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Methylene blue photosensitized oxidation of hypotaurine in the presence of azide generates reactive nitrogen species: formation of nitrotyrosine
BBRC Biochemical and Biophysical Research Communications 301 (2003) 411–416 www.elsevier.com/locate/ybbrc
Methylene blue photosensitized oxidation of hypotaurine in the presence of azide generates reactive nitrogen species: formation of nitrotyrosineq Laura Pecci,* Gabriella Montefoschi, Antonio Antonucci, Mara Costa, and Doriano Cavallini Dipartimento di Scienze Biochimiche ‘‘A. Rossi Fanelli’’ and Centro di Studio sulla Biologia Molecolare del CNR, Universit a di Roma ’’La Sapienza’’Piazzale A. Moro 5, 00185 Rome, Italy Received 23 December 2002
Abstract In our previous study on the hypotaurine (HTAU) oxidation by methylene blue (MB) photochemically generated singlet oxygen (1 O2 ) we found that azide, usually used as 1 O2 quencher, produced, instead, an evident enhancing effect on the oxidation rate [L. Pecci, M. Costa, G. Montefoschi, A. Antonucci, D. Cavallini, Biochem. Biophys. Res. Commun. 254 (1999) 661–665]. We show here that this effect is strongly dependent on pH, with a maximum at approximately pH 5.7. When the MB photochemical system containing HTAU and azide was performed in the presence of tyrosine, 3-nitrotyrosine was produced with maximum yield at pH 5.7, suggesting that azide, by the combined action of HTAU and singlet oxygen, generates nitrogen species which contribute to tyrosine nitration. In addition to HTAU, cysteine sulfinic acid, and sulfite were found to induce the formation of 3-nitrotyrosine. No detectable tyrosine nitration was observed using taurine, the oxidation product of HTAU, or thiol compounds such as cysteine and glutathione. It is shown that during the MB photooxidation of HTAU in the presence of azide, nitrite, and nitrate are produced. Evidences are presented, indicating that nitrite represents the nitrogen species involved in the production of 3-nitrotyrosine. A possible mechanism accounting for the enhancing effect of azide on the photochemical oxidation of HTAU and the production of nitrogen species is proposed. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Hypotaurine; Taurine; Azide; Singlet oxygen; Nitrotyrosine; Nitrite; Peroxynitrite
Sodium azide (N 3 ) is known as a quencher of singlet oxygen (1 O2 ) [1] and has been widely used to demonstrate the participation of 1 O2 in the oxidation of several biological compounds [2–4]. We previously reported that the oxidation rate of hypotaurine (HTAU) and other sulfinates by methylene blue (MB) photochemically generated 1 O2 is enhanced by addition of azide [5,6]. This unusual effect of azide has been ascribed to the oneelectron oxidant azidyl radical (N3 ) generated by interaction of azide with singlet oxygen [7]. Azidyl radical formation has been also demonstrated on the oxidation of azide by catalase and various perq
Abbreviations: MB, methylene blue; 1 O2 , singlet oxygen; HTAU, hypotaurine; TAU, taurine; NO2 Tyr, 3-nitrotyrosine; ONOO , peroxynitrite; DTPA, diethylenetriaminepentaacetic acid. * Corresponding author. Fax: +39-064-440-062. E-mail address: [email protected] (L. Pecci).
oxidases in the presence of H2 O2 [8–11]. Subsequent reactions of N3 generate nitrogen (N2 ) and nitrogen oxides such as nitrous oxide (N2 O) and nitric oxide (NO) [8]. In a recent paper, it has been reported that oxidation of azide, catalyzed by catalase and H2 O2 , generates unknown nitrogen intermediates which react with tyrosine to form 3-nitrotyrosine [12]. Nitration of tyrosine has been extensively used as a marker for the action of peroxynitrite (ONOO ) and other nitric oxide-derived species [13–15]. In this regard, formation of 3-nitrotyrosine has been demonstrated in systems such as hypochlorite/nitrite [16,17], peroxidase/nitrite [18–20], and singlet oxygen/ nitrite [21]. This study was undertaken to investigate whether the enhancing effect of azide on the MB photooxidation of hypotaurine is associated with the production of reactive nitrogen species with oxidant and nitrating properties.
