Peroxynitrite oxidation of tubulin sulfhydryls inhibits microtubule polymerization

Considerable evidence both in vitro and in vivo implicates protein damage by peroxynitrite as a probable mechanism of cell death. Herein, we report that treatment of bovine brain microtubule protein, composed of tubulin and microtubule-associated proteins, with peroxynitrite led to a dose-dependent inhibition of microtubule polymerization. The extent of cysteine oxidation induced by peroxynitrite correlated well with inhibition of microtubule polymerization. Disulfide bonds between the subunits of the tubulin heterodimer were detected by Western blot as a result of peroxynitrite-induced cysteine oxidation. Addition of disulfide reducing agents including dithiothreitol and beta-mercaptoethanol restored a significant portion of the polymerization activity that was lost following peroxynitrite addition. Thus, peroxynitrite-induced disulfide bonds are at least partially responsible for the observed inhibition of polymerization. Sodium bicarbonate protected microtubule protein from the peroxynitrite-induced inhibition of polymerization. Tyrosine nitration of microtubule protein by 1 mM peroxynitrite increased approximately twofold when sodium bicarbonate was present whereas the extent of cysteine oxidation decreased from 7.5 to 6.3 mol cysteine/mol tubulin. These results indicate that cysteine oxidation of tubulin by peroxynitrite, rather than tyrosine nitration, is the primary mechanism of inhibition of microtubule polymerization.     

Peroxynitrite-mediated oxidative modifications of myosin and implications on structure and function

Abstract

The peroxynitrite-induced functional impairment of myosin was studied in different reaction conditions, known to alter the oxidative chemistry of peroxynitrite, to better understand the molecular mechanisms of this interaction. It is shown that peroxynitrite is able to enhance the basal MgATPase activity up to 2-fold while inhibiting the actin-stimulated ATPase activity of myosin and that the extent of these functional alterations is dependent on the reaction medium. The observed changes in the stimulation of the MgATPase activity correlate with the extent of carbonyl formation in myosin. The enzyme inhibition is more potent in conditions where the efficiency of tyrosine nitration and peroxynitrite reactivity towards sulphydryls are lower. Together with the observation that reversion of sulphydryl oxidation did not lead to the recovery of myosin functional and structural impairments, these results point out to the importance of protein carbonylation as a post-translational modification in the peroxynitrite-induced myosin functional impairment.     

Functional and structural analysis of catalase oxidized by singlet oxygen

Purified catalase-1 (CAT-1) from Neurospora crassa asexual spores is oxidized by singlet oxygen giving rise to active enzyme forms with different electrophoretic mobility. These enzyme forms are detected in vivo under stress conditions and during development at the start of the asexual morphogenetic transitions. CAT-1 heme b is oxidized to heme d by singlet oxygen. Here, we describe functional and structural comparisons of the non-oxidized enzyme with the fully oxidized one. Using a broad H(2)O(2) concentration range (0.01-3.0 M), non-hyperbolic saturation kinetics was found in both enzymes, indicating that kinetic complexity does not arise from heme oxidation. The kinetics was consistent with the existence of two kinds of active sites differing more than 10-times in substrate affinity. Positive cooperativity for one or both of the saturation curves is possible. Kinetic constants obtained at 22 degrees C varied slightly and apparent activation energies for the reaction of both components are not significantly different. Protein fluorescence and circular dicroism of the two enzymes were nearly identical, indicating no gross conformational change with oxidation. Increased sensitivity to inhibition by cyanide indicated a local change at the active site in the oxidized catalase. Oxidized catalase was less resistant to high temperatures, high guanidinium ion concentration, and digestion with subtilisin. It was also less stable than the non-oxidized enzyme at an acid pH. The overall data show that the oxidized enzyme is structurally different from the non-oxidized one, although it conserves most of the remarkable stability and catalytic efficiency of the non-oxidized enzyme. Because the enzyme in the cell can be oxidized under physiological conditions, preservation of functional and structural properties of catalase could have been selected through evolution to assure an active enzyme under oxidative stress conditions.     

