carbon dioxide reactivity toward albumin and cells from protein–tyrosine nitration to protein–cysteine nitrosation

Free Radical Biology & Medicine 38 (2005) 189 – 200 www.elsevier.com/locate/freeradbiomed

Original Contribution

Tempol diverts peroxynitrite/carbon dioxide reactivity toward albumin and cells from protein–tyrosine nitration to protein–cysteine nitrosation Denise C. Fernandes, Danilo B. Medinas, Maria Ju´lia M. Alves, Ohara Augusto* Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, CP 26077, CEP 05513-970, Sa˜o Paulo, SP, Brazil Received 21 July 2004; accepted 21 September 2004 Available online 10 November 2004

Abstract Tempol has been shown to protect experimental animals from injuries associated with excessive nitric oxide production. In parallel, tempol decreased the levels of protein-3-nitrotyrosine in the injured tissues, suggesting that it interacted with nitric oxide-derived oxidants such as nitrogen dioxide and peroxynitrite. Relevantly, a few recent studies have shown that tempol catalytically diverts peroxynitrite/carbon dioxide reactivity toward phenol from nitration to nitrosation. To examine whether this shift occurs in biological environments, we studied the effects of tempol (10–100 AM) on peroxynitrite/carbon dioxide (1 mM/2 mM) reactivity toward proteins, native bovine serum albumin (BSA) (0.5–0.7 cys/mol) and reductively denatured BSA (7–19 cys/mol), and cells (J774 macrophages). Although not a true catalyst, tempol strongly inhibited protein–tyrosine nitration (70–90%) and protein–cysteine oxidation (20–50%) caused by peroxynitrite/carbon dioxide in BSA, denatured BSA, and cells while increasing protein–cysteine nitrosation (200–400%). Tempol consumption was attributed mainly to its reaction with protein–cysteinyl radicals. Most of the tempol, however, reacted with the radicals produced from peroxynitrite/carbon dioxide, that is, nitrogen dioxide and carbonate radical anion. Accordingly, tempol decreased the yields of BSA–cysteinyl and BSA–tyrosyl/ tryptophanyl radicals, as well their decay products such as protein–3-nitrotyrosine. The parallel increase in protein–nitrosocysteine yields demonstrated that part of the peroxynitrite is oxidized to nitric oxide by the oxammonium cation produced from tempol oxidation by peroxynitrite/carbon dioxide-derived radicals. Protein–nitrosocysteine formation was shown to occur by radical and nonradical mechanisms in studies with a protein–cysteinyl radical trapper. These studies may contribute to the understanding of the protective effects of tempol in animal models of inflammation. D 2004 Elsevier Inc. All rights reserved. Keywords: Peroxynitrite; Tempol; Nitric oxide; Carbonate radical anion; Nitrogen dioxide; Protein nitration; Protein nitrosation; Free radicals

Introduction Tempol is a membrane permeable nitroxide radical that has long been known to protect laboratory animals and bacterial and mammalian cells from the injury associated

Abbreviations: BSA, bovine serum albumin; dBSA, reductively denatured bovine serum albumin; DBNBS, 3,5-dibromo-4-nitrosobenzenesulfonic acid; PBN, phenyl-N-t-butylnitrone; Peroxynitrite, the sum of peroxynitrite anion (ONOO
, oxoperoxonitrate (-1)) and peroxynitrous acid (ONOOH, hydrogen oxoperoxonitrate) unless specified; TEMPOL and TP-NO , 4-hydroxy-2,2,6,6,-tetramethyl-1-piperidinyloxy. * Corresponding author. Fax: +55 11 30912186, +55 11 3815 5579. E-mail address: [email protected] (O. Augusto).

S

0891-5849/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2004.09.027

with oxidative stress conditions [1,2]. Nitroxide antioxidant mechanisms have been shown to include dismutation of superoxide anion, detoxification of ferryl-heme species, and termination of alkyl, alkoxyl, and peroxyl radical chain reactions [1,2]. More recently, the nitroxide tempol has been shown to protect experimental animals from injuries associated with excessive nitric oxide production such as those resulting from multiple organ failure, transient cerebral ischemia, carrageenan-induced pleurisy, and dinitrobenzenesulfonic acid-induced colitis [3–6]. In all of these models, tempol decreased the levels of protein–3-nitrotyrosine residues in the injured tissues. The formation of protein–3-nitrotyrosine under conditions of nitric oxide overproduction is likely to depend on

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nitric oxide-derived oxidants such as nitrogen dioxide and peroxynitrite [7–11]. The better elucidated nitration mechanism in vitro supports a free radical process with participation of both nitrogen dioxide and protein–tyrosyl radicals that recombine to produce 3-nitrotyrosine residues. As nitrogen dioxide itself is only a moderately strong oxidizing agent (E = 0.99 V, pH 7.4), it is not surprising that efficient tyrosine nitration has been demonstrated so far with systems that produce either directly or indirectly tyrosyl radicals in addition to nitrogen dioxide [8–11 and references therein]. Examples of such systems are heme peroxidase/hydrogen peroxide/nitrite and peroxynitrite/carbon dioxide. Heme peroxidases such as horseradish peroxidase and myeloperoxidase in the presence of hydrogen peroxide form compounds I and II, which can oxidize nitrite and solvent-exposed protein–tyrosine residues to nitrogen dioxide and tyrosyl radicals, respectively. In the case of the peroxynitrite/carbon dioxide pair, they react fast to produce nitrogen dioxide and the carbonate radical anion: ONOO
þ CO2 Y 0:65 NO
3 þ 0:65 CO2