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(02)03063-2
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Materials and methods Methylene blue (MB), hypotaurine (HTAU), cysteine sulfinic acid, taurine (TAU) L -tyrosine, 3-nitrotyrosine (NO2 Tyr), and sodium azide were obtained from Sigma Chem. Cadmium powder (-100 mesh) was purchased from Aldrich Chem. Co. All other reagents were of the highest purity commercially available. Oxygen consumption was measured at 37 °C using a water-jacketed cell (1.85 ml) and Clark type electrode connected to a Gilson 5/6 oxygen analyzer. The reaction was started by illumination with a 200 W tungsten lamp at a distance of about 10 cm from the cell. For the quantitative analyses of products, usually 10 ml of the reaction solution was placed in a beaker (25 ml), with a surface diameter of 4 cm, thermostated at 37 °C and illuminated, under stirring, with the lamp standing 10 cm above the beaker. A layer of 0.5 cm water contained in a petri dish was interposed to shield from infrared radiation. HTAU and TAU were determined by HPLC using o-phthaldialdehyde precolumn derivatization [22]. Tyrosine and 3-nitrotyrosine were determined by HPLC, using a 4 lm Nova-pak C18 (3:9 mm 150 mm) column (Waters) and UV detection at 274 and 360 nm, respectively [21]. Nitrite and nitrate determinations were made by the batch Griess assay [23]. In brief: 200 ll of the reaction mixture was added with 50 ll of 1 M glycine/NaOH buffer, pH 9.0, and 0.75 ml of Griess reagent. Following 10 min at room temperature, absorbance readings at 540 nm were used to determine nitrite levels. Nitrate levels were estimated after its reduction to nitrite by copper-plated cadmium granules. To accomplish this conversion 400 ll of the reaction mixture was added with 100 ll of 1 M glycine/NaOH, pH 9.0, and with 0.5 g of copperized cadmium. After 1 h at room temperature under shaking, the samples were centrifuged at 14,000 rpm for 15 min and 250 ll of supernatant was treated with 0.75 ml of Griess reagent as above for nitrite determinations. In each assay, a sample of the incubation mixture was spiked with a known amount of nitrate (usually 5 nmol in 10 ll) to verify the extent of the reduction to nitrite. Nitrite and nitrate solutions used for standard curves contained azide at the same concentration present in the incubation mixtures.
Results and discussion The activating effect of azide on the photooxidation of hypotaurine In a previous work, we studied the oxidation of hypotaurine (HTAU) to taurine (TAU) by singlet oxygen (1 O2 ), generated using MB as photosensitizer and visible light [5]. It was found that addition of sodium azide 1 (N 3 ), a known quencher of O2 , produced an unusual enhancing effect on the MB photooxidation rate of HTAU. Fig. 1 shows that this effect, measured by oxygen consumption rate, is strongly dependent on pH, with a maximum at approximately pH 5.7. Oxygen uptake measurements and HPLC analyses of the reaction mixtures at pH 3.5, 5.7, and 7.0, either in the absence or in the presence of azide, indicate the overall reaction stoichiometry of 1 mol of HTAU oxidized to 1 mol of TAU per 0.5 mol of oxygen consumed. We previously proposed that the enhancing effect of azide on the singlet oxygen dependent oxidation of HTAU could be attributed to the strong oxidant properties of the azidyl radical, N3 , generated by interaction of azide with 1 O2 [7].
Fig. 1. The MB photooxidation of HTAU in the absence and in the presence of azide as a function of pH. The reaction mixtures containing 500 lM HTAU and 10 lM MB in 50 mM phosphate buffer, adjusted to the indicated pH with 10% NaOH or with 5% H3 PO4 , were placed in the oxygraph chamber at 25 °C and the reaction was started by illumination. Oxygen consumption rate in the absence of azide (d) and in the presence of 2 mM azide (j). Results are means SEM of three or more separate experiments.
Nitration of tyrosine In addition to the oxidant properties, it has been shown that azidyl radical, generated by the catalase/ H2 O2 system or by the Fenton reaction, produces unknown reactive nitrogen species able to nitrate tyrosine forming 3-nitrotyrosine [12]. Fig. 2 shows that when tyrosine (100 lM) is added to the MB photochemical system containing 1 mM HTAU and 10 mM azide (hereafter referred to as HTAU/ 1 N 3 = O2 system) at pH 5.7, 3-nitrotyrosine (NO2 Tyr) is produced, the yield increasing almost linearly with the illumination time. No detectable tyrosine nitration was observed in dark controls or in illuminated controls lacking HTAU or azide. Moreover the formation of 3-
1 Fig. 2. Time course of tyrosine nitration by HTAU/N 3 = O2 system. The reaction mixture contained 100 lM tyrosine, 1 mM HTAU, 10 mM azide, and 10 lM MB in 50 mM phosphate buffer, including 100 lM DTPA, pH 5.7. The reaction was started by exposure to light and allowed to occur at 37 °C under stirring. At the indicated time intervals 3-nitrotyrosine formation was measured by HPLC as described under Materials and methods. Results are means SEM of at least three separate experiments.