Peroxynitrite induces F-actin depolymerization and blockade of myosin ATPase stimulation

Treatment of F-actin with the peroxynitrite-releasing agent 3-morpholinosydnonimine (SIN-1) produced a dose-dependent F-actin depolymerization. This is due to released peroxynitrite because it is not produced by 'decomposed SIN-1', and it is prevented by superoxide dismutase concentrations efficiently preventing peroxynitrite formation. F-actin depolymerization has been found to be very sensitive to peroxynitrite, as exposure to fluxes as low as 50-100nM peroxynitrite leads to nearly 50% depolymerization in about 1h. G-actin polymerization is also impaired by peroxynitrite although with nearly 2-fold lower sensitivity. Exposure of F-actin to submicromolar fluxes of peroxynitrite produced cysteine oxidation and also a blockade of the ability of actin to stimulate myosin ATPase activity. Our results suggest that an imbalance of the F-actin/G-actin equilibrium can account for the observed structural and functional impairment of myofibrils under the peroxynitrite-mediated oxidative stress reported for some pathophysiological conditions.     

Metal-catalyzed oxidation of Escherichia coli glutamine synthetase: correlation of structural and functional changes

Metal-catalyzed oxidation of proteins has been implicated in a variety of biological processes, particularly in the marking of proteins for subsequent proteolytic degradation. The metal-catalyzed oxidation of bacterial glutamine synthetase causes conformational, covalent, and functional alterations in the protein. To understand the structural basis of the functional changes, the time course of oxidative modification of glutamine synthetase was studied utilizing a nonenzymic model oxidation system consisting of ascorbate, oxygen, and iron. The structural modifications induced included: decreased thermal stability; weakening of subunit interactions; decrease in isoelectric point; introduction of carbonyl groups into amino acid side chains; and loss of two histidine residues. These changes did not denature the protein, but instead induced relatively subtle changes. Indeed, even the most extensively modified protein had a sedimentation velocity which was identical to that of the native enzyme. Comparison of the time courses of the structural and functional changes established that: (i) Loss of the metal binding site and of catalytic activity occurred with loss of one histidine per subunit; (ii) increased susceptibility to proteolysis occurred with loss of two histidine residues per subunit. Thus, oxidation at one site suffices to inactivate the enzyme, but two sites must be modified to induce susceptibility to proteolysis. The limited and specific changes induced by metal-catalyzed oxidation are consistent with a site-specific free radical mechanism.     

Role of the disulphide bridge in folding, stability and function of porcine odorant binding protein: spectroscopic equilibrium studies on C63A/C155A double mutant

Porcine odorant binding protein (pOBP) contains a single disulphide bridge linking residues Cys63 and Cys155. In order to get information on the role played by this crosslink in determining the structural and functional properties of the protein, we substituted these two Cys residues with two Ala residues by site directed mutagenesis and investigated the changes in folding, stability and functional features, as detected by fluorescence and circular dichroism measurements. In particular, we studied both chemical and thermal unfolding/refolding processes under equilibrium conditions, the first induced by guanidinium hydrochloride and the second by raising the temperature from 15 to 90 degrees C. Chemical unfolding curves, as obtained from intrinsic fluorescence and far-UV circular dichroism data, can be fitted by a simple two-state cooperative sigmoidal function; however, their partial overlap (C(1/2)=0.57+/-0.05 from fluorescence and 0.66+/-0.03 from CD) suggests the formation of an intermediate, which lacks tertiary structural features. Thermal unfolding was found to be reversible if the protein was heated up to 65 degrees C, but irreversible above that temperature because of aggregation. The thermodynamic unfolding parameters of this double mutant protein, when compared to those of the wild type protein, clearly point out the important role played by the disulphide bridge on the stability and function of this protein family and probably of many other lipocalins.     

EGCG has Dual and Opposing Effects on the N-terminal Region of Self-associating α-synuclein Oligomers

Oligomers of the protein α-synuclein (α-syn) are thought to be a major toxic species in Parkinson’s disease, particularly through their ability to permeabilize cell membranes. The green tea polyphenol epigallocatechin gallate (EGCG) has been found to reduce this ability. We have analyzed α-syn oligomer dynamics and interconversion by H/D exchange monitored by mass spectrometry (HDX-MS). Our results show that the two oligomers OI and OII co-exist in equilibrium; OI is a multimer of OII and its dissociation can be followed by HDX-MS by virtue of the correlated exchange of the N-terminal region. Urea destabilizes the α-syn oligomers, dissociating OI to OII and monomers. Oligomers exposed to EGCG undergo Met oxidation. Intriguingly, EGCG induces an oxidation-dependent effect on the structure of the N-terminal region. For the non-oxidized N-terminal region, EGCG increases the stability of the folded structure as measured by a higher level of protection against H/D exchange. In contrast, protection is clearly abrogated in the Met oxidized N-terminal region. Having a non-oxidized and disordered N-terminal region is known to be essential for efficient membrane binding. Therefore, our results suggest that the combined effect of a structural stabilization of the non-oxidized N-terminal region and the presence of a disordered oxidized N-terminal region renders the oligomers less cytotoxic by decreasing the ability of the N-terminal region to bind to cell membranes and facilitate their permeabilization.     