S S þ 0:35 NO2 þ 0:35 CO3
:

ð1Þ

Although produced in substoichiometric amounts, the simultaneous flux of these radicals is very efficient in nitrating free tyrosine because the highly oxidizing carbonate radical anion (E = 1.78 V at pH 7.4) produces the tyrosyl radical that recombines with nitrogen dioxide [8,11]. Despite the accumulated information on physiological mechanisms of protein nitration [7–11], it is far from clear how tempol can affect it in vivo because there are few studies of the interaction of tempol with nitric oxide-derived oxidants [12–15]. Relevantly, previous studies have demonstrated that tempol catalytically diverts peroxynitrite/carbon dioxide reactivity toward phenol from phenol nitration to phenol nitrosation [12,13]. We evidenced that this shift occurred because the peroxynitrite-derived carbonate radical anion (Eq. 1) oxidized tempol to the corresponding oxammonium cation ! ! þ CO 3
þ TPNO þ Hþ Y HCO
3 þ TPNO ;

or decayed to nitrite [13], þ N2 O3 þ H2 O Y 2NO
2 þ 2H :

ð6Þ

More recently, the rate constants of the oxidation of tempol by both the carbonate radical anion (k 2 = 4  108 M-1 s-1) (Eq. 3) and nitrogen dioxide (k 7 = 8.7  108 M-1 s-1; k –7 = 2.7  105 M-1 s-1) (Eq. 7) have been determined and shown to be similar [14,15]: ! ! þ
NO2 þ TPNO Y ð7Þ p NO 2 þ TPNO : Thus, both radicals derived from peroxynitrite/carbon dioxide can oxidize tempol although, in some circumstances, the reversibility of nitrogen dioxide-mediated oxidation may favor tempol oxidation by the carbonate radical anion. To contribute to the understanding of the effects of tempol in vivo, it is important to examine whether peroxynitrite/carbon dioxide reactivity toward proteins is altered by tempol (Fig. 1) as shown in the case of phenol [12–15]. This is a chemical target that can be both nitrated and nitrosated by nitric oxide-derived oxidants. In cells, the main targets of these oxidants are protein–metal centers and protein residues, in particular, cysteine, tyrosine, tryptophan, and amino groups (Fig. 1). The characterization of the resulting protein products has been under active investigation [8–11,16] and nitrosotryptophan and 6-nitrotryptophan are among those recently identified in vitro [17,18]. Undoubtedly, many studies will be required to characterize all protein products and to establish markers to demonstrate their occurrence in vivo. Nevertheless, it is already known

ð2Þ

which, in turn, oxidized remaining peroxynitrite to oxygen and nitric oxide to regenerate tempol, TPNOþ þ ONOO
Y TPNO þ NO þ O 2 : !

!

ð3Þ

Reaction of nitric oxide with peroxynitrite-derived nitrogen dioxide (Eq. 1), a nitrating species, produced dinitrogen trioxide, !

! NO þ NO2 Y p N2 O 3 ;

ð4Þ

a nitrosating species that either attacked phenol, N2 O3 þ PhO
Y nitrosophenol þ NO
2 ;

ð5Þ

Fig. 1. Schematic representation of the possible competing paths for peroxynitrite/carbon dioxide interaction with proteins in the absence and presence of tempol. P represents a generic protein and PNNO represents a generic protein nitrosated at the nitrogen of amino groups or at tryptophan residues. The reactions were not balanced but as shown in the text (Eq. 1), the yield of nitrogen dioxide and carbonate radical anion from peroxynitrite/carbon dioxide is 35%. The scheme summarizes the known reactions of tempol with peroxynitrite/carbon dioxide-derived radicals [12–15] and the main reactions of proteins with nitrogen dioxide, carbonate radical anion, dinitrogen trioxide and nitric oxide [8,11,21]. Double arrows specify reactions whose equilibrium constants (K) have been determined [14,15,21]. Inset: Alternative mechanism for P-cysteine nitrosation, that is, the recombination of nitric oxide with P-cysteinyl radicals [19–21].

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that protein–3-nitrotyrosine and protein–nitrosocysteine are produced in vitro and in vivo from nitric oxide and its derived oxidants [8–11,16,19–22]. Likewise, consensual methodologies are available to quantify these products. Thus, protein–3-nitrotyrosine and protein–nitrosocysteine can be useful markers of the effects and mechanisms of tempol in biological environments (Fig. 1). Here, we examine the effects of tempol on peroxynitrite/carbon dioxide-mediated protein–tyrosine nitration and protein– cysteine nitrosation using as targets a model protein, bovine serum albumin (BSA), and cells, J774 macrophages.