L. Pecci et al. / Biochemical and Biophysical Research Communications 301 (2003) 411–416
nitrotyrosine requires oxygen as indicated by the finding that in anaerobic conditions, no NO2 Tyr was detected (not shown). As reported in Fig. 3, the yield of 3-nitrotyrosine is dependent on HTAU and azide concentration, with maximal formation at 2 mM HTAU and 10 mM azide. Fig. 4 shows that the yield of 3-nitrotyrosine is strongly dependent on pH, with a maximum at pH 5.7. In order to investigate the importance of the sulfuroxidation state in the reaction of tyrosine nitration, the MB photochemical system containing different sulfur compounds, azide and tyrosine at pH 5.7, was analyzed for the production of 3-nitrotyrosine. As shown in Table 1, in addition to HTAU, only cysteine sulfinic acid and sulfite are able to induce the formation of 3-nitrotyrosine. No detectable tyrosine nitration was observed using taurine, the oxidation product of HTAU, or thiol compounds such as cysteine and glutathione. Of interest is the finding that 3-nitrotyrosine is formed during the 1 O2 -dependent oxidation of sulfinates where azide exerts an activating effect [5,6], but not with thiols where azide, acting as 1 O2 quencher, has an inhibitory effect [24].
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1 Fig. 4. Tyrosine nitration by the HTAU/N 3 = O2 system as a function of pH. The reaction mixtures containing 100 lM tyrosine, 1 mM HTAU, 10 mM azide, and 10 lM MB in 50 mM phosphate buffer plus 100 lM DTPA, adjusted to the indicated pH with 10% NaOH or with 5% H3 PO4 , were exposed to light for 30 min at 37 °C and 3-nitrotyrosine formation was determined by HPLC. The data have been corrected for 3-nitrotyrosine decomposition occurring at pH > 6.0, as determined using authentic 3-nitrotyrosine exposed to light and MB in the presence of 10 mM azide. Results are means SEM of three separate experiments.
Table 1 3-Nitrotyrosine formation by MB photooxidation of various sulfur compounds in the presence of azide Compounda
3-Nitrotyrosineb (lM)
Hypotaurine Cysteine sulfinic acid Sulfite Taurine Cysteine Glutathione
1:45 0:06 0:49 0:07 0:55 0:07 n.d.c n.d.c n.d.c
a The reaction mixtures containing 1 mM of the indicated sulfur compound, 100 lM tyrosine, 10 mM azide, and 10 lM MB in 50 mM phosphate buffer plus 100 lM DTPA, pH 5.7, were exposed to light for 30 min at 37 °C. b Results are means SEM of three or more separate experiments. c Not detected.
The nitrating species
1 Fig. 3. Tyrosine nitration by HTAU/N 3 = O2 system as a function of HTAU and azide concentration. The reaction mixtures containing tyrosine, HTAU, azide, and 10 lM MB in 50 mM phosphate buffer plus 100 lM DTPA, pH 5.7, were exposed to light for 30 min at 37 °C and 3-nitrotyrosine formation was measured by HPLC. (A) 100 lM tyrosine in the presence of 10 mM azide and the indicated concentrations of HTAU. (B) 100 lM tyrosine in the presence of 1 mM HTAU and the indicated concentrations of azide. Results are means SEM of three separate experiments.
In a recent study, we have reported the MB photosensitized oxidation of tyrosine in the presence of nitrite produces 3-nitrotyrosine [21]. In order to check the possible involvement of this pathway in the formation of 3-nitrotyrosine observed under the experimental conditions reported in Fig. 2, the production of nitrite (NO 2) 1 by the HTAU/N = O system at pH 5.7 was investi2 3 gated. Fig. 5 shows that the production of NO 2 proceeds as long as HTAU is present and stops after about 20 min, when HTAU is almost completely oxidized to TAU. The simultaneous production of nitrate (NO 3 ) is also shown in Fig. 5. The yield of nitrate was about 2-fold higher than that of nitrite. No detectable production of NO 2 =NO3 was observed in dark controls or in illuminated controls lacking HTAU or azide. Direct oxidation of nitrite to nitrate by the MB photochemical system was
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Fig. 5. Nitrite and nitrate production in the course of HTAU photooxidation in the presence of azide. The reaction mixture contained 1 mM HTAU, 10 mM azide, and 10 lM MB in 50 mM phosphate buffer plus 100 lM DTPA, pH 5.7. The reaction was started by illumination and allowed to occur at 37 °C under stirring. At the indicated time intervals, aliquots were withdrawn and analyzed for nitrite (j), nitrate (), HTAU (.), and TAU (N) by the procedures described under Materials and methods. Results are means SEM of three or more separate experiments.