Oxidation of methionine residues in recombinant human interleukin-1 receptor antagonist: implications of conformational stability on protein oxidation kinetics

Oxidation of methionine residues is involved in several biochemical processes and in degradation of therapeutic proteins. The relationship between conformational stability and methionine oxidation in recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was investigated to document how thermodynamics of unfolding affect methionine oxidation in proteins. Conformational stability of rhIL-1ra was monitored by equilibrium urea denaturation, and thermodynamic parameters of unfolding (DeltaGH2O, m, and Cm) were estimated at different temperatures. Methionine oxidation induced by hydrogen peroxide at varying temperatures was monitored during "coincubation" of rhIL-1ra with peptides mimicking specific regions of the reactive methionine residues in the protein. The coincubation study allowed estimation of oxidation rates in protein and peptide at each temperature from which normalized oxidation rate constants and activation energies were calculated. The rate constants for buried Met-11 in the protein were lower than for methionine in the peptide with an associated increase in activation energy. The rate constants and activation energy of solvent exposed methionines in protein and peptide were similar. The results showed that conformational stability, monitored using the Cm value, has an effect on oxidation rates of buried methionines. The rate constant of buried Met-11 correlated well with the Cm value but not DeltaGH2O. No correlation was observed for the oxidation rates of solvent-exposed methionines with any thermodynamic parameters of unfolding. The findings presented have implications in protein engineering, in design of accelerated stability studies for protein formulation development, and in understanding disease conditions involving protein oxidation.     

Inhibition of tubulin polymerization by hypochlorous acid and chloramines

Protein thiol oxidation and modification by nitric oxide and glutathione are emerging as common mechanisms to regulate protein function and to modify protein structure. Also, thiol oxidation is a probable outcome of cellular oxidative stress and is linked to degenerative disease progression. We assessed the effect of the oxidants hypochlorous acid and chloramines on the cytoskeletal protein tubulin. Total cysteine oxidation by the oxidants was monitored by labeling tubulin with the thiol-selective reagent 5-iodoacetamidofluorescein; by reaction with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid); and by detecting interchain tubulin disulfides by Western blot under nonreducing conditions. Whereas HOCl induced both cysteine and methionine oxidation of tubulin, chloramines were predominantly cysteine oxidants. Cysteine oxidation of tubulin, rather than methionine oxidation, was associated with loss of microtubule polymerization activity, and treatment of oxidized tubulin with disulfide reducing agents restored a considerable portion of the polymerization activity that was lost after oxidation. By comparing the reactivity of hypochlorous acid and chloramines with the previously characterized oxidants, peroxynitrite and the nitroxyl donor Angeli's salt, we have identified tubulin thiol oxidation, not methionine oxidation or tyrosine nitration, as a common outcome responsible for decreased polymerization activity.     

Peroxynitrite protects neurons against nitric oxide-mediated apoptosis. A key role for glucose-6-phosphate dehydrogenase activity in neuroprotection

Peroxynitrite is thought to be a nitric oxide-derived neurotoxic effector molecule involved in the disruption of key energy-related metabolic targets. To assess the consequences of such interference in cellular glucose metabolism and viability, we studied the possible modulatory role played by peroxynitrite in glucose oxidation in neurons and astrocytes in primary culture. Here, we report that peroxynitrite triggered rapid stimulation of pentose phosphate pathway (PPP) activity and the accumulation of NADPH, an essential cofactor for glutathione regeneration. In contrast to peroxynitrite, nitric oxide elicited NADPH depletion, glutathione oxidation, and apoptotic cell death in neurons, but not in astrocytes. These events were noticeably counteracted by pretreatment of neurons with peroxynitrite. In an attempt to elucidate the mechanism responsible for this PPP stimulation and neuroprotection, we found evidence consistent with both exogenous and endogenous peroxynitrite-mediated activation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that catalyzes the first rate-limiting step in the PPP. Moreover, functional overexpression of the G6PD gene in stably transformed PC12 cells induced NADPH accumulation and offered remarkable resistance against nitric oxide-mediated apoptosis, whereas G6PD gene-targeted antisense inhibition depleted NADPH levels and exacerbated cellular vulnerability. In light of these results, we suggest that G6PD activation represents a novel role for peroxynitrite in neuroprotection against nitric oxide-mediated apoptosis.     

Hemin-H2O2-NO2(-) induced protein oxidation and tyrosine nitration are different from those of SIN-1: a study on glutamate dehydrogenase nitrative/oxidative modification

Protein oxidation and tyrosine nitration are two major post-translational modifications of protein by reactive nitrogen oxide species, which are mainly produced by peroxynitrite and heme peroxidases (hemin)-H(2)O(2)-NO(2)(-) system. We report herein some novel phenomena between hemin-H(2)O(2)-NO(2)(-) and 3-morpholinosydnonimine hydrochloride (SIN-1)-mediated oxidation and nitration reactions of glutamate dehydrogenase (GDH). Hemin-H(2)O(2) could effectively induce GDH protein oxidation and reduce its activity. Although the addition of low concentration of nitrite promoted protein oxidation, protein oxidation was weakened with the increase of nitrite concentration, meanwhile, tyrosine nitration was increased and the enzyme activity was partially restored. However, with the increase of SIN-1 concentration, protein oxidation and tyrosine nitration were increased, enzyme activity was decreased. The presence of desferrioxamine and/or catechin inhibit tyrosine nitration both in hemin-H(2)O(2)-NO(2)(-) and in SIN-1, but they promoted protein oxidation and reduced the enzyme activity in hemin-H(2)O(2)-NO(2)(-) system, while inhibited protein oxidation and recover the enzyme activity in SIN-1 system. These results reveal both hemin-H(2)O(2)-NO(2)(-) and SIN-1 can cause inactivation of GDH through protein oxidation and tyrosine nitration, but the impact of the effect of protein oxidation (not thiol oxidation) on enzyme activity is stronger than that of protein tyrosine nitration. Moreover, mass spectrometric analysis indicated that nitrated tyrosine residues by hemin-H(2)O(2)-NO(2)(-) were Tyr262 and Tyr471 while by SIN-1 were Tyr401 and Tyr493. It meant that protein oxidation and tyrosine nitration of GDH induced by hemin-H(2)O(2)-NO(2)(-) were different from those induced by SIN-1.     

Denatured State Structural Property Determines Protein Stabilization by Macromolecular Crowding: A Thermodynamic and Structural Approach

Understanding of protein structure and stability gained to date has been acquired through investigations made under dilute conditions where total macromolecular concentration never surpasses 10 g l⁻¹. However, biological macromolecules are known to evolve and function under crowded intracellular environments that comprises of proteins, nucleic acids, ribosomes and carbohydrates etc. Crowded environment is known to result in altered biological properties including thermodynamic, structural and functional aspect of macromolecules as compared to the macromolecules present in our commonly used experimental dilute buffers (for example, Tris HCl or phosphate buffer). In this study, we have investigated the thermodynamic and structural consequences of synthetic crowding agent (Ficoll 70) on three different proteins (Ribonuclease-A, lysozyme and holo α-lactalbumin) at different pH values. We report here that the effect of crowding is protein dependent in terms of protein thermal stability and structure. We also observed that the structural characteristics of the denatured state determines if crowding will have an effect or not on the protein stability.     

Protein tyrosine nitration of mitochondrial carbamoyl phosphate synthetase 1 and its functional consequences

Mitochondria are the primary locus for the generation of reactive nitrogen species including peroxynitrite and subsequent protein tyrosine nitration. Protein tyrosine nitration may have important functional and biological consequences such as alteration of enzyme catalytic activity. In the present study, mouse liver mitochondria were incubated with peroxynitrite, and the mitochondrial proteins were separated by 1D and 2D gel electrophoresis. Nitrotyrosinylated proteins were detected with an anti-nitrotyrosine antibody. One of the major proteins nitrated by peroxynitrite was carbamoyl phosphate synthetase 1 (CPS1) as identified by LC-MS protein analysis and Western blotting. The band intensity of nitration normalized to CPS1 was increased in a peroxynitrite concentration-dependent manner. In addition, CPS1 activity was decreased by treatment with peroxynitrite in a peroxynitrite concentration- and time-dependent manner. The decreased CPS1 activity was not recovered by treatment with reduced glutathione, suggesting that the decrease of the CPS1 activity is due to tyrosine nitration rather than cysteine oxidation. LC-MS analysis of in-gel digested samples, and a Popitam-based modification search located 5 out of 36 tyrosine residues in CPS1 that were nitrated. Taken together with previous findings regarding CPS1 structure and function, homology modeling of mouse CPS1 suggested that nitration at Y1450 in an α-helix of allosteric domain prevents activation of CPS1 by its activator, N-acetyl-l-glutamate. In conclusion, this study demonstrated the tyrosine nitration of CPS1 by peroxynitrite and its functional consequence. Since CPS1 is responsible for ammonia removal in the urea cycle, nitration of CPS1 with attenuated function might be involved in some diseases and drug-induced toxicities associated with mitochondrial dysfunction.     

Metallothionein inhibits peroxynitrite-induced DNA and lipoprotein damage

Previous studies have demonstrated that metallothionein functions as an antioxidant that protects against oxidative DNA, protein, and lipid damage induced by superoxide anion, hydrogen peroxide, hydroxyl radical, and nitric oxide. The present study was undertaken to test the hypothesis that metallothionein also protects from DNA and lipoprotein damage induced by peroxynitrite, an important reactive nitrogen species that causes a diversity of pathological processes. A cell-free system was used. DNA damage was detected by the mobility of plasmid DNA in electrophoresis. Oxidation of low density lipoprotein was measured by a thiobarbituric acid-reactive substance, which was confirmed by lipid hydroperoxide assay. Plasmid DNA damage and low density lipoprotein oxidation were induced by 3-morpholinosydnomine, which produces peroxynitrite through the reaction between nitric oxide and superoxide anion or by synthesized peroxynitrite directly. DNA damage by 3-morpholinosydnomine was prevented by both metallothionein and superoxide dismutase, whereas the damage caused by peroxynitrite was prevented by metallothionein only. The oxidation of low density lipoprotein by 3-morpholinosydnomine and peroxynitrite was also significantly inhibited by metallothionein. This study thus demonstrates that metallothionein may react directly with peroxynitrite to prevent DNA and lipoprotein damage induced by this pathological reactive nitrogen species.     

Differential effects of peroxynitrite on human mitochondrial creatine kinase isoenzymes. Inactivation,octamer destabilization,and identification of involved residues

Creatine kinase isoenzymes are very susceptible to free radical damage and are inactivated by superoxide radicals and peroxynitrite. In this study, we have analyzed the effects of peroxynitrite on enzymatic activity and octamer stability of the two human mitochondrial isoenzymes (ubiquitous mitochondrial creatine kinase (uMtCK) and sarcomeric mitochondrial creatine kinase (sMtCK)), as well as of chicken sMtCK, and identified the involved residues. Inactivation by peroxynitrite was concentration-dependent and similar for both types of MtCK isoenzymes. Because peroxynitrite did not lower the residual activity of a sMtCK mutant missing the active site cysteine (C278G), oxidation of this residue is sufficient to explain MtCK inactivation. Mass spectrometric analysis confirmed oxidation of Cys-278 and further revealed oxidation of the C-terminal Cys-358, possibly involved in MtCK/membrane interaction. Peroxynitrite also led to concentration-dependent dissociation of MtCK octamers into dimers. In this study, ubiquitous uMtCK was much more stable than sarcomeric sMtCK. Mass spectrometric analysis revealed chemical modifications in peptide Gly-263-Arg-271 located at the dimer/dimer interface, including oxidation of Met-267 and nitration of Trp-268 and/or Trp-264, the latter being a very critical residue for octamer stability. These data demonstrate that peroxynitrite affects the octameric state of MtCK and confirms human sMtCK as the generally more susceptible isoenzyme. The results provide a molecular explanation of how oxidative damage can lead to inactivation and decreased octamer/dimer ratio of MtCK, as seen in neurodegenerative diseases and heart pathology, respectively.     

Bicarbonate enhances peroxidase activity of Cu,Zn-superoxide dismutase. Role of carbonate anion radical and scavenging of carbonate anion radical by metalloporphyrin antioxidant enzyme mimetics.

Much evidence exists for the increased peroxidase activity of copper, zinc superoxide dismutase (SOD1) in oxidant-induced diseases. In this study, we measured the peroxidase activity of SOD1 by monitoring the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF). Bicarbonate dramatically enhanced DCFH oxidation to DCF in a SOD1/H(2)O(2)/DCFH system. Peroxidase activity could be measured at a lower H(2)O(2) concentration ( approximately 1 microm). We propose that DCFH oxidation to DCF is a sensitive index for measuring the peroxidase activity of SOD1 and familial amyotrophic lateral sclerosis SOD1 mutants and that the carbonate radical anion (CO(3)) is responsible for oxidation of DCFH to DCF in the SOD1/H(2)O(2)/bicarbonate system. Bicarbonate enhanced H(2)O(2)-dependent oxidation of DCFH to DCF by spinal cord extracts of transgenic mice expressing SOD1(G93A). The SOD1/H(2)O(2)/HCO(3)(-)-dependent oxidation was mimicked by photolysis of an inorganic cobalt carbonato complex that generates CO(3). Metalloporphyrin antioxidants that are usually considered as SOD1 mimetic or peroxynitrite dismutase effectively scavenged the CO(3) radical. Implications of this reaction as a plausible protective mechanism in inflammatory cellular damage induced by peroxynitrite are discussed.     

Osmophobic effect of glycerol on irreversible thermal denaturation of rabbit creatine kinase

Protein stability plays an extremely important role not only in its biological function but also in medical science and protein engineering. Osmolytes provide a general method to protect proteins from the unfolding and aggregation induced by extreme environmental stress. In this study, the effect of glycerol on protection of the model enzyme creatine kinase (CK) against heat stress was investigated by a combination of spectroscopic method and thermodynamic analysis. Glycerol could prevent CK from thermal inactivation and aggregation in a concentration-dependent manner. The spectroscopic measurements suggested that the protective effect of glycerol was a result of enhancing the structural stability of native CK. A further thermodynamic analysis using the activated-complex theory suggested that the effect of glycerol on preventing CK against aggregation was consistent with those previously established mechanisms in reversible systems. The osmophobic effect of glycerol, which preferentially raised the free energy of the activated complex, shifted the equilibrium between the native state and the activated complex in favor of the native state. A comparison of the inactivation rate and the denaturation rate suggested that the protection of enzyme activity by glycerol should be attributed to the enhancement of the structural stability of the whole protein rather than the flexible active site.     

Inhibitory effect of isoflavones on peroxynitrite-mediated low-density lipoprotein oxidation

Peroxynitrite, a potent oxidant formed in vivo from the reaction of nitric oxide with superoxide, can mediate low-density liprotein (LDL) oxidation which is thought to increase the risk of atherosclerosis. This study investigates the inhibitory effect of the isoflavones, genistein and daidzein, together with their glycosidic forms, genistin and daidzin, on the peroxynitrite-mediated LDL oxidation and nitration of tyrosine. Genistein and daidzein were observed to dose-dependently inhibit peroxynitrite-mediated LDL oxidation, while their glucoside conjugates showed less activity. Moreover, all the isoflavones used in this study were found to be potent peroxynitrite scavengers, preventing the nitration of tyrosine. The ability of the isoflavones at 50 microM to decrease the tyrosine nitration induced by peroxynitrite (1 mM) was in the ratios of genistein (49%), daidzein (40%), daidzin (41%) and genistin (42%) when compared to the control (tyrosine incubated only with peroxynitrite). These results suggest that an intake of isoflavones could contribute to protecting against cardiovascular diseases and chronic inflammatory diseases.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100–300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( ? )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Peroxynitrite-mediated oxidation of fibrinogen inhibits clot formation

The clotting activity of human fibrinogen was fully inhibited in vitro by peroxynitrite. The decrease of activity followed an exponential function and the concentration of peroxynitrite needed to inhibit 50% of fibrinogen clotting was 22 microM at 25 degrees C. The oxidative modification(s) induced by the peroxynitrite system (i.e. ONOO-, ONOOH and ONOOH*) appeared specifically to affect fibrin clot formation (through the inhibition of fibrinogen polymerization) since the interaction of peroxynitrite-modified fibrinogen with thrombin appeared to be unaffected. The addition of NaHCO3 decreased the peroxynitrite effect on fibrinogen clotting, suggesting that the reactive species formed by the reaction of CO2 with peroxynitrite are less efficient oxidants of peroxynitrite itself. Similar effects were observed after addition of bilirubin, which also exerted a significant protection against peroxynitrite-mediated modification of fibrinogen.