Experimental procedures Materials All reagents were purchased from Sigma, Merck, or Fisher and were analytical grade or better. Peroxynitrite was synthesized from sodium nitrite (0.6 M) and hydrogen peroxide (0.65 M) in a quenched-flow reactor; excess hydrogen peroxide was used to minimize nitrite contamination. To eliminate excess hydrogen peroxide, the peroxynitrite solution was treated with manganese dioxide [23]. Synthesized peroxynitrite contained low levels of contaminating hydrogen peroxide (b1%) and nitrite (10–40%) that were determined as previously described [23] by the titanyl method and by absorbance measurements at 354 nm (q = 24.6 M-1 cm-1), respectively. The concentration of peroxynitrite stock solutions was determined spectrophotometrically at 302 nm (q = 1670 M-1 cm-1) [23]. Commercial BSA was reduced with h-mercaptoethanol (10 times excess) overnight at 48C, followed by dialysis against phosphate buffer (50 mM, pH 7.4). Denatured bovine serum albumin (dBSA) was obtained by treatment with dithiothreitol (1000 times excess) in phosphate buffer (50 mM, pH 7.4) for 2 h at 08C, followed by dialysis against 0.1 M HCl [24]. Prior to use, the pH of the samples of dBSA was increased up to 9.0 by the addition of NaOH before adjustment of the pH to about 7.0 by HCl addition; such a procedure was necessary to avoid dBSA precipitation. Concentrations of BSA solutions were determined from absorbance at 280 nm (q = 43,600 M-1 cm-1) [25]. The concentrations of BSA and dBSA used were typically 40– 50 AM and contained 0.5–0.8 BSA–cysSH/BSA) and 7–19 dBSA–cysSH/dBSA, respectively. Carbon dioxide concentration was calculated from the added bicarbonate concentrations by using pK a = 6.4 [13]. Buffers were pretreated with Chelex-100 to remove contaminant metal ions. All solutions were prepared with distilled water purified with a Millipore Milli-Q system.

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[Ru(III)(NH3)4(SO4)4pic]+ and molybdenum oxide as previously described [26]. Solid sodium bicarbonate was transferred to the electrode cell and purged with argon before addition of the other incubation components. The latter were purged with argon and added to the electrode cell with a Hamilton gastight syringe. The electrode was calibrated with diluted solutions of nitric oxide [26]. BSA treatment and analysis of thiol, nitrosocystein, and 3-nitrotyrosine BSA or dBSA (40–50 AM) was treated with 1 mM peroxynitrite in the absence or presence of tempol (10–100 AM) in 50 mM phosphate buffer, pH 7.4, containing 20 mM bicarbonate for 5 min at room temperature (25 F 28C). Three aliquots were then removed for analysis of remaining thiol and produced 3-nitrotyrosine and nitrosocysteine in both BSA and dBSA samples. In the case of the aliquot used for nitrosocysteine analysis, it was immediately treated with N-ethylmaleimide (10 time excess over initial thiol concentration) to block remaining thiols [16,27,28]. After 10 min at room temperature, it was centrifuged (seven or eight times at 15,000g for 20 min) through Centricon filters (5 kDa cutoff) to eliminate contaminant nitrite. After nitrite reached a concentration below the detection limit of the Griess assay (V1 AM), the protein sample was divided into two equal aliquots that were diluted with water containing and not containing mercurium chloride (0.05%). After a further 10 min incubation at room temperature, nitrite content was determined by the Griess assay and by comparison with a standard curve obtained with known concentrations of authentic nitrite [16]. The concentration of nitrosocysteine produced corresponded to the difference in nitrite concentration obtained in the samples untreated and treated with mercurium chloride [16,27,28]. The level of nitrosothiol produced in native BSA treated with peroxynitrite was shown to be close to the detection limit of the Griess method and the sample was analyzed by chemiluminescence (see, below) [16]. The aliquot of the incubation mixture used to determine the remaining thiol was diluted with phosphate buffer, pH 8.0, containing 5,5V-dithiobis-2-nitrobenzoic acid (1 mM). After a further 20 min incubation, thiol was determined by spectrophotometric quantitation of 5-thio-2nitrobenzoate at 412 nm (q = 13,600 M-1 cm-1) [29]. Oxidized thiol was calculated from the difference between initial and final thiol concentrations. In the third aliquot, 3nitrotyrosine formation was determined from the increase in absorbance at 430 nm (q430 4400 M-1 cm-1) after addition of 0.1 vol of NaOH (1M) [30]. Macrophage culture and treatment

Nitric oxide analysis Nitric oxide was determined electrochemically with a home-built gold electrode modified by a film of trans-

The mouse macrophage cell line J744 [31] was routinely grown in DME medium supplemented with 44 mM NaHCO3, 100 mg/ml streptomycin, 25 mg/ml ampicillin, and 10% fetal

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calf serum in an atmosphere containing 5% CO2. Immediately before the experiments, the cells were collected by centrifugation, washed, and resuspended (1  107 cells/ml) in modified PBS (0.14 M NaCl, 2.7 mM KCl, 50 mM Na2HPO4, 1.4 mM KH2PO4, 0.39 mM MgCl2, and 0.9 mM CaCl2), pH 7.4 [32]. Then, sodium bicarbonate (20 mM) and tempol (0–100 AM) were added, the pH was adjusted to 7.4, and peroxynitrite (1 mM) was added 5 min later. After a further 5 min incubation at room temperature (25 F 28C), the cell suspension was centrifuged (200g for 10 min), the supernatant was removed, and the cells were washed and resuspended in PBS containing N-ethylmaleimide (10 mM) and DTPA (1 mM). The cells were then lysed by three cycles of freeze–thawing and Nonidet P-40 (1%) was added. The lysates were centrifuged at 1000g for 10 min to remove cell debris and the supernatant was removed and maintained at
808C until analysis of total protein, protein–3-nitrotyrosine, nitrite, and nitrosocysteine. The latter were always analyzed within 3 days [16]. Total protein in the lysates was determined by the Bradford method with a Bio-Rad kit. Analysis of nitrite and nitrosocysteine in cell lysates by chemiluminescence These analyses were performed in a Sievers NOA nitric oxide analyzer by following the procedure optimized by Feelisch and co-workers [16]. Briefly, the analysis solution responsible for reducing nitrite and nitroso products present in the samples to nitric oxide contained potassium iodide (45 mM), iodine (10 mM), decanol (ca. 5%) in glacial acetic acid and was maintained at 608C. This solution was used for 3–5 to 20 sample injections depending on sample preincubation or not with sulfanilamide [16]. Each cell lysate sample was divided into three aliquots. One was directly injected into the analyzer and corresponded to the contents of nitrite plus nitrosocysteine plus mercury-resistant nitroso compounds such as nitrosoamines and iron–nitrosyl complexes. The other aliquot was treated with sulfanilamide (0.5%, final concentration) and HCl (0.05 M, final concentration) and incubated for 15 min at room temperature in the dark before injection. This treatment eliminates nitrite from the sample. The third aliquot was treated with sulfanilamide (0.5%, final concentration), HCl (0.05 M, final concentration), and mercurium chloride (0.2%, final concentration) and incubated for 30 min in the dark before injection. This treatment eliminates nitrosocysteine from the samples, and the remaining peak would correspond to mercury-resistant nitroso compounds such as heme nitrosyl and nitrosoamines [16]. These, however, were not detectable under our experimental conditions. The peak areas of sample aliquots (400 Al) were obtained with Liquid.exe (Sievers) software. The analyzer was calibrated daily with a freshly prepared nitrite solution (0.01–2 AM).

Analysis of 3-nitrotyrosine in cell lysates by dot immunoblotting Cell lysates (0.5 Ag protein) were transferred onto a nitrocellulose membrane through a dot-blot system (Apelex), and the membrane was blocked with 5% powdered milk for 2 h, and exposed to primary antibody solution for 1 h (anti-nitrotyrosine 1/5000, Oxis). The primary antibody was detected with a secondary antibody labeled with horseradish peroxidase (HRP). Quantification of 3-nitrotyrosine was performed by densitometry with the ImageQuant V5.2 software (Molecular Dynamics) and comparison with BSA nitrated by peroxynitrite whose 3nitrotyrosine content was determined by absorbance at 430 nm (see, also, above) [30]. Detection of 3-nitrotyrosine and nitrosocysteine by immunocytochemistry Cells (1  106 cells/ml) were treated with 1 mM peroxynitrite/carbon dioxide as described above. After 5 min incubation, the cells were centrifuged, washed with PBS, and fixed with paraformaldehyde (4%) in glass coverslips. These were incubated with blocking solution (BSA 3%, saponine 0.1%, PBS) for 40 min and washed twice with PBS. Then, they were incubated with primary anti-nitrotyrosine antibody (1/100, Oxis) for 1 h and with secondary anti-sheep rhodamine-conjugated antibody (1/ 200, Calbiochem) containing 4V,6-diamidino-2-phenylindole (50 AM) for 1 h. After being washed and treated with Fluorsave (Calbiochem), the slides were examined under a microscope (Olympus BX 40) with reflected light fluorescence attachment (BX-FLA). A similar protocol was employed for nitrosocysteine detection [28] by using as primary antibody anti-nitrosocysteine (1/100, A. G. Scientific), and as secondary antibody, an FITC conjugated antirabbit antibody (1/300) (Sigma). EPR experiments EPR spectra were recorded at room temperature on a Bruker ER 200D-SRC upgraded to an EMX instrument. The incubation mixtures and instrumental conditions are described in the legends to the figures.

Results Effects of tempol on peroxynitrite/carbon dioxide-mediated BSA alterations The demonstration that nitric oxide and oxygen are produced from peroxynitrite/carbon dioxide in the presence of tempol constituted early evidence to support the reactions of tempol with the radicals derived from peroxynitrite/ carbon dioxide (Fig. 1) [13]. Here, we confirmed that 1 mM

D.C. Fernandes et al. / Free Radical Biology & Medicine 38 (2005) 189–200

Fig. 2. Production of nitric oxide from 1 mM peroxynitrite/2 mM carbon dioxide in the presence of 20 AM tempol and of 20 AM tempol plus 46 AM BSA (0.7 BSA–cysSH/BSA) in 65 mM phosphate buffer, pH 7.4, at 258C. At the time shown by the arrow, 1 mM peroxynitrite was added to equilibrated solutions of the buffer and the specified components. In the absence of tempol, nitric oxide was not detected in the absence or presence of BSA. Nitric oxide was detected electrochemically as described under Experimental procedures.

peroxynitrite/carbon dioxide at pH 7.4 produces detectable levels of nitric oxide (around 25 AM) in the presence of 20 AM tempol (Fig. 2). When 46 AM BSA is also present, nitric oxide levels decrease to about 13 AM, indicating that BSA is a target of peroxynitrite/carbon dioxide even in the presence of tempol (Fig. 2). To compare BSA alterations promoted by peroxynitrite/ carbon dioxide in the presence and absence of tempol, both native BSA (b1 free cysteine (cys34) and 20 tyrosine residues) and reductively denatured BSA (dBSA) were examined. The latter treatment increases free cysteine residues [24] and, hence, increases one of the main biological targets of nitric oxide-derived oxidants [21]. BSA alterations

193

promoted by peroxynitrite/carbon dioxide have been previously shown to include production of BSA–cysteinyl radicals [33–35], BSA–sulfenic acid and BSA-3–nitrotyrosine [36], among other products [18] (Fig. 1). As shown in Fig. 3, 100 AM tempol was very effective in inhibiting production of 3-nitrotyrosine (70 – 90% inhibition) and thiol oxidation (20–50% inhibition) promoted by 1 mM peroxynitrite/2 mM carbon dioxide in different BSA samples (containing from 0.5 BSA–cysSH/BSA to 19 BSA–cysSH/ BSA). In parallel, tempol increased BSA–cysteine nitrosation from 200 to 400% (Fig. 3). The data in Fig. 3 confirm that BSA–cysSH residues are the main targets of peroxynitrite/carbon dioxide-derived radicals, being oxidized by 85 to 100% in the different BSA samples. Considering that under our experimental conditions, in the absence of tempol, total radical yields (nitrogen dioxide and carbonate radical) were around 700 AM (Eq. 1), it is noteworthy that about 500 AM thiol was oxidized in dBSA containing 570 AM thiol (Fig. 3). In contrast, the yield of both 3-nitrotyrosine and nitrosocysteine varied from 1 to 10% in the presence and absence of tempol (Figs. 3 and 4). The effects of tempol on thiol oxidation, tyrosine nitration, and thiol nitrosation of BSA or dBSA samples by 1 mM peroxynitrite/carbon dioxide were concentrationdependent and detectable at concentrations equal to or higher than 10 AM (Fig. 4). These results suggested that tempol was not acting as a true catalyst, in contrast with its behavior in the phenol/peroxynitrite/carbon dioxide system [12,13]. To examine this point, tempol consumption was monitored by EPR (Fig. 5).The results showed that tempol was barely consumed in incubations containing native BSA (Figs. 5A, 5B) but it was extensively consumed (about 90%) in incubations containing dBSA (Fig. 4C), independent of its concentration in the range 10 to 100 AM (data not shown). Tempol was consumed only in the presence of the

Fig. 3. Effects of 100 AM tempol on BSA–tyr nitration, BSA–cys oxidation, and BSA–cys nitrosation during the interaction of 1 mM peroxynitrite/2 mM carbon dioxide with 45 AM native and dBSA in 50 mM phosphate buffer, pH 7.4, at 258C. Incubation conditions and methods are described under Experimental procedures; as specified there, nitrosothiol analysis was performed by chemiluminescence in the case of native BSA. The samples used contained 0.5 and 7 and 12.6 BSA–cysSH/BSA for the native and denatured samples, respectively; total initial thiol corresponded to 22.5, 315 and 517 AM, respectively. The values shown are means F SD of three independent determinations.

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Production of 3-nitrotyrosine in BSA treated with peroxynitrite/carbon dioxide (Figs. 3 and 4) [36] indicates that BSA–tyrosyl radicals are also produced during the interaction between BSA and peroxynitrite/carbon dioxide, although they have not been previously characterized by EPR. Instead, production of BSA–tryptophanyl radical has previously been proposed by EPR spin trapping experiments with DBNBS [37]. Here, we show that treatment of BSA and dBSA with peroxynitrite/carbon dioxide in the presence of the spin trap DBNBS produces a DBNBS/!carbon–BSA radical adduct as characterized by the EPR spectrum of an immobilized triplet (Figs. 7A, 7C, spectrum labeled ). In the case of native BSA, a mobile triplet that can be attributed to an oxidation product of DBNBS itself was also detected (Fig. 7A, spectrum labeled as ) [32]. The yield of DBNBS radical adduct was about the same for native and dBSA, in agreement with the similar 3-nitrotyrosine yield obtained (Fig. 3). This suggests that the trapped radical is a BSA–tyrosyl radical, although the EPR parameters of DBNBS radical adducts cannot discriminate protein–tyrosyl from protein–tryptophanyl radicals [38]. The above results demonstrate that tempol inhibits the yields of both BSA–cysteinyl- and BSA–carbon-centered radical adducts (Figs. 6 and 7). This could be anticipated because tempol reacts with the primary radicals produced from peroxynitrite/carbon dioxide (nitrogen dioxide and

!

Fig. 4. Concentration-dependent effect of tempol on BSA–tyr nitration, BSA–cys oxidation, and BSA–cys nitrosation during the interaction of 1 mM peroxynitrite/2 mM carbon dioxide with 45 AM dBSA and native BSA in 50 mM phosphate buffer, pH 7.4, at 258C. (A–C) dBSA sample; (D) native BSA. Incubation conditions and methods are described under Experimental procedures; as specified there, nitrosothiol analysis was performed by chemiluminescence in the case of native BSA. The samples used contained 0.6 and 12.6 BSA-cysSH/BSA for the native and denatured samples, respectively. The values shown are means F SD of three independent determinations.

complete system (data not shown). This suggested that some of the tempol was reacting with the BSA radicals produced from BSA/peroxynitrite/carbon dioxide [33–35]. To confirm this hypothesis, we used the EPR spin-trapping technique. Effects of tempol on BSA-derived radicals The BSA–cysteinyl radical has been previously detected as the corresponding PBN radical adduct on treatment of native BSA with peroxynitrite [33]. Here, production of PBN/!Scys–BSA radical adducts was confirmed for both BSA and dBSA (Figs. 6A, 6C). Higher yields of radical adducts were obtained with dBSA as expected from its higher content of free cysteine. The presence of 10 AM tempol in the incubation mixtures greatly decreased the yield of PBN/!Scys–BSA radical adducts as shown by the decrease in the intensity of the first, broad, protein radical adduct peak (Fig. 6, spectrum labeled ). This is the only peak that can be clearly compared in the experiments with and without tempol because it does not overlap with the peaks of tempol itself (Fig. 6).

!

Fig. 5. EPR spectra of 100 AM tempol before and after incubation with 45 AM native or dBSA and 1 mM peroxynitrite/2 mM carbon dioxide in 50 mM phosphate buffer, pH 7.4, at 258C. (A) Tempol; (B) Tempol incubated with native BSA and peroxynitrite/carbon dioxide; (C) tempol incubated with dBSA and peroxynitrite/carbon dioxide. Spectra were obtained 5 min after peroxynitrite addition. The samples used contained 0.7 and 11 BSAcysSH/BSA for the native and denatured samples, respectively. Instrumental conditions: microwave power, 20 mW; time constant, 327 ms; scan rate, 0.3 G/s; modulation amplitude, 1 G; gain, 8.93  103.

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195

consumed mainly in the presence of thiol-rich dBSA (Fig. 5), which produced higher yields of BSA–cysteinyl radicals (Fig. 6). Despite its consumption, tempol in substoichiometric amounts (10–100 AM) strongly inhibited BSA and dBSA thiol oxidation and tyrosine nitration promoted by 1 mM peroxynitrite/carbon dioxide (Figs. 3 and 4). In parallel, tempol increased BSA– and dBSA– cysteine nitrosation, a fact that argues for its transformation into the oxammonium cation to oxidize peroxynitrite to nitric oxide (Eq. 3) (Figs. 1 and 2).

Fig. 6. EPR spectra of the PBN/!Scys–BSA radical adduct obtained from 1 min incubation of 45 AM native or dBSA with 1 mM peroxynitrite/2 mM carbon dioxide/25 mM PBN in 50 mM phosphate buffer, pH 7.4, at 258C. (A) Native BSA in the absence of tempol; (B) the same as (A) in the presence of 10 AM tempol; (C) dBSA in the absence of tempol; (D) the same as (C) in the presence of 10 AM tempol. The labeled ( ) and out-ofscale peaks correspond to those of the protein radical adduct and tempol spectrum, respectively. Instrumental conditions: microwave power, 20 mW; time constant, 327 ms; scan rate, 0.3 G/s; modulation amplitude, 2.5 G; gain, 8.93  104. Inset: Effect of PBN on the yields of dBSA–cys nitrosation produced from incubations of peroxynitrite (1 mM)/carbon dioxide (2 mM)/tempol (100 AM) with 45 AM dBSA (19 BSA-cysSH/BSA) in 50 mM phosphate buffer, pH 7.4, at 258C. Incubation conditions and methods are described under Experimental procedures. The values shown are the means F SD of three independent determinations.

!

carbonate radical anion) that, as a consequence, cannot oxidize BSA to radicals (Fig. 1) [13–15]. The latter reactions, however, cannot explain the observed tempol consumption (Fig. 5). This fraction of tempol should be reacting with the BSA radicals produced and, indeed, nitroxides are known to react with both carbon [1,39,40]and sulfur [41,42]-centered radicals. Reaction with the latter is likely to be more important because tempol was

Fig. 7. EPR spectra of the DBNBS/!carbon-centered BSA radical adduct obtained from 1 min incubation of 45 AM native or dBSA with 1 mM peroxynitrite/2 mM carbon dioxide/10 mM DBNBS in 50 mM phosphate buffer, pH 7.4, at 258C. (A) Native BSA in the absence of tempol; (B) same as (A) in the presence of 10 AM tempol; (C) dBSA in the absence of tempol; (D) same as (C) in the presence of 10 AM tempol. The labeled ( , ) and out-of-scale peaks correspond to those of the protein radical adduct, the DBNBS oxidation product, and tempol spectrum, respectively. Instrumental conditions: microwave power, 20 mW; time constant, 327 ms; scan rate, 0.3 G/s; modulation amplitude, 2.5 G; gain, 8.93  104.

!

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BSA nitrosation mechanisms Although the above results demonstrate production of nitric oxide from peroxynitrite/carbon dioxide/tempol, even in the presence of proteins (Figs. 2–4), they do not provide information about the nitrosation mechanism. Formation of BSA– and dBSA–nitrosocysteine may occur by reaction of protein–cysteine residues with dinitrogen trioxide [43–45], by recombination of protein–cysteinyl radicals with nitric oxide [19–21], or by both mechanisms (Fig. 1). To examine the participation of protein–cysteinyl radicals in the nitrosation process, we examined the effects of PBN, which efficiently trapped these radicals (Fig. 6), on the yields of dBSA–nitrosocysteine. All tested PBN concentrations (12.5, 25, and 50 mM PBN) inhibited the yield of nitrosocysteine to a similar extent, that is, by about 40% as shown for two of the PBN concentrations used in the inset to Fig. 5. This saturation effect indicates that nitrosocysteine is produced by both radical and nonradical mechanisms. In contrast, PBN did not affect the yields of protein–3nitrotyrosine and inhibited by about 10% the yields of

total oxidized thiol (around 600 AM). These results indicate that PBN trapped a small fraction of the resulting protein–cysteinyl radicals as expected from the many possible reactions of protein–cysteinyl radicals [35], particularly the intramolecular reactions with themselves and with the several protein–cysteine residues present in dBSA. Effects of tempol on peroxynitrite/carbon dioxide-mediated alterations of macrophage proteins The above studies with BSA and dBSA showed that tempol, although not a true catalyst, shifted peroxynitrite/ carbon dioxide reactivity toward proteins from nitration to nitrosation mechanisms (Figs. 1–7). To examine whether the process occurs in cells, we treated macrophages with 1 mM peroxynitrite/carbon dioxide in the absence and presence of tempol (10–100 AM) and monitored cell protein–tyrosine nitration and protein–cysteine nitrosation (Figs. 8 and 9). First, we performed immunocytochemistry studies. Although qualitative, the results clearly showed that

Fig. 8. Representative immunocytochemistry of macrophages treated with 1 mM peroxynitrite/carbon dioxide in the presence and absence of 100 AM tempol. Incubation and staining conditions are described under Experimental procedures. (A–C) 3-Nitrotyrosine residues (red staining), DNA (blue staining), and merging of the latter in cells treated with peroxynitrite/carbon dioxide in the absence of tempol. (D–F) Same as (A–C) for cells treated in the presence of 100 AM tempol. (G–I) Nitrocysteine residues (green staining), DNA (blue staining) and merging of the latter in cells treated with peroxynitrite/carbon dioxide in the absence of tempol. Panels J-L the same as G-I for cells treated in the presence of 100 AM tempol.

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Also, under our experimental conditions it was not possible to detect mercurium-resistant thiol compounds such as iron– nitrosyl complexes and nitrosoamines (see Experimental procedures). The effects of tempol on cells treated with peroxynitrite/ carbon dioxide were shown to be concentration-dependent (Fig. 9) as was the case for BSA (Fig. 4). In the case of cells, tempol was likely to act both intra- and extracellularly. In fact, the presence of intracellular tempol was detectable by the EPR spectra of lysates of extensively washed cells when the tempol concentrations used were higher than 50 AM (Fig. 10). In addition, it has been demonstrated that peroxynitrite diffuses into cells even in the presence of extracellular carbon dioxide [46,47]. Intra- and extracellular oxidation of peroxynitrite to nitric oxide by the oxammonium cation of tempol is expected to increase nitrite yields as observed (Fig. 9B) because nitrite is a decay product of dinitrogen trioxide (Eqs. 5 and 6) [13]. Although most extracellular nitrite should have been eliminated before analysis by chemiluminescence, we cannot exclude that some of it contributed to the values determined in the lysates of washed cells (Fig. 9B). Also, we cannot exclude that part of the detected nitrosocysteine (Fig. 9B) corresponded to low-molecular-weight compounds, in particular nitrosoglutathione, because glutathione is an important cellular target of peroxynitrite-derived radicals [21,32].

Fig. 9. Production of 3-nitrotyrosine residues, nitrosocysteine residues, and intracellular nitrite in macrophages treated with 1 mM peroxynitrite/carbon dioxide in the presence of different tempol concentrations. Incubation conditions and methods are described under Experimental procedures. (A) Yields of 3-nitrotyrosine residues; obtained. Inset: staining of protein and 3nitrotyrosine in a representative dot blot. (B) Yields of intracellular nitrite and nitrosocysteine obtained; the values shown for dPN were those obtained for cells treated with previously decomposed peroxynitrite/carbon dioxide. Values are means F SD of three independent determinations.

100 AM tempol decreased macrophage–protein nitration mediated by 1 mM peroxynitrite/carbon dioxide while increasing macrophage protein–cysteine nitrosation (Fig. 8). These results were then confirmed by quantitative experiments where macrophage protein–tyrosine nitration was quantified by densitometry of dot immunoblots (Fig. 9A) and cellular nitrite and nitrosocysteine were quantified by reduction to nitric oxide and chemiluminescent analysis (Fig. 9B) (see Experimental procedures). It is important to note that in these experiments we measured total nitrosocysteines, that is, low- and high-molecular-weight compounds. Indeed, the available anti-nitrosocysteine antibody did not provide reliable dot immunoblots, in contrast with results previously reported with a different antibody [28].

Fig. 10. EPR spectra of intracellular tempol obtained from lysates of macrophages treated with 1 mM peroxynitrite/carbon dioxide in the presence and absence of tempol. Incubation conditions and methods are described under Experimental procedures. (A) Lysates of macrophages treated with peroxynitrite/carbon dioxide; (B) lysates of macrophages treated with peroxynitrite/carbon dioxide in the presence of 25 AM tempol; (C) same as (B) in the presence of 50 AM tempol; (D) same as (B) in the presence of 100 AM tempol.

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Discussion Our results extend previous observations by demonstrating that tempol diverts peroxynitrite/carbon dioxide decomposition from nitrating to nitrosating species [12–15] even in the presence of proteins (Figs. 2–4) and cells (Figs. 8 and 9). The basic mechanism proposed for the peroxynitrite/carbon dioxide/phenol system (Eqs. 1–7) [12–15] can account for the results described here if allowance is made for the many reactions in which the reactive species produced from peroxynitrite/carbon dioxide/tempol can engage in the presence of proteins. According to our results, tempol preferentially reacted with nitrogen dioxide and carbonate radical anion probably because a flux of these radicals is produced a few milliseconds after the addition of peroxynitrite to the carbon dioxide containing incubations (Fig. 1) [8,11,46,47]. The oxammonium cation produced oxidized remaining peroxynitrite to nitric oxide (Fig. 2), whereas most nitrogen dioxide and carbonate radical anion that escaped reaction with both tempol and nitric oxide or with tempol, respectively, oxidized target proteins (Fig. 1). In the case of BSA and dBSA, they were oxidized to radicals characterized as BSA–cysteinyl (Fig. 6) and BSA–tyrosyl/ trytophanyl radicals (Fig. 7; see, also Results) that decayed to two-electron oxidation products. BSA-derived radicals can also react with tempol with rate constants (k ~ 109 M-1 s-1) [38–41,48] even higher than those of tempol with peroxynitrite-derived radicals (k ~ 108 M-1 s-1) [14,15] but the small initial concentration of protein radicals should have minimized their reaction with tempol. Reactions other than those summarized in Fig. 1, however, occurred to some extent as evidenced by tempol consumption (Fig. 5). This was attributed mainly to its reaction with the protein–cysteinyl radicals produced because tempol consumption was more pronounced in the presence of dBSA (Fig. 5), which contained high cysteine levels (7–19 BSA–cysSH/BSA) and produced higher yields of dBSA–cysteinyl radicals (Fig. 6). Although not a true catalyst, 100 AM tempol strongly inhibited protein–tyrosine nitration (70–90%) and protein– cysteine oxidation (20–50%) caused by 1 mM peroxynitrite/carbon dioxide in BSA, dBSA, and cells while increasing protein–cysteine nitrosation (200–400%) (Figs. 3, 4, and 9). In cells, part of the nitrosocysteine detected may be due to low-molecular-weight compounds, particularly nitrosoglutathione [19–22,32]. Most of the expected decay products of protein radicals formed from nitrogen dioxide and carbonate radical anion (Fig. 1) were detected such as cysteine oxidation products, most likely protein sulfenic acid and protein disulfide [34– 36] and protein nitrotyrosine (Figs. 2, 3, 7, 8) [7–11]. Interestingly, the results demonstrated that about half of the protein–nitrosocysteine produced was formed by a radical mechanism. Indeed, addition of BSA to peroxynitrite/ carbon dioxide/tempol inhibited in about 50% the yield of

detectable nitric oxide (Fig. 2). Also, PBN, which was an efficient trap of BSA–cysteinyl radicals produced (Fig. 6) [33,34], inhibited BSA–nitrosocysteine production by about 40% and displayed a saturation effect (Fig. 6, inset). The remaining half of the BSA–nitrosocysteine produced most likely resulted from the reaction of BSA–cysteine with dinitrogen trioxide, which is formed from the reaction between nitric oxide and nitrogen dioxide (Eq. 4) (Fig. 1) [13,19,43–45]. Production of dinitrogen trioxide under our experimental conditions was also supported by the increased levels of nitrite measured in cells treated with peroxynitrite/ carbon dioxide in the presence of tempol (Fig. 9B; see also Results). The demonstration that part of the protein–nitrosocysteine produced under our experimental conditions resulted from recombination of protein–cysteinyl radicals with nitric oxide is particularly relevant (Figs. 2 and 6). Indeed, protein–nitrosocysteines have been receiving increasing attention as mediators of cell signaling [49,50], but most of the mechanisms proposed for their biological production, with a few exceptions [45,51–53], do not include the intermediacy of free radicals [43,44,54,55]. Two recent studies evidenced the production of nitrosoglutathione from nitric oxide under physiologically relevant oxygen tensions by a free radical mechanism [19,20] and our results on BSA–nitrosocysteine formation (Fig. 6) add to them. The occurrence of such a mechanism in vivo would argue for the possible participation of glutathiyl and protein–cysteinyl radicals in signaling pathways [21], a provocative hypothesis that remains to be explored. In summary, our results demonstrated that tempol reacts with peroxynitrite-derived radicals even in biological environments, and diverts their reactivity from protein– cysteine oxidation and protein–tyrosine nitration to protein–cysteine nitrosation. On the one hand, protein– cysteine oxidation and nitrosation count with well-known enzymatic repair systems [49,50,56–58], whereas the systems responsible for the repair/proteolysis of nitrated proteins remain obscure [11,59, and cited references]. On the other hand, tissue injury protection by tempol has been shown to be paralleled by inhibition of tissue protein nitration [3–6]. In this context, our results may explain some of the protective effects of tempol in experimental models of inflammation.

Acknowledgments We thank Ms. Andrea Oliveira for some preliminary immunocytochemistry experiments and Dr. D. S. P Abdalla and Dr. M. Bertotti for providing access to nitric oxide analyzers. This work was supported by grants from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), and Financiadora de Estudos e Projetos (FINEP).

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