not observed. These findings indicate that, under our experimental conditions, azide produces nitrite and nitrate only by the combined action of singlet oxygen and HTAU. Hence, it appeared likely that nitrite formed during the MB photooxidation of HTAU in the presence of azide could have been the nitrogen species involved in the production of 3-nitrotyrosine seen in Fig. 2. However, the question that arises is whether nitrite and nitrate are end-products of an intermediate nitrogen species. A likely candidate which responds to this feature could be peroxynitrite (ONOO ). Indeed, ONOO yields nitrite and nitrate in a ratio which depends on pH and on the presence of oxidizable compounds [25–27]. It is well known that peroxynitrite reacts with tyrosine to form 3-nitrotyrosine [28], therefore, if ONOO is gen1 erated by the HTAU/N 3 = O2 system, it could represent a potential nitrating agent which contributes to the production of 3-nitrotyrosine. This hypothesis could not be verified exploiting ONOO scavengers such as thiols, uric acid, and methionine, since all these compounds also react with singlet oxygen [24,29,30]. On the other hand, addition of 25 mM bicarbonate, which is known to increase the ONOO -dependent nitration of tyrosine [31], resulted without effect (data not shown), suggesting that the possible intermediate generation of peroxynitrite is not associated with the formation of NO2 Tyr. To obtain further insights into nitrogen species involved in the 3-nitrotyrosine formation, tyrosine was added to 1 the HTAU/N 3 = O2 system at pH 5.7 after 30 min illumination (i.e., at the end of HTAU oxidation and NO 2= NO formation, as seen in Fig. 5) and the resulting re3 action mixture was exposed to light for further 30 min. It was found that the 3-nitrotyrosine yield was 1:5 0:05 lM (n ¼ 3). Since peroxynitrite is a short lived species (t1=2 ¼ 1 s, at physiological pH, 37 °C and even
shorter at acidic pH) [32], it appeared likely that, under these experimental conditions, the only nitrating agent should have been nitrite accumulated during the pre1 incubation of the HTAU/N 3 = O2 system. According to this, no detectable production of 3-nitrotyrosine was observed when the reaction mixture resulting from the addition of tyrosine to the pre-incubated HTAU/ 1 N 3 = O2 system was not exposed to light. Indeed, as previously demonstrated, the reaction of nitrite with tyrosine to form 3-nitrotyrosine requires singlet oxygen [21]. It should be noted that the yield of NO2 Tyr produced after 30 min illumination of the reaction mixture resulting from the addition of tyrosine to the 1 pre-incubated HTAU/N 3 = O2 system was identical, within the error, to that found when tyrosine was co1 incubated with the HTAU/N 3 = O2 system (Fig. 2, at 30 min and Table 1, first row). These findings strongly support the conclusion that nitrite represents the main nitrogen species responsible for the production of 3nitrotyrosine also in the latter conditions.
Conclusions The data presented in this paper indicate that the enhancing effect of azide on the MB photooxidation of HTAU is accompanied by the production of nitrite and nitrate and that nitrite is the nitrogen species responsible of the production of 3-nitrotyrosine observed 1 when tyrosine is added to the HTAU/N 3 = O2 system. We previously suggested that azide could stimulate the singlet oxygen dependent oxidation of HTAU by a mechanism involving the strong one-electron oxidant azidyl radical, N3 , which dissociates from the chargetransfer complex generated during the quenching of 1 O2 by azide [7]: 1 N 3 þ O2 ! ðN3 . . . :O2 Þ ! N3 þ O2
It was proposed that the azidyl radical could act as HTAU oxidant N3 þ HTAU ! HTAU þ N 3 generating the HTAU radical to be converted into TAU by further reactions. This interpretation, however, does not account for the production of NO 2 =NO3 (Fig. 5). Therefore, consistent with the present results, it is more likely that HTAU could interact, via one-electron transfer, with the charge-transfer complex in such a way as to cause production of an azide-derived intermediate nitrogen species (NOx ) which subsequently generates nitrite and nitrate: HTAU þ ðN3 . . . :O 2 Þ ! HTAU þ NOx # #
TAU
NO 2 þ NO3
L. Pecci et al. / Biochemical and Biophysical Research Communications 301 (2003) 411–416
The finding that the yield of nitrate is higher than that of nitrite (Fig. 5) suggests the formation of peroxynitrite as intermediate nitrogen species. The spontaneous decay of peroxynitrite produces nitrite and nitrate in yields which depends on pH: at pH 5.7 peroxynitrite undergoes protonation to give peroxynitrous acid (ONOOH, pka ¼ 6:8) which isomerizes almost completely to nitrate [25]. In the presence of compounds which react with ONOOH, nitrite is also formed and the NO 2 =NO3 yields appear to be dependent on the mechanism of oxidation [26,27]. Since HTAU can be oxidized to TAU by peroxynitrite in a reaction accompanied by a fast oxygen consumption (Fontana et al., work in progress), the possible occurrence of this reaction in the HTAU/ 1 N 3 = O2 system could account for the product distribution of nitrite and nitrate and for the increase of the oxidation rate of HTAU observed in the presence of azide. In this interpretation HTAU acts either as the reactive compound able to generate peroxynitrite by its interaction with the azide/singlet oxygen complex, or the target molecule oxidizable by ONOOH. Therefore, although we do not know at present the mechanism of the oxidation of HTAU by peroxynitrous acid, the following reactions can be proposed: ONOOH þ HTAU ! HTAU þ NO 2 þ NO3
HTAU þ O2 !! TAU In addition to oxidant properties peroxynitrite is also a nitrating species able to nitrate tyrosine [28]. However, under our experimental conditions, the competitive reaction of ONOOH with HTAU (1 mM) should prevent the nitration of tyrosine (100 lM). Accordingly, our results indicate that the formation of 3-nitrotyrosine, observed when tyrosine was added to the HTAU/ 1 N 3 = O2 system, involves only nitrite through the previously reported pathway [21]: 1 NO 2 þ Tyr þ O2 ! NO2 Tyr þ O2 1 The nitration of tyrosine by the HTAU/N 3 = O2 system indicates the occurrence of another mechanism of production of azide-derived nitrogen reactive species to be added to that involving peroxidase-catalyzed oxidation of azide in the presence of H2 O2 [12]. Since azide is widely used either as quencher of singlet oxygen or inhibitor of metalloenzymes, its possible contribution to generate nitrating species deserves further studies which will be undertaken in the future.
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Methylene blue photosensitized oxidation of hypotaurine in the presence of azide generates reactive nitrogen species: formation of nitrotyrosineq Laura Pecci,* Gabriella Montefoschi, Antonio Antonucci, Mara Costa, and Doriano Cavallini Dipartimento di Scienze Biochimiche ‘‘A. Rossi Fanelli’’ and Centro di Studio sulla Biologia Molecolare del CNR, Universit a di Roma ’’La Sapienza’’Piazzale A. Moro 5, 00185 Rome, Italy Received 23 December 2002
Abstract In our previous study on the hypotaurine (HTAU) oxidation by methylene blue (MB) photochemically generated singlet oxygen (1 O2 ) we found that azide, usually used as 1 O2 quencher, produced, instead, an evident enhancing effect on the oxidation rate [L. Pecci, M. Costa, G. Montefoschi, A. Antonucci, D. Cavallini, Biochem. Biophys. Res. Commun. 254 (1999) 661–665]. We show here that this effect is strongly dependent on pH, with a maximum at approximately pH 5.7. When the MB photochemical system containing HTAU and azide was performed in the presence of tyrosine, 3-nitrotyrosine was produced with maximum yield at pH 5.7, suggesting that azide, by the combined action of HTAU and singlet oxygen, generates nitrogen species which contribute to tyrosine nitration. In addition to HTAU, cysteine sulfinic acid, and sulfite were found to induce the formation of 3-nitrotyrosine. No detectable tyrosine nitration was observed using taurine, the oxidation product of HTAU, or thiol compounds such as cysteine and glutathione. It is shown that during the MB photooxidation of HTAU in the presence of azide, nitrite, and nitrate are produced. Evidences are presented, indicating that nitrite represents the nitrogen species involved in the production of 3-nitrotyrosine. A possible mechanism accounting for the enhancing effect of azide on the photochemical oxidation of HTAU and the production of nitrogen species is proposed. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Hypotaurine; Taurine; Azide; Singlet oxygen; Nitrotyrosine; Nitrite; Peroxynitrite
Sodium azide (N 3 ) is known as a quencher of singlet oxygen (1 O2 ) [1] and has been widely used to demonstrate the participation of 1 O2 in the oxidation of several biological compounds [2–4]. We previously reported that the oxidation rate of hypotaurine (HTAU) and other sulfinates by methylene blue (MB) photochemically generated 1 O2 is enhanced by addition of azide [5,6]. This unusual effect of azide has been ascribed to the oneelectron oxidant azidyl radical (N3 ) generated by interaction of azide with singlet oxygen [7]. Azidyl radical formation has been also demonstrated on the oxidation of azide by catalase and various perq
Abbreviations: MB, methylene blue; 1 O2 , singlet oxygen; HTAU, hypotaurine; TAU, taurine; NO2 Tyr, 3-nitrotyrosine; ONOO , peroxynitrite; DTPA, diethylenetriaminepentaacetic acid. * Corresponding author. Fax: +39-064-440-062. E-mail address: [email protected] (L. Pecci).
oxidases in the presence of H2 O2 [8–11]. Subsequent reactions of N3 generate nitrogen (N2 ) and nitrogen oxides such as nitrous oxide (N2 O) and nitric oxide (NO) [8]. In a recent paper, it has been reported that oxidation of azide, catalyzed by catalase and H2 O2 , generates unknown nitrogen intermediates which react with tyrosine to form 3-nitrotyrosine [12]. Nitration of tyrosine has been extensively used as a marker for the action of peroxynitrite (ONOO ) and other nitric oxide-derived species [13–15]. In this regard, formation of 3-nitrotyrosine has been demonstrated in systems such as hypochlorite/nitrite [16,17], peroxidase/nitrite [18–20], and singlet oxygen/ nitrite [21]. This study was undertaken to investigate whether the enhancing effect of azide on the MB photooxidation of hypotaurine is associated with the production of reactive nitrogen species with oxidant and nitrating properties.
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Materials and methods Methylene blue (MB), hypotaurine (HTAU), cysteine sulfinic acid, taurine (TAU) L -tyrosine, 3-nitrotyrosine (NO2 Tyr), and sodium azide were obtained from Sigma Chem. Cadmium powder (-100 mesh) was purchased from Aldrich Chem. Co. All other reagents were of the highest purity commercially available. Oxygen consumption was measured at 37 °C using a water-jacketed cell (1.85 ml) and Clark type electrode connected to a Gilson 5/6 oxygen analyzer. The reaction was started by illumination with a 200 W tungsten lamp at a distance of about 10 cm from the cell. For the quantitative analyses of products, usually 10 ml of the reaction solution was placed in a beaker (25 ml), with a surface diameter of 4 cm, thermostated at 37 °C and illuminated, under stirring, with the lamp standing 10 cm above the beaker. A layer of 0.5 cm water contained in a petri dish was interposed to shield from infrared radiation. HTAU and TAU were determined by HPLC using o-phthaldialdehyde precolumn derivatization [22]. Tyrosine and 3-nitrotyrosine were determined by HPLC, using a 4 lm Nova-pak C18 (3:9 mm 150 mm) column (Waters) and UV detection at 274 and 360 nm, respectively [21]. Nitrite and nitrate determinations were made by the batch Griess assay [23]. In brief: 200 ll of the reaction mixture was added with 50 ll of 1 M glycine/NaOH buffer, pH 9.0, and 0.75 ml of Griess reagent. Following 10 min at room temperature, absorbance readings at 540 nm were used to determine nitrite levels. Nitrate levels were estimated after its reduction to nitrite by copper-plated cadmium granules. To accomplish this conversion 400 ll of the reaction mixture was added with 100 ll of 1 M glycine/NaOH, pH 9.0, and with 0.5 g of copperized cadmium. After 1 h at room temperature under shaking, the samples were centrifuged at 14,000 rpm for 15 min and 250 ll of supernatant was treated with 0.75 ml of Griess reagent as above for nitrite determinations. In each assay, a sample of the incubation mixture was spiked with a known amount of nitrate (usually 5 nmol in 10 ll) to verify the extent of the reduction to nitrite. Nitrite and nitrate solutions used for standard curves contained azide at the same concentration present in the incubation mixtures.
Results and discussion The activating effect of azide on the photooxidation of hypotaurine In a previous work, we studied the oxidation of hypotaurine (HTAU) to taurine (TAU) by singlet oxygen (1 O2 ), generated using MB as photosensitizer and visible light [5]. It was found that addition of sodium azide 1 (N 3 ), a known quencher of O2 , produced an unusual enhancing effect on the MB photooxidation rate of HTAU. Fig. 1 shows that this effect, measured by oxygen consumption rate, is strongly dependent on pH, with a maximum at approximately pH 5.7. Oxygen uptake measurements and HPLC analyses of the reaction mixtures at pH 3.5, 5.7, and 7.0, either in the absence or in the presence of azide, indicate the overall reaction stoichiometry of 1 mol of HTAU oxidized to 1 mol of TAU per 0.5 mol of oxygen consumed. We previously proposed that the enhancing effect of azide on the singlet oxygen dependent oxidation of HTAU could be attributed to the strong oxidant properties of the azidyl radical, N3 , generated by interaction of azide with 1 O2 [7].
Fig. 1. The MB photooxidation of HTAU in the absence and in the presence of azide as a function of pH. The reaction mixtures containing 500 lM HTAU and 10 lM MB in 50 mM phosphate buffer, adjusted to the indicated pH with 10% NaOH or with 5% H3 PO4 , were placed in the oxygraph chamber at 25 °C and the reaction was started by illumination. Oxygen consumption rate in the absence of azide (d) and in the presence of 2 mM azide (j). Results are means SEM of three or more separate experiments.
Nitration of tyrosine In addition to the oxidant properties, it has been shown that azidyl radical, generated by the catalase/ H2 O2 system or by the Fenton reaction, produces unknown reactive nitrogen species able to nitrate tyrosine forming 3-nitrotyrosine [12]. Fig. 2 shows that when tyrosine (100 lM) is added to the MB photochemical system containing 1 mM HTAU and 10 mM azide (hereafter referred to as HTAU/ 1 N 3 = O2 system) at pH 5.7, 3-nitrotyrosine (NO2 Tyr) is produced, the yield increasing almost linearly with the illumination time. No detectable tyrosine nitration was observed in dark controls or in illuminated controls lacking HTAU or azide. Moreover the formation of 3-
1 Fig. 2. Time course of tyrosine nitration by HTAU/N 3 = O2 system. The reaction mixture contained 100 lM tyrosine, 1 mM HTAU, 10 mM azide, and 10 lM MB in 50 mM phosphate buffer, including 100 lM DTPA, pH 5.7. The reaction was started by exposure to light and allowed to occur at 37 °C under stirring. At the indicated time intervals 3-nitrotyrosine formation was measured by HPLC as described under Materials and methods. Results are means SEM of at least three separate experiments.
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nitrotyrosine requires oxygen as indicated by the finding that in anaerobic conditions, no NO2 Tyr was detected (not shown). As reported in Fig. 3, the yield of 3-nitrotyrosine is dependent on HTAU and azide concentration, with maximal formation at 2 mM HTAU and 10 mM azide. Fig. 4 shows that the yield of 3-nitrotyrosine is strongly dependent on pH, with a maximum at pH 5.7. In order to investigate the importance of the sulfuroxidation state in the reaction of tyrosine nitration, the MB photochemical system containing different sulfur compounds, azide and tyrosine at pH 5.7, was analyzed for the production of 3-nitrotyrosine. As shown in Table 1, in addition to HTAU, only cysteine sulfinic acid and sulfite are able to induce the formation of 3-nitrotyrosine. No detectable tyrosine nitration was observed using taurine, the oxidation product of HTAU, or thiol compounds such as cysteine and glutathione. Of interest is the finding that 3-nitrotyrosine is formed during the 1 O2 -dependent oxidation of sulfinates where azide exerts an activating effect [5,6], but not with thiols where azide, acting as 1 O2 quencher, has an inhibitory effect [24].
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1 Fig. 4. Tyrosine nitration by the HTAU/N 3 = O2 system as a function of pH. The reaction mixtures containing 100 lM tyrosine, 1 mM HTAU, 10 mM azide, and 10 lM MB in 50 mM phosphate buffer plus 100 lM DTPA, adjusted to the indicated pH with 10% NaOH or with 5% H3 PO4 , were exposed to light for 30 min at 37 °C and 3-nitrotyrosine formation was determined by HPLC. The data have been corrected for 3-nitrotyrosine decomposition occurring at pH > 6.0, as determined using authentic 3-nitrotyrosine exposed to light and MB in the presence of 10 mM azide. Results are means SEM of three separate experiments.
Table 1 3-Nitrotyrosine formation by MB photooxidation of various sulfur compounds in the presence of azide Compounda
3-Nitrotyrosineb (lM)
Hypotaurine Cysteine sulfinic acid Sulfite Taurine Cysteine Glutathione
1:45 0:06 0:49 0:07 0:55 0:07 n.d.c n.d.c n.d.c
a The reaction mixtures containing 1 mM of the indicated sulfur compound, 100 lM tyrosine, 10 mM azide, and 10 lM MB in 50 mM phosphate buffer plus 100 lM DTPA, pH 5.7, were exposed to light for 30 min at 37 °C. b Results are means SEM of three or more separate experiments. c Not detected.
The nitrating species
1 Fig. 3. Tyrosine nitration by HTAU/N 3 = O2 system as a function of HTAU and azide concentration. The reaction mixtures containing tyrosine, HTAU, azide, and 10 lM MB in 50 mM phosphate buffer plus 100 lM DTPA, pH 5.7, were exposed to light for 30 min at 37 °C and 3-nitrotyrosine formation was measured by HPLC. (A) 100 lM tyrosine in the presence of 10 mM azide and the indicated concentrations of HTAU. (B) 100 lM tyrosine in the presence of 1 mM HTAU and the indicated concentrations of azide. Results are means SEM of three separate experiments.
In a recent study, we have reported the MB photosensitized oxidation of tyrosine in the presence of nitrite produces 3-nitrotyrosine [21]. In order to check the possible involvement of this pathway in the formation of 3-nitrotyrosine observed under the experimental conditions reported in Fig. 2, the production of nitrite (NO 2) 1 by the HTAU/N = O system at pH 5.7 was investi2 3 gated. Fig. 5 shows that the production of NO 2 proceeds as long as HTAU is present and stops after about 20 min, when HTAU is almost completely oxidized to TAU. The simultaneous production of nitrate (NO 3 ) is also shown in Fig. 5. The yield of nitrate was about 2-fold higher than that of nitrite. No detectable production of NO 2 =NO3 was observed in dark controls or in illuminated controls lacking HTAU or azide. Direct oxidation of nitrite to nitrate by the MB photochemical system was
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Fig. 5. Nitrite and nitrate production in the course of HTAU photooxidation in the presence of azide. The reaction mixture contained 1 mM HTAU, 10 mM azide, and 10 lM MB in 50 mM phosphate buffer plus 100 lM DTPA, pH 5.7. The reaction was started by illumination and allowed to occur at 37 °C under stirring. At the indicated time intervals, aliquots were withdrawn and analyzed for nitrite (j), nitrate (), HTAU (.), and TAU (N) by the procedures described under Materials and methods. Results are means SEM of three or more separate experiments.
not observed. These findings indicate that, under our experimental conditions, azide produces nitrite and nitrate only by the combined action of singlet oxygen and HTAU. Hence, it appeared likely that nitrite formed during the MB photooxidation of HTAU in the presence of azide could have been the nitrogen species involved in the production of 3-nitrotyrosine seen in Fig. 2. However, the question that arises is whether nitrite and nitrate are end-products of an intermediate nitrogen species. A likely candidate which responds to this feature could be peroxynitrite (ONOO ). Indeed, ONOO yields nitrite and nitrate in a ratio which depends on pH and on the presence of oxidizable compounds [25–27]. It is well known that peroxynitrite reacts with tyrosine to form 3-nitrotyrosine [28], therefore, if ONOO is gen1 erated by the HTAU/N 3 = O2 system, it could represent a potential nitrating agent which contributes to the production of 3-nitrotyrosine. This hypothesis could not be verified exploiting ONOO scavengers such as thiols, uric acid, and methionine, since all these compounds also react with singlet oxygen [24,29,30]. On the other hand, addition of 25 mM bicarbonate, which is known to increase the ONOO -dependent nitration of tyrosine [31], resulted without effect (data not shown), suggesting that the possible intermediate generation of peroxynitrite is not associated with the formation of NO2 Tyr. To obtain further insights into nitrogen species involved in the 3-nitrotyrosine formation, tyrosine was added to 1 the HTAU/N 3 = O2 system at pH 5.7 after 30 min illumination (i.e., at the end of HTAU oxidation and NO 2= NO formation, as seen in Fig. 5) and the resulting re3 action mixture was exposed to light for further 30 min. It was found that the 3-nitrotyrosine yield was 1:5 0:05 lM (n ¼ 3). Since peroxynitrite is a short lived species (t1=2 ¼ 1 s, at physiological pH, 37 °C and even
shorter at acidic pH) [32], it appeared likely that, under these experimental conditions, the only nitrating agent should have been nitrite accumulated during the pre1 incubation of the HTAU/N 3 = O2 system. According to this, no detectable production of 3-nitrotyrosine was observed when the reaction mixture resulting from the addition of tyrosine to the pre-incubated HTAU/ 1 N 3 = O2 system was not exposed to light. Indeed, as previously demonstrated, the reaction of nitrite with tyrosine to form 3-nitrotyrosine requires singlet oxygen [21]. It should be noted that the yield of NO2 Tyr produced after 30 min illumination of the reaction mixture resulting from the addition of tyrosine to the 1 pre-incubated HTAU/N 3 = O2 system was identical, within the error, to that found when tyrosine was co1 incubated with the HTAU/N 3 = O2 system (Fig. 2, at 30 min and Table 1, first row). These findings strongly support the conclusion that nitrite represents the main nitrogen species responsible for the production of 3nitrotyrosine also in the latter conditions.
Conclusions The data presented in this paper indicate that the enhancing effