bronchoalveolar lavage fluid (BALF) at 4 hours post treatment. Neutrophil influx into airways coincided with increases in cytokine expression (TNFα, CINC2β, and MIP3α), and increased activity of the p65 subunit of NF-κB in nuclear extracts derived from lung homogenates. Treatment of rats with saline alone, or human serum albumin or oxidized rhTrx at equal molarities, did not significantly increase BALF neutrophil or cytokine levels. Similarly, deposition of CF sputum (50 μl) into the lung immediately prior to reduced rhTrx delivery attenuated these responses. These observations suggest that rhTrx is reducing disulfide bond substrates present in CF sputum, limiting the reduction of other
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lung constituents. Taken together, these findings indicate that the observed chemotactic and cytokine responses are an effect of the reducing potential of the rhTrx preparation.
16 On the Modeling of Heme-Dependent, O2Oxidation of Thiols by Ru-Porphyrins Júlio S. Rebouças1, and Brian R. James2 1 University of British Columbia; Duke University Medical Center, 2 University of British Columbia Heme-proteins play key roles in some aspects of the oxidation of biological thiols. for instance, the activity of heme-regulated eIF2α kinase is dictated by a delicate thiol/disulfide balance (J.J. Chen et al., J. Biol. Chem. 1989, 264, 9559); and the hemecontaining enzymes horseradish peroxidase, myeloperoxidase, and lactoperoxidase, which usually employ H2O2 as oxygen donor, are remarkably able to catalyze the O2-oxidation of thiols, without added H2O2 (B.E. Svensson et al., Free Radic. Biol. Med. 1993, 14, 167; L.S. Harman et al., J. Biol. Chem. 1984, 259, 5606; J. Olsen et al., Biochim. Biophys. Acta 1976, 445, 324). a well-documented dysfunction associated with ‘free’ heme toxicity is the heme-dependent oxidation of membrane-protein thiols in sickle red blood cells (R.P. Hebbel et al., Blood 1988, 71, 876). the synthesis, transport, storage, and degradation of heme occur under very tight cellular control but, under stress or pathological conditions, the heme moiety may be freed from a heme-protein and may then mediate, catalyze, or participate in reactions that hold little (if any) resemblance to those of the parent protein; these reactions may in fact resemble more closely those of ‘protein-free’ model systems. Little is known about the chemistry of those oxidations, and porphyrin-based models for O2-oxidation of thiol are scarce. We describe here detailed kinetic and spectroscopic studies on the O2-oxidation of aromatic and aliphatic thiols catalyzed by ‘protein-free’ Ru-porphyrin species. the kinetic of these oxidations vary with the nature of the thiol and show rate-dependence on thiol, O2, H2O, and Ru. the involvement of dioxo-, oxo-, thiol-, thiolate-, and disulfide-Ru species in the 1 catalysis were examined by H NMR spectroscopy. a marked inhibitory effect of CO indicate that the role of the Ru species is not that of a radical-chain initiator, and that metal centered-redox processes are likely involved in the catalysis. a tentative, RSOHbased mechanism that accommodates generally the kinetic ratedependences, SH/SD kinetic isotope effect, and spectroscopic data is presented. the difficulties in establishing unequivocally the participation of either thiyl or sulfenic acid intermediates are also addressed. (Supported by: NSERC, CAPES, and UBC)
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18 Deoxymyoglobin is a Nitrite Reductase that Generates NO Sruti Shiva1, Zhi Huang1, Rozalina Grubina1, Lorna A. Ringwood1, Peter Macarthur1, and Mark T. Gladwin1 1 Vascular Medicine Branch, NHLBI, National Institutes of Health Previous studies have demonstrated that nitrite is an endocrine reservoir of nitric oxide (NO) that participates in hypoxic vasodilation. Nitrite can be reduced to NO enzymatically by deoxyhemoglobin in a reaction that is under allosteric control with a maximum reaction rate near the hemoglobin P50. in this study, we characterized the nitrite reductase activity of deoxygenated myoglobin, which reduces nitrite approximately 50 times faster than deoxyhemoglobin due to its lower heme redox potential. Spectrophotometric and chemiluminescent measurements show the deoxymyoglobin-nitrite reaction produces NO in a second order reaction that is linearly dependent on deoxymyoglobin, nitrite and proton concentration, with a bimolecular rate constant -1 -1 of 14 M s at pH 7.4 at 37°C. from a pathophysiological standpoint, the IC50 for NO-dependent inhibition of mitochondrial respiration is approximately 100 nM at physiological oxygen tensions (5-10 μM) in the working heart; we therefore hypothesized that the myoglobin-dependent reduction of nitrite could modulate mitochondrial respiration. Indeed, the addition of deoxymyoglobin and nitrite to isolated rat heart and liver mitochondria resulted in the inhibition of respiration, while myoglobin or nitrite alone had no effect. the addition of nitrite to rat heart homogenate, which contains both myoglobin and mitochondria, resulted in NO gas generation and inhibition of mitochondrial respiration; these effects were blocked by myoglobin oxidation with ferricyanide but not by allopurinol, a xanthine oxidoreductase inhibitor. These data expand upon the novel paradigm that heme-globins conserve and generate NO via nitrite reduction along physiological oxygen gradients, and further demonstrate that NO generation from nitrite reduction can escape heme autocapture to mediate fundamental biological responses to hypoxia.
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Nitric Oxide Regulates MMP-9 Activity at the Protein Level Lisa Ridnour1, Alisha Windhausen1, Jeffery Isenberg2, Nolan Yeung3, Michael Vitek3, David Roberts2, and David Wink1 1 Radiation Biology Branch, NCI, 2Laboratory of Pathology, NCI, 3 Duke University Interest in redox involvement in the regulation of extracellular matrix is beginning to emerge. Matrix reorganization is a dynamic process that occurs during both normal physiology and disease, and is mediated by proteolytic enzymes including matrix metalloproteinases (MMPs). MMPs are tightly regulated at multiple levels including transcription, post-transcription, and posttranslation. in addition, these enzymes are distinguished by a highly conserved catalytic domain containing a zinc ion as well as a pro-domain, which regulates enzyme activation by modulation of the cysteine switch. NO and RNOS derived from NO target zinc finger motifs as well as cysteine residues. Toward this end, our laboratory has identified a biphasic regulatory affect of NO, released from spermine NONOate, on the activities of MMPs secreted from ANA-1 murine macrophages, as well as purified
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MMP-9 enzyme, suggesting that NO activation of MMP-9 occurs, in part, at the protein level. the NO-mediated biphasic affect observed in the conditioned media of ANA-1 cells was reversed by the sGC inhibitor ODQ, which modulated TIMP-1 protein. Moreover, basal MMP activity levels in the conditioned media of ANA-1 cells was suppressed by LNAME, suggesting that low levels of constitutively produced NO modulate MMP activity. These results demonstrate the ability of NO to regulate MMP activities, which occurs at least in part, at the protein level via NO/RNOS protein modification and/or TIMP1 modulation.
Double Edge Redox-Implications for the Interaction Between Endogenous Thiols and Copper Ions: In VitroStudies Hernan C. Speisky1, Catalina P. Carrasco-Pozo1, Edgar N. Pastene1, Maritza D. Gomez1, and Claudio A.Oolea1 1 University of Chile The presence of a thiol moiety on the structure of glutathione, γglutamyl-cysteine, cysteinyl-glycine, cysteine and homocysteine, endows these compounds with the ability to exert, in the presence 2+ of Cu ions, a double-edge redox action. They could act either as antioxidants, by donating an electron or a hydrogen atom to a free 2+ into radical, or as pro-oxidants by reducing Cu the redox-active + Cu specie. We addressed here the redox-consequences 2+ associated with the interaction between these thiols and Cu , investigating their free radical-scavenging, O2•− -generating, and ascorbate-oxidizing properties. Upon a brief pre-incubation, the 2+ thiols/Cu interaction led to a decrease in their free radical-
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scavenging properties, and to an apparently unrelated loss of thiol density. Both effects were, however, only partial, even in the 2+ presence of a large molar Cu -excess, and were unaffected by increasing the pre-incubation time. at equimolar concentrations, the mixtures contained Cu(II)-complexes (EPR-detected), that were time-stable and ascorbate-reducible, but redox-inactive towards oxygen. at a slight molar thiol-excess (3:1), the mixtures generated time-stable Cu(I)-complexes that were unreactive to ascorbate and oxygen. a major exception was the 2+ GSH/Cu mixture which displayed a O2•− -generating capacity, as ·evidenced through its ability to generate an O2 -EPR spectrum, to reduce cytochrome-c, and to continually consume oxygen (in the 2+ presence of SOD). the O2•− -generating ability of the GSH/Cu mixture remained unaltered–at least- during 16 hr of preincubation. We conclude that the interaction between GSH and 2+ Cu ions leads to the formation of redox-active complexes which are capable of promoting a pro-oxidant action. Funded by Fondecyt #1040736.
20 Sublethal Hyperoxia Alters Pulmonary and Hepatic Thioredoxin-1 Levels in Neonatal Mice Trent E Tipple1, Lynette K Rogers1, Charles V Smith2, and Stephen E Welty1 1 Columbus Children's Research Institute, 2Seattle Children's Hospital and Regional Medical Center Prolonged exposure of neonatal mice to sublethal hyperoxia causes functional and pathological changes similar to those seen in infants with bronchopulmonary dysplasia (BPD). Expression of thioredoxin-1 (Trx1), a dithiol disulfide oxidoreductase important in antioxidant defense, regulation of cellular proliferation, and regulation of gene expression, is altered by changes in ambient oxygen tension in a baboon model of BPD. the objective of the present studies was to test the hypothesis that neonatal mice exposed to sublethal hyperoxia will exhibit alterations in normal developmental pulmonary and hepatic levels of Trx1. Within 24 h of birth, C3H/HeN mice were exposed to sublethal hyperoxia (FiO2 = 0.85) or were kept in room air (FiO2 = 0.21). the dams nursing the pups were rotated between hyperoxia and room air every 24 hours. at 1, 3, and 7 d, mice were euthanized, lungs and livers removed, and Trx1 levels determined by western blotting. No time-dependent effects on Trx1 levels in the lungs and livers of room air controls were observed. on days 1, 3, and 7, Trx1 levels in lungs of pups exposed to hyperoxia were less than 25% of the levels in room air controls. on days 1 and 3, hepatic Trx1 levels were not different between room air and hyperoxia, but on day 7, hepatic Trx1 levels in hyperoxic pups were three times the levels in room air controls. the data indicate that tissue levels of Trx1 in the livers and lungs of newborn mice are altered by changes in ambient oxygen tension. the alterations of Trx1 caused by exposure of newborn mice to sublethal hyperoxia may contribute to altered lung developmental maturation in this model. Similar effects in prematurely born human infants similarly could contribute to altered lung development that is characteristic of BPD.
21 Copper Dependence of the Biotin Switch Assay Xunde Wang1, Geng Liu1, Peter Macarthur1, Neil Hogg2, and Mark T. Gladwin1 1 Vascular Medicine Branch, NHLBI; NIH, Bethesda, MD 20892, USA, 2 Medical College of Wisconsin, Milwaukee WI 53226 Studies have shown that modification of critical cysteine residues in proteins leads to the regulation of protein function. These modifications include disulfide bond formation, glutathiolation, sulfenic and sulfinic acid modifications, and S-nitrosation. the biotin switch assay was developed to detect S-nitrosation (Jaffrey et al., Nat. Cell Biol. 3 (2001), pp. 193–197). Specifically, proteins
are denatured with SDS in the presence of methyl methanethiosulfonate (MMTS) to alkylate free thiols. the SDS and temperature of 50°C are used to expose and facilitate alkylation of buried thiols. After acetone precipitation or Sephadex G25 separation to remove excess MMTS, HPDP-biotin and 1 mM ascorbate are added to reduce the S-nitrosothiol bond and modify the reduced thiol with HPDP-biotin. the proteins are then separated on SDS-PAGE and quantified using either streptavidinHRP or anti-biotin HRP conjugate. Interestingly, we have found that different levels of S-nitrosothiol are obtained for the same protein using different buffers and with the inclusion of different chelators (DTPA vs. EDTA). Using purified S-nitrosated albumin, we have found that “contaminating” copper is required to degrade S-nitrothiol before ascorbate and biotin-HPDP can react with the free thiol; ascorbate itself does not reduce the S-nitrosothiol. Removal of copper from buffers by DTPA and other copper chelators preserves approximately 90% of the S-nitrosothiol, while including copper and ascorbate completely eliminates the Snitrosothiol in the preparation and increases the biotin labeling. These biotin switch experiments were confirmed using triiodide based reductive chemiluminascence. the results indicate that degradation of S-nitrosothiol in the standard biotin switch assay is copper dependent and that experimental variability in S-NO yields using this assay occur secondary to the inclusion of copper chelators and variable copper contamination in buffers and labware.
22 The Role of the N-Terminal Octarepeat Domain in the Antioxidant Activity of Mouse Prion Protein Yasuko Asano Watanabe1, Osamu Inanami1, Tetsu Inoue1, Daisuke Iizuka1, and Mikinori Kuwabara1 1 Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University C The cellular prion protein (PrP ) contains the octarepeat domain 2+ (OR domain) in the N-terminal region that acts as the Cu 2+ binding domain. Cu is considered to modulate various biological C functions of PrP such as the cellular enzymatic activity of superoxide dismutase (SOD) and conversion to the scrapie isoform of PrP. in this study, to clarify further physiological C C function of PrP as an antioxidant, the susceptibility of PrP overexpressed NIH3T3 cells to various oxidants was evaluated. in addition, we prepared recombinat mouse PrP (rmoPrP(23-231)) and truncated PrP (rmoPrP(91-231)), lacking OR domain, and then the O2− -scavenging activity of rmoPrP and the inhibiting activity of 2+ 2+ rmoPrP against Cu /H2O2-catalyzed oxidative reaction or Cu t catalyzed oxidative reaction of BuOOH were measured by spintrapping technique using electron spin resonance spectroscopy 2+ (ESR). the Cu - or H2O2-induced cell death was significantly C inhibited by overexpression of wild-type PrP , but radiation2+ induced cell death was not. the oxidative reaction of Cu with 2+ t H2O2 or Cu -catalyzed oxidative reaction of BuOOH was also shown to be significantly reduced by the addition of rmoPrP(23-231) but not rmoPrP(91-231) by spin-trapping experiments. Whereas, in the assay using spin-trapping technique, the O2− -scavenging 2+ activity of rmoPrP(23-231)/Cu complex was about two times higher 2+ 3 than that of Cu alone, but 10 times lower than that of Cu/ZnC SOD. These results suggested that PrP inhibited the cellular 2+ damage induced by Cu - and H2O2-oxidative stress thorough OR domain of PrP.
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01: Metals, Metalloproteins and Redox Reactions 1 Mn Porphyrin-Based SOD Mimic, MnTe-2-PyP5+ (AEOL-10113), Targets Mouse Heart Mitochondria After Single 10 Mg/Kg IP Administration Ivan Spasojevic1, Ines Batinic-Haberle1, Yiqun Yu1, Yumin Chen2, Teresa J Noel2, Marsha P Cole3, Yunfeng Zhao4, and Daret St. Clair2 1 Duke University Medical Center, 2University of Kentucky, Lexington, 3 University of Pittsburgh, 4Louisiana State University The Mn(III) tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-25+ PyP (AEOL 10113) has been proven effective in treating oxidative stress-induced diseases including cancer, radiation, diabetes, and injuries of the central nervous system. in addition to 5+ elimination of O2•− , MnTE-2-PyP is equally or more effective in − reducing levels of ONOO and CO3− , respectively. Thus decreasing levels of these reactive species is considered its mode of action and may be involved in redox-regulation of signaling pathways as well. Recently, Ferrer-Sueta at al (Free Radic Biol. Med. 2006) have shown with submitochondrial 5+ particles that MnTE-2-PyP was able to protect components of the mitochondrial electron transport chain from peroxynitritemediated damage at μM levels. Our study complements their data in showing, for the first time, that μM mitochondrial concentrations 5+ of MnTE-2-PyP are obtainable in vivo. for this study we have 5+ developed a new and sensitive method for in vivo MnTE-2-PyP 2+ detection based on the exchange of porphyrin Mn metal site 2+ with Zn , followed by the HPLC/fluorescence detection of ZnTE4+ 2-PyP . at 3 and 6 hours after a single 10 mg/kg IP 5+ administration of MnTE-2-PyP , the mice (11 in total) were sacrificed and mitochondria isolated using Mela and Seitz method 5+ (Methods Enzymol. 1979). We found that MnTE-2-PyP localizes in heart mitochondria, on average, at 3.0 ng/mg protein levels. Assuming a mitochondrial volume of 0.2 μL/mg protein, the 5+ concentration of MnTE-2-PyP in mitochondriawas found to be 15.5 μM which is sufficient to protect mitochondria from oxidative damage. at that concentration Mn porphyrin can compete in equality for ONOO− with at least 3 mM CO2 and can additionally intercept a substantial amount of CO3•− (Ferrer-Sueta et al, Chem. Res Toxicol. 1999; Ferrer-Sueta et al, J. Biol. Chem. 2003).
2 Radical Production from Hydrogen Peroxide/ Bicarbonate:Modulation by Cu(II) Complexes Giselle Cerchiaro1, and Ohara Augusto1 1 Departamento de Bioquímica, Universidade de São Paulo, Brazil The bicarbonate/carbon dioxide pair has been long known to accelerate transition metal ion-catalyzed peroxidation of several biotargets by mechanisms that remain under scrutiny. on the other hand, our previous studies showed that stable Cu(II) II 2complexes of different imine ligands ([Cu L) ] that cycle between Cu(I) and Cu(II) display diverse apoptotic effects on cell cultures II (1). Also, it has been reported that Cu(II)-tetraglicine ([Cu (H-2G4)] − ) forms a stable Cu(III) complex. Thus, it became interesting to compare radical generation from H2O2 and H2O2/HCO3− in the presence of these different complexes in phosphate buffer pH 7.4. Hydroxyl and CO3•− formation was monitored by spin trapping experiments in the presence of DMPO and in the presence and absence of DMSO ( 2); also ABTS oxidation was used to probe CO3•− formation. in parallel, O2•− formation and oxygen evolution was monitored by reduction of tetranitromethane and oxymetry, respectively. Taken together, the results showed that Cu(II) imine complexes produce predominantly O2•− whereas Cu(II) tetraglicine
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produces mainly •OH. in the presence of H2O2/HCO3− , the Cu(II) tetraglicine produces mainly the CO3•− . in contrast, the Cu(II) imine complexes increased DMPO/•OH production by a Fentonand carbonate radical-independent mechanism. the produced oxidant remains to be identified but it is likely to be important in vivo because its formation correlates with apoptotic effects. (1) Cerchiaro, G., Aquilano, K., Filomeni, G., Rotilio, G., Ciriolo, M.R., Ferreira, A.M.D. J. Inorg. Biochem. (2005) 99, 1433. (2) Zhang, H., Joseph, J., Felix, C., Kalyanaraman, B. J. Biol. Chem. (2000) 275, 14038.
3 Role of Lipoic Acid in the Redox Regulation of Mitochondrial Aldehyde Dehydrogenase (ALDH2) Activity: Implications for Mitochondrial Oxidative Stress and Nitrate Tolerance Andreas Daiber1, Philip Wenzel1, Matthias Oelze1, Ulrich Hink1, Karin Schäuble2, Stefan Schildknecht3, Markus Bachschmid3, and Thomas Münzel1 1 University Hospital Mainz, 2University Konstanz, 3Boston University School of Medicine Background: the chronic therapy with nitroglycerin (GTN) results in a rapid development of nitrate tolerance which is associated with an increased production of reactive oxygen species (ROS). We have recently shown that mitochondria are an important source of GTN-induced ROS and that the GTN-bioactivating mitochondrial aldehyde dehydrogenase (ALDH-2) is oxidatively inactivated in the setting of tolerance. Here we investigated the effect of various oxidants on ALDH-2 activity and restoration of dehydrogenase activity by dihydrolipoic acid. Methods: in vivo tolerance in Wistar rats was induced by infusion of GTN (6.6 µg/kg/min for 4 d). Vascular reactivity was measured by isometric tension studies of isolated aortic rings in response to GTN. Aldehyde dehydrogenase (ALDH-2) activity in isolated mitochondria was determined by an HPLC-based assay and the activity of yeast ALDH was measured spectrophotometrically. Results: Chronic GTN infusion lead to impaired vascular responses to GTN and decreased ALDH-2 activity which was corrected by dihydrolipoic acid coincubation. Superoxide, peroxynitrite and GTN itself were highly efficient in inhibiting ALDH-2 and yeast ALDH activity which was restored by dithiol compounds such as dihydrolipoic acid and dithiothreitol. Hydrogen peroxide and nitric oxide were rather insensitive inhibitors. Conclusions: Our observations indicate that mitochondrial oxidative stress (especially superoxide and peroxynitrite) in response to organic nitrate treatment may inactivate ALDH-2 thereby leading to nitrate tolerance. Furthermore, the present data demonstrate that the mitochondrial dithiol compound dihydrolipoic acid restores ALDH-2 dehydrogenase activity via reduction of a disulfide at the active site and thereby improves nitrate tolerance.
4 Nitroxyl Formation from NH2OH and NOHA Oxidation Sonia Donzelli1, Christopher H Switzer1, Michael G Espey1, Douglas D Thomas1, Lisa a Ridnour1, Katrina M Miranda2, Carlo G Tocchetti3, Giuseppe Lazzarino4, Nazareno Paolocci5, and David a Wink1 1 NIH/NCI, 2University of Arizona, 3Johns-Hopkins University, 4 University of Catania, 5Johns Hopkins University School of Medicine The interactions of nitric oxide (NO) with heme proteins such as soluble guanylate cyclase and oxyhemoglobin are well established. in contrast, the chemistry between other nitrogen oxides and heme/metal proteins is still under appreciated while mechanisms for such reactions are not fully elicited yet. as such, products of nitric oxide syntase like the decoupled intermediate
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N -hydroxy-L-arginine (NOHA) and hydroxylamine (NH2OH) can be oxidized by hypervalent heme species through a two-electron oxidation resulting nitroxyl (HNO) production. Here we sought to determine whether the formation of free diffusible HNO combined with GSH ultimately forms sulfinamide [GS(O)NH2]. If so, then the detection of GS(O)NH2 may serve as useful marker for tracing, and perhaps quantifying, free HNO production in biological settings. to this purpose, GSH was incubated with NOHA/NH2OH, different heme proteins (metmyoglobin (metMb), methemoglobin) and H2O2 in phosphate buffer. to confirm that this reaction occurs also with peroxidases we tested GSH reaction towards horseradish peroxidase, catalase and myeloperoxidase. in our model, the highest yield of GS(O)NH2 was observed with GSH reaction with NH2OH and metMb. We tested next how the yield of GS(O)NH2 formation varies as a function of the concentrations of mtMb, NH2OH and H2O2. in general, the formation of GS(O)NH2 was dose-dependence with all these reagents. Present evidence shows that under our experimental conditions measurable levels of GS(O)NH2 are formed, strongly suggesting that significant HNO amount could be generated and can freely diffuse from its generation site (i.e. the heme metMb pocket) to reach the medium (or likely cellular cytoplasm) where it can directly interact with biological targets, such as free thiols. Our data provides first, critical ground of knowledge to explore the relevance of GSH and NH2OH reaction with heme-proteins to generate and trace HNO formation in intact cells or in vivo systems.
5 Novel Copper(II) Macrocyclic Complexes with Superoxide Scavenging Activity Ana Sofia Fernandes1, M. Fátima Cabral1, José Rueff2, Cátia Caneiras1, Jorge Gaspar2, Matilde Castro1, Judite Costa1, and Nuno G. Oliveira1 1 Fac. Pharmacy - Univ. of Lisbon, 2Fac. Medical Sciences - New Univ. of Lisbon Several human pathologies involve the overproduction of reactive oxygen species, namely superoxide anion radical. Preclinical studies have revealed that superoxide dismutase (SOD) enzymes play a protective role in animal models of several diseases. Synthetic SOD mimetics have thus emerged as a potential novel class of drugs. Among these agents, copper(II) complexes with macrocyclic ligands may constitute an important group. in this work we synthesized the copper(II) complexes of five macrocyclic ligands: [9]aneN2O, ac2[9]aneN2O, [15]aneN4O, [15]aneN3O2 and ac4[15]aneN4O. the ability of these complexes to scavenge the superoxide anion generated by the xanthine-xanthine oxidase system was evaluated using two different methods: the classical nitroblue tetrazolium (NBT) reduction assay (colorimetric) and the dihydroethidium (DHE) oxidation assay (fluorimetric). All the macrocyclic complexes tested, with the exception of [15]aneN3O2, revealed an effective superoxide anion scavenging activity, showing IC50 in the low micromolar range. a very good correlation 2 between the two methods was observed (r =0.985), being the IC50 values obtained with the DHE method about 3-fold lower than those from the NBT method. Cytotoxicity was assessed in V79 Chinese hamster cells using the MTT method. the results revealed that the copper complexes were not considerably toxic in the range of concentrations tested in the SOD assays. in summary, these novel macrocyclic copper(II) complexes, specially those with a high stability constant and low IC50 appear to be highly promising superoxide scavenger agents. Acknowledgements: the authors acknowledge FCT (Project POCTI/49114/QUI/2002) for the financial support.
6 Ferritin Heavy Chain, an Antioxidant/Stress Response/ Iron Storage Protein, Induces a Phenotypic Cure for Sickle Cell Disease in Eythroid Precursor Cells from Sickle Cell Patients Robert H. Broyles1, Visar Belegu2, Austin C. Roth1, Emily J. Clarkson1, Kelly S. Williamson1, Charles A. Stewart1, Quentin N. Pyr1, Robert A. Floyd1, Klodiana Jani3, Marie Trudel3, Paolo Santambrogio4, Sonia Levi4, Paolo Arosio5, and Joan Parkhurst Cain1 1
Oklahoma Medical Research Foundation, & University of Oklahoma Health Sciences Center, Oklahoma City, 2Kennedy Krieger Institute, Johns Hopkins University School of Medicine, Baltimore, 3Institut de Reserches Clinques de Montreal, 4Instituto di Ricera Biologica e Tecnologica, H. San Raffaele, Milano, 5University of Brescia Ferritin heavy chain (FtH), an antioxidant/stress response/ironstorage protein, localizes to the nucleus in K562 cells and represses the human adult beta-globin promoter (Broyles et al., PNAS 98: 9145, 2001). Since other work indicates FtH is an activator of fetal-globin genes, we hypothesize that FtH is a developmental hemoglobin (Hb) switching factor and that delivery of FtH to human adult erythroid cell precursors will reverse the phenotype to HbF, offering a phenotypic cure for sickle cell disease (SCD). Chromatin immunoprecipitation (ChIP) assays, antisense treatments, and an FtH transgenic mouse have confirmed that FtH is a globin gene regulatory protein in vivo. with erythroid precursor cells isolated from blood of pediatric SCD patients, under an IRB-approved protocol, we have used a twophase system for in vitro maturation of erythroid cells in the presence of FtH, delivered as pure protein, as an expression plasmid, or as a priority inducer compound that activates the endogenous FtH gene. in each mode of delivery, FtH stimulates a complete switch from HbS to HbF in five experiments using erythroid precursor cells from three different SCD donors. Fluorescently-labeled FtH protein is taken into red cell precursors in culture, suggesting that the purified protein can be directly delivered without gene therapy. This method of producing a phenotypic cure in SCD patients should be easy and inexpensive to deliver in vivo.
7 Redox Reactions of Nitrite with Oxy- and DeoxyHemoglobin and Myoglobin Along the Physiological Oxygen Gradient Rozalina Grubina1, Zhi Huang1, Sruti Shiva1, and Mark T. Gladwin1 1
National Institutes of Health The complex autocatalytic reaction of nitrite with oxyhemoglobin has been studied for a century; this reaction ultimately oxidizes nitrite to nitrate and oxyhemoglobin to methemoglobin. in contrast, the anaerobic reaction of nitrite with deoxyhemoglobin has recently been characterized as a unique allosteric autocatalytic reaction with the potential to generate bioavailable NO. Interestingly, the physiologically relevant reaction of nitrite simultaneously with both oxy- and deoxyhemoglobin at various hemoglobin oxygen saturations has never been characterized. Using least squares deconvolution absorption spectroscopy and electron paramagnetic resonance spectrometry of experiments evaluating the reaction of nitrite with both hemoglobin and myoglobin at various oxygen partial pressures, we find that the reaction of nitrite with both deoxyhemoglobin and deoxymyoglobin inhibits the autocatalytic phase of the nitrite oxy-hemoglobin and oxymyoglobin reactions. the inhibition of the oxy-reactions occurs at oxymyoglobin saturations below approximately 70% and oxyhemoglobin saturations below 60%. from a mechanistic standpoint, we consider that the NO produced from the reduction of nitrite by deoxyhemoglobin and deoxymyoglobin reacts with
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and inactivates nitrogen dioxide and ferryl-hemoglobin, both necessary intermediates in the catalytic phase of the nitriteoxyglobin reactions. the nitrite reductase activity of deoxyglobins is further potentiated by intra-cellular reductants, while the autcatalytic phase of the nitrite-oxyglobin reactions is inhibited in this reductive milieu. These chemistries determine the oxygendependent disposition of nitrite in red cells and tissue by limiting the oxidative inactivation of nitrite by oxyhemoglobin and oxymyoglobin and promoting nitrite reduction to NO during globin deoxygenation.
8 Oxidative Potential and Sources of Endogenous “Free” Iron in Rats Subjected to Endotoxic Shock Johanna Catharina Duvigneau1, Christina Piskernik2, Susanne Haindl2, Romana Hartl1, Ingeborg Kehrer2, Thomas Ebel1, Manfred Gemainer1, Heinz Redl2, and Andrey V. Kozlov2 1 Institute for Medical Chemistry, University of Veterinary Medicine, Vienna, Austria, 2Ludwig Boltzmann Institute for Experimental and Clinical Traumatology Iron ions multiply damaging potential of reactive oxygen species catalyzing Fenton reaction. the aim of this study was to clarify whether bacterial endotoxin (lipopolysaccharide) influence iron metabolism in a manner which increases the intracellular levels of potentially dangerous “free” iron in liver. “Free” iron levels in liver (EPR-spectroscopy) were increased at 4h after endotoxin challenge. Expression of transferrin receptor (reverse transcription polymerase chain reaction) was decreased, suggesting that the observed increase in “free” iron levels is not a consequence of increased iron uptake. the expression of heme oxygenase-1 (HO-1) was increased, while that of ferritin was decreased. HO-1 degrades heme thereby liberating iron, and can thus increase intracellular iron levels. Cytochrome p450, a potential substrate for HO-1, is known to degrade in endotoxic shock. We have found a positive correlation between HO-1 expression and “free” iron levels and a negative correlation between HO-1 expression and respiratory function of mitochondria. In-vitro we have shown that within all products of HO-1 only free iron reduced the performance of mitochondria. Our data suggest that endotoxin induces up-regulation of HO-1, leading to degradation of P450 and subsequent increase in intracellular free iron levels. the latter possibly contributes to liver damage.
9 Identification of Protein Radicals Formed in the Human Neuroglobin-H2O2 Reaction Olivier M Lardinois1, Leesa J Deterding1, Kenneth B Tomer1, and Ronald P Mason1 1 National Institute of Environmental Health Sciences Neuroglobin (Ngb) is a newly discovered protein that belongs to the globin family of proteins. Ngb is predominantly expressed in the brain, the retina and other nerve tissues. Although the functional role of Ngb remains unclear, Ngb contains a hemebinding domain and may participate in diverse processes such as oxygen transport, oxygen storage, nitric oxide detoxification, or modulation of terminal oxidase activity. We have utilized the immuno-spin trapping technique to investigate whether a protein radical intermediate is formed in the Ngb-H2O2 reaction. Immuno-spin trapping uses polyclonal antiserum raised against the nitrone spin trap 5,5-dimethyl-1pyrroline-N-oxide (DMPO) to detect the presence of DMPO covalently attached to proteins, an attachment which occurs only subsequent to DMPO trapping of a radical species. When DMPO spin trapping in the presence of H2O2 was combined with ELISA, Ngb/DMPO adducts were detected. the absence of H2O2 or its elimination by catalase results in nearly complete loss of trapping.
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To determine the actual site of DMPO covalent attachment to the protein moiety, the spin-trapped protein adducts were digested with trypsin, the peptides from the enzymatic digest were separated by HPLC, and the fractions eluted from the column were analyzed by ELISA. Only one fraction showed a strong positive ELISA signal and was further characterized by tandem mass spectrometry. An abundant ion that corresponds in mass to tryptic peptide T8 (amino acids residues 67-93) plus a DMPO molecule was observed. Sequencing by collision-induced dissociation revealed Tyr 87 to be the site of modification by DMPO. Liquid chromatography/peptide mapping in combination with immuno-spin trapping is an extremely powerful technique for determining the location of radical formation in proteins; it makes MS analysis substantially easier allowing greater certainty in assignment.
10 The Inactivation of Cu, Zn Superoxide Dismutase by Hydrogen Peroxide:The Mechanism of Protection Stefan I Liochev1, Andrew B Goldstone2, and Irwin Fridovich1 1 Duke University Medical Center, 2Duke University The Cu,Zn SOD is known to be inactivated by HO2− and to be protected against that inactivation by a number of small molecules including formate, imidazole, and urate. This inactivation has been shown to be due to oxidation of a ligand field histidine residue by a bound oxidant formed by reaction of the active site Cu(II) with HO2− . We now report that formate protects more effectively than does NADH and that the protective action of both increase as the pH was raised in the range 8.0 – 9.5 . This is taken to indicate increased accessibility of the Cu site with rising pH. Formate appears to act as a sacrificial substrate that protects by competing with the endogenous histidine residue for reaction with the bound oxidant, or that repairs the damage by reducing the histidyl radical intermediate
11 Relative Free Radical Scavenging Ability, GPx Activity and In Vitro Radioprotection by Two Selenocysteine Derivatives Indira Priyadarsini1, Beena Mishra1, Amit Kunwar1, Santosh Kumar2, Leeladhar Kumbhar1, and Vimal K Jain1 1 Bhabha Atomic Research Centre, Mumbai-400085, India, 2Deccan Medical College, Hyderabad, India Selenium is an essential trace element. It is present in the active site of glutathione peroxidase (GPx) enzyme of eukaryotic cells in the form of selenocysteine Selenium compounds show antioxidant activity and are being explored as new class of radioprotectors. with this aim, two diselenides, selenocysteine(SeC), and bis-selenopropanoic acid (SeP) were examined for free radical reactions and in vitro radioprotection studies. SeC was purchased from Sigma, USA and SeP was synthesized and purified in our laboratory. Reactions of hydroxyl radicals and peroxyl radicals with the compounds were studied using nanosecond pulse radiolysis, which indicated formation of selenium centered radical cations, stabilized by the oxygen and nitrogen atoms at γ-position. SeP showed better reactivity with peroxyl radicals than SeC. Inhibition of γ-radiation induced lipid peroxidation was studied in phasphatidyl choline liposomes and at a constant dose of 210 Gy, IC50 value for inhibition of lipid peroxidation was found to be, 300 and 110 μM respectively for SeC and SeP. Further γ-radiation (50 Gy) induced pBR322 plasmid DNA strand breakage studies revealed SeP to be very effective in inhibiting the DNA damage. the compounds exhibit GPx like activity and this activity is being correlated with their free radical induced redox reactions and radioprotection.
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12 Chromium(VI) Reduction by Human Cytochrome B5 Generates Hydroxyl Radical and Superoxide Griselda R.. Borthiry1, William E. Antholine1, B. Kalyanaraman1, Judith M. Myers1, and Charles R. Myers1 1
Medical College of Wisconsin The reduction of hexavalent chromium, Cr(VI), can generate reactive Cr intermediates and oxidative stress. the potential role of human microsomal enzymes in free radical generation was examined using reconstituted proteoliposomes (PLs) containing purified cytochrome b5 and NADPH:P450 reductase. Under room air, the reduction of Cr(VI) to Cr(V) by the PLs was confirmed by 53 ESR using isotopically pure Cr(VI). When 5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) was included as a spin trap, a very prominent signal for the hydroxyl radical (HO•) adduct was observed as well as a smaller signal for the superoxide (O2•− ) adduct. These adducts were observed even at very low Cr(VI) concentrations (10 µM). NADPH, Cr(VI), O2 and the PLs were all required for significant HO• generation. Superoxide dismutase eliminated the O2•− adduct, but resulted in a 30% increase in the HO• adduct. Catalase largely diminished the HO• adduct signal indicating its dependence on H2O2. Some sources of catalase were found to have Cr(VI)-reducing contaminants which could confound results, but a source of catalase free of these contaminants was used for these studies. Exogenous H2O2 was not needed, indicating that the PLs generated sufficient H2O2. Adding exogenous H2O2, however, did increase the amount of DEPMPO/HO• adduct. the inclusion of formate yielded the carbon dioxide radical adduct of DEPMPO. Experiments with dimethylsulfoxide (DMSO) plus the spin trap αphenyl-N-tert-butylnitrone (PBN) yielded the methoxy and methyl radical adducts of PBN, confirming the generation of HO•. Quantification of the various species over time was consistent with a stoichiometric excess of HO• relative to the net amount of Cr(VI) reduced. This represents the first demonstration of a role for cytochrome b5 in the generation of HO•. Overall, the simultaneous generation of Cr(V) and H2O2 by the PLs and the resulting generation of HO• at low Cr(VI) concentrations could have important implications for human Cr(VI) toxicity.
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0.010 ± 0.009% at Time 0 and 0.011 ± 0.010% at 30 hr. Unlike previous studies with a polymeric iron dextran complex that destabilized these admixtures and produced very high PFAT5 values, the novel iron salt used in this study did not affect emulsion stability. Conclusions. Iron is often necessary in patients receiving long-term PN therapy, but poses significant safety issues if given as a separate infusion or as part of the PN regimen. the use of a novel iron compound results in stable lipid emulsion-containing PN admixtures during their 30-hr period of clinical use.
14 Modification of Cysteine 111 in Human Cu, ZnSuperoxide Dismutase Ayako Okado-Matsumoto1, and Irwin Fridovich2 1 Osaka University Graduate School of Medicine, 2Duke University Medical Center Human Cu,Zn-superoxide dismutase (hSOD1) has 4 cysteines per subunit. Cys57 and Cys148 are involved in an intrasubunit disulfide bond, while Cys6 and Cys111 are free. Cys6 is buried within the protein while Cys111 is on the surface, near the dimer interface. We examined by liquid chromatography-mass spectrometry the commercially purchased hSOD1 isolated from erythrocytes as well as hSOD1s isolated from human erythrocytes, brain, and hSOD1 expressed in Sf9, yeast, and E. coli. Our goal was to ascertain whether the Cys111 modification occurred naturally in vivo. Only the Sigma erythrocyte hSOD1 appeared to contain a trisulfide crosslink between the Cys111 residues. Thus it failed to react with N-ethylmaleimide, showed absorbtion at 325nm that was eliminated by 2-mercaptoethanol, and had a mass 30 units more than expected for the native dimer. We examined the possibility that different purification methods might cause this modification in erythrocyte hSOD1. None of the procedures examined for hSOD1 purification produced such a trisulfide. in disagreement with Liu et al. [Biochemistry, 2000, 39, 8125-8132], complete derivitization of both Cys111s of hSOD1 from Sf9 cells with N-ethylmaleimide, 4-vinylpyridine, and by 5,5’dithiobis(2-nitrobenzoic acid) were readily achieved; indicating that steric hindrance was not a problem.
15 Pro-Inflammatory Impact of Reduced Thioredoxin on Rat Airways: Modulation by Airway Mucus
Stability of Lipid Emulsion-Based Parenteral Nutrition (PN) Admixtures Containing a Novel Iron Salt Deanna J. Nelson1, David Driscoll2, and Ajay Gupta3 1 BioLink LIfe Sciences, Inc., 2BIDMC/Harvard Medical School, 3 Drew Univ Medical School Purpose. to assess the effects of a novel iron compound on the stability of PN admixtures by applying the measures and limits proposed in USP <729>. Methods. the USP proposes two criteria to verify the stability of lipid emulsions. the first criterion, the intensity-weighted mean droplet size (MDS), expressed in nm, was determined using dynamic light scattering. the second measure, the large-diameter tail of the globule size distribution (GSD), expressed as the volume-weighted percent of fat > 5 µm or PFAT5, was determined using light extinction employing a single-particle optical sensing technique. the upper USP limits for these measures are 500 nm and 0.05%, respectively. Twenty clinically relevant PNs containing variable amounts of soluble ferric pyrophosphate were prepared in triplicate (n=60) under aseptic conditions and studied over 30 hr at room temperature. the MDS values were determined at the beginning (Time 0) and end of study (Time 30), while PFAT5 levels were determined at Time 0, and at 6, 24 and 30 hr. Results. the MDS at Time 0 was 275.0 ± 2.9 nm and 276.8 ± 13.8 nm at Time 30 hr. PFAT5 was
Ray C Rancourt1, Rees L Lee2, Heidi C O'Neill1, Frank J Accurso3, and Carl W White1 1 National Jewish Medical & Research Center, Denver CO, 2Pediatric Pulmonary Medicine, Navy, 3The Mike McMorris Cystic Fibrosis Research and Treatment Center;The Childrens Hospital, Denver CO. Thioredoxin (Trx) has previously been shown to decrease the viscosity of cystic fibrosis (CF) sputum. Since Trx intended for use as a mucolytic would be administered into the lung, this study examined the effects of intratracheal instillation of reduced recombinant human thioredoxin (rhTrx) in naïve rat airways. Delivery of reduced rhTrx (1.2 mg in 100 μl of 0.9% NaCl) resulted in significant increases in the number of neutrophils in bronchoalveolar lavage fluid (BALF) at 4 hours post treatment. Neutrophil influx into airways coincided with increases in cytokine expression (TNFα, CINC2β, and MIP3α), and increased activity of the p65 subunit of NF-κB in nuclear extracts derived from lung homogenates. Treatment of rats with saline alone, or human serum albumin or oxidized rhTrx at equal molarities, did not significantly increase BALF neutrophil or cytokine levels. Similarly, deposition of CF sputum (50 μl) into the lung immediately prior to reduced rhTrx delivery attenuated these responses. These observations suggest that rhTrx is reducing disulfide bond substrates present in CF sputum, limiting the reduction of other
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lung constituents. Taken together, these findings indicate that the observed chemotactic and cytokine responses are an effect of the reducing potential of the rhTrx preparation.
16 On the Modeling of Heme-Dependent, O2Oxidation of Thiols by Ru-Porphyrins Júlio S. Rebouças1, and Brian R. James2 1 University of British Columbia; Duke University Medical Center, 2 University of British Columbia Heme-proteins play key roles in some aspects of the oxidation of biological thiols. for instance, the activity of heme-regulated eIF2α kinase is dictated by a delicate thiol/disulfide balance (J.J. Chen et al., J. Biol. Chem. 1989, 264, 9559); and the hemecontaining enzymes horseradish peroxidase, myeloperoxidase, and lactoperoxidase, which usually employ H2O2 as oxygen donor, are remarkably able to catalyze the O2-oxidation of thiols, without added H2O2 (B.E. Svensson et al., Free Radic. Biol. Med. 1993, 14, 167; L.S. Harman et al., J. Biol. Chem. 1984, 259, 5606; J. Olsen et al., Biochim. Biophys. Acta 1976, 445, 324). a well-documented dysfunction associated with ‘free’ heme toxicity is the heme-dependent oxidation of membrane-protein thiols in sickle red blood cells (R.P. Hebbel et al., Blood 1988, 71, 876). the synthesis, transport, storage, and degradation of heme occur under very tight cellular control but, under stress or pathological conditions, the heme moiety may be freed from a heme-protein and may then mediate, catalyze, or participate in reactions that hold little (if any) resemblance to those of the parent protein; these reactions may in fact resemble more closely those of ‘protein-free’ model systems. Little is known about the chemistry of those oxidations, and porphyrin-based models for O2-oxidation of thiol are scarce. We describe here detailed kinetic and spectroscopic studies on the O2-oxidation of aromatic and aliphatic thiols catalyzed by ‘protein-free’ Ru-porphyrin species. the kinetic of these oxidations vary with the nature of the thiol and show rate-dependence on thiol, O2, H2O, and Ru. the involvement of dioxo-, oxo-, thiol-, thiolate-, and disulfide-Ru species in the 1 catalysis were examined by H NMR spectroscopy. a marked inhibitory effect of CO indicate that the role of the Ru species is not that of a radical-chain initiator, and that metal centered-redox processes are likely involved in the catalysis. a tentative, RSOHbased mechanism that accommodates generally the kinetic ratedependences, SH/SD kinetic isotope effect, and spectroscopic data is presented. the difficulties in establishing unequivocally the participation of either thiyl or sulfenic acid intermediates are also addressed. (Supported by: NSERC, CAPES, and UBC)
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18 Deoxymyoglobin is a Nitrite Reductase that Generates NO Sruti Shiva1, Zhi Huang1, Rozalina Grubina1, Lorna A. Ringwood1, Peter Macarthur1, and Mark T. Gladwin1 1 Vascular Medicine Branch, NHLBI, National Institutes of Health Previous studies have demonstrated that nitrite is an endocrine reservoir of nitric oxide (NO) that participates in hypoxic vasodilation. Nitrite can be reduced to NO enzymatically by deoxyhemoglobin in a reaction that is under allosteric control with a maximum reaction rate near the hemoglobin P50. in this study, we characterized the nitrite reductase activity of deoxygenated myoglobin, which reduces nitrite approximately 50 times faster than deoxyhemoglobin due to its lower heme redox potential. Spectrophotometric and chemiluminescent measurements show the deoxymyoglobin-nitrite reaction produces NO in a second order reaction that is linearly dependent on deoxymyoglobin, nitrite and proton concentration, with a bimolecular rate constant -1 -1 of 14 M s at pH 7.4 at 37°C. from a pathophysiological standpoint, the IC50 for NO-dependent inhibition of mitochondrial respiration is approximately 100 nM at physiological oxygen tensions (5-10 μM) in the working heart; we therefore hypothesized that the myoglobin-dependent reduction of nitrite could modulate mitochondrial respiration. Indeed, the addition of deoxymyoglobin and nitrite to isolated rat heart and liver mitochondria resulted in the inhibition of respiration, while myoglobin or nitrite alone had no effect. the addition of nitrite to rat heart homogenate, which contains both myoglobin and mitochondria, resulted in NO gas generation and inhibition of mitochondrial respiration; these effects were blocked by myoglobin oxidation with ferricyanide but not by allopurinol, a xanthine oxidoreductase inhibitor. These data expand upon the novel paradigm that heme-globins conserve and generate NO via nitrite reduction along physiological oxygen gradients, and further demonstrate that NO generation from nitrite reduction can escape heme autocapture to mediate fundamental biological responses to hypoxia.
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Nitric Oxide Regulates MMP-9 Activity at the Protein Level Lisa Ridnour1, Alisha Windhausen1, Jeffery Isenberg2, Nolan Yeung3, Michael Vitek3, David Roberts2, and David Wink1 1 Radiation Biology Branch, NCI, 2Laboratory of Pathology, NCI, 3 Duke University Interest in redox involvement in the regulation of extracellular matrix is beginning to emerge. Matrix reorganization is a dynamic process that occurs during both normal physiology and disease, and is mediated by proteolytic enzymes including matrix metalloproteinases (MMPs). MMPs are tightly regulated at multiple levels including transcription, post-transcription, and posttranslation. in addition, these enzymes are distinguished by a highly conserved catalytic domain containing a zinc ion as well as a pro-domain, which regulates enzyme activation by modulation of the cysteine switch. NO and RNOS derived from NO target zinc finger motifs as well as cysteine residues. Toward this end, our laboratory has identified a biphasic regulatory affect of NO, released from spermine NONOate, on the activities of MMPs secreted from ANA-1 murine macrophages, as well as purified
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MMP-9 enzyme, suggesting that NO activation of MMP-9 occurs, in part, at the protein level. the NO-mediated biphasic affect observed in the conditioned media of ANA-1 cells was reversed by the sGC inhibitor ODQ, which modulated TIMP-1 protein. Moreover, basal MMP activity levels in the conditioned media of ANA-1 cells was suppressed by LNAME, suggesting that low levels of constitutively produced NO modulate MMP activity. These results demonstrate the ability of NO to regulate MMP activities, which occurs at least in part, at the protein level via NO/RNOS protein modification and/or TIMP1 modulation.
Double Edge Redox-Implications for the Interaction Between Endogenous Thiols and Copper Ions: In VitroStudies Hernan C. Speisky1, Catalina P. Carrasco-Pozo1, Edgar N. Pastene1, Maritza D. Gomez1, and Claudio A.Oolea1 1 University of Chile The presence of a thiol moiety on the structure of glutathione, γglutamyl-cysteine, cysteinyl-glycine, cysteine and homocysteine, endows these compounds with the ability to exert, in the presence 2+ of Cu ions, a double-edge redox action. They could act either as antioxidants, by donating an electron or a hydrogen atom to a free 2+ into radical, or as pro-oxidants by reducing Cu the redox-active + Cu specie. We addressed here the redox-consequences 2+ associated with the interaction between these thiols and Cu , investigating their free radical-scavenging, O2•− -generating, and ascorbate-oxidizing properties. Upon a brief pre-incubation, the 2+ thiols/Cu interaction led to a decrease in their free radical-
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scavenging properties, and to an apparently unrelated loss of thiol density. Both effects were, however, only partial, even in the 2+ presence of a large molar Cu -excess, and were unaffected by increasing the pre-incubation time. at equimolar concentrations, the mixtures contained Cu(II)-complexes (EPR-detected), that were time-stable and ascorbate-reducible, but redox-inactive towards oxygen. at a slight molar thiol-excess (3:1), the mixtures generated time-stable Cu(I)-complexes that were unreactive to ascorbate and oxygen. a major exception was the 2+ GSH/Cu mixture which displayed a O2•− -generating capacity, as ·evidenced through its ability to generate an O2 -EPR spectrum, to reduce cytochrome-c, and to continually consume oxygen (in the 2+ presence of SOD). the O2•− -generating ability of the GSH/Cu mixture remained unaltered–at least- during 16 hr of preincubation. We conclude that the interaction between GSH and 2+ Cu ions leads to the formation of redox-active complexes which are capable of promoting a pro-oxidant action. Funded by Fondecyt #1040736.
20 Sublethal Hyperoxia Alters Pulmonary and Hepatic Thioredoxin-1 Levels in Neonatal Mice Trent E Tipple1, Lynette K Rogers1, Charles V Smith2, and Stephen E Welty1 1 Columbus Children's Research Institute, 2Seattle Children's Hospital and Regional Medical Center Prolonged exposure of neonatal mice to sublethal hyperoxia causes functional and pathological changes similar to those seen in infants with bronchopulmonary dysplasia (BPD). Expression of thioredoxin-1 (Trx1), a dithiol disulfide oxidoreductase important in antioxidant defense, regulation of cellular proliferation, and regulation of gene expression, is altered by changes in ambient oxygen tension in a baboon model of BPD. the objective of the present studies was to test the hypothesis that neonatal mice exposed to sublethal hyperoxia will exhibit alterations in normal developmental pulmonary and hepatic levels of Trx1. Within 24 h of birth, C3H/HeN mice were exposed to sublethal hyperoxia (FiO2 = 0.85) or were kept in room air (FiO2 = 0.21). the dams nursing the pups were rotated between hyperoxia and room air every 24 hours. at 1, 3, and 7 d, mice were euthanized, lungs and livers removed, and Trx1 levels determined by western blotting. No time-dependent effects on Trx1 levels in the lungs and livers of room air controls were observed. on days 1, 3, and 7, Trx1 levels in lungs of pups exposed to hyperoxia were less than 25% of the levels in room air controls. on days 1 and 3, hepatic Trx1 levels were not different between room air and hyperoxia, but on day 7, hepatic Trx1 levels in hyperoxic pups were three times the levels in room air controls. the data indicate that tissue levels of Trx1 in the livers and lungs of newborn mice are altered by changes in ambient oxygen tension. the alterations of Trx1 caused by exposure of newborn mice to sublethal hyperoxia may contribute to altered lung developmental maturation in this model. Similar effects in prematurely born human infants similarly could contribute to altered lung development that is characteristic of BPD.
21 Copper Dependence of the Biotin Switch Assay Xunde Wang1, Geng Liu1, Peter Macarthur1, Neil Hogg2, and Mark T. Gladwin1 1 Vascular Medicine Branch, NHLBI; NIH, Bethesda, MD 20892, USA, 2 Medical College of Wisconsin, Milwaukee WI 53226 Studies have shown that modification of critical cysteine residues in proteins leads to the regulation of protein function. These modifications include disulfide bond formation, glutathiolation, sulfenic and sulfinic acid modifications, and S-nitrosation. the biotin switch assay was developed to detect S-nitrosation (Jaffrey et al., Nat. Cell Biol. 3 (2001), pp. 193–197). Specifically, proteins
are denatured with SDS in the presence of methyl methanethiosulfonate (MMTS) to alkylate free thiols. the SDS and temperature of 50°C are used to expose and facilitate alkylation of buried thiols. After acetone precipitation or Sephadex G25 separation to remove excess MMTS, HPDP-biotin and 1 mM ascorbate are added to reduce the S-nitrosothiol bond and modify the reduced thiol with HPDP-biotin. the proteins are then separated on SDS-PAGE and quantified using either streptavidinHRP or anti-biotin HRP conjugate. Interestingly, we have found that different levels of S-nitrosothiol are obtained for the same protein using different buffers and with the inclusion of different chelators (DTPA vs. EDTA). Using purified S-nitrosated albumin, we have found that “contaminating” copper is required to degrade S-nitrothiol before ascorbate and biotin-HPDP can react with the free thiol; ascorbate itself does not reduce the S-nitrosothiol. Removal of copper from buffers by DTPA and other copper chelators preserves approximately 90% of the S-nitrosothiol, while including copper and ascorbate completely eliminates the Snitrosothiol in the preparation and increases the biotin labeling. These biotin switch experiments were confirmed using triiodide based reductive chemiluminascence. the results indicate that degradation of S-nitrosothiol in the standard biotin switch assay is copper dependent and that experimental variability in S-NO yields using this assay occur secondary to the inclusion of copper chelators and variable copper contamination in buffers and labware.
22 The Role of the N-Terminal Octarepeat Domain in the Antioxidant Activity of Mouse Prion Protein Yasuko Asano Watanabe1, Osamu Inanami1, Tetsu Inoue1, Daisuke Iizuka1, and Mikinori Kuwabara1 1 Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University C The cellular prion protein (PrP ) contains the octarepeat domain 2+ (OR domain) in the N-terminal region that acts as the Cu 2+ binding domain. Cu is considered to modulate various biological C functions of PrP such as the cellular enzymatic activity of superoxide dismutase (SOD) and conversion to the scrapie isoform of PrP. in this study, to clarify further physiological C C function of PrP as an antioxidant, the susceptibility of PrP overexpressed NIH3T3 cells to various oxidants was evaluated. in addition, we prepared recombinat mouse PrP (rmoPrP(23-231)) and truncated PrP (rmoPrP(91-231)), lacking OR domain, and then the O2− -scavenging activity of rmoPrP and the inhibiting activity of 2+ 2+ rmoPrP against Cu /H2O2-catalyzed oxidative reaction or Cu t catalyzed oxidative reaction of BuOOH were measured by spintrapping technique using electron spin resonance spectroscopy 2+ (ESR). the Cu - or H2O2-induced cell death was significantly C inhibited by overexpression of wild-type PrP , but radiation2+ induced cell death was not. the oxidative reaction of Cu with 2+ t H2O2 or Cu -catalyzed oxidative reaction of BuOOH was also shown to be significantly reduced by the addition of rmoPrP(23-231) but not rmoPrP(91-231) by spin-trapping experiments. Whereas, in the assay using spin-trapping technique, the O2− -scavenging 2+ activity of rmoPrP(23-231)/Cu complex was about two times higher 2+ 3 than that of Cu alone, but 10 times lower than that of Cu/ZnC SOD. These results suggested that PrP inhibited the cellular 2+ damage induced by Cu - and H2O2-oxidative stress thorough OR domain of PrP.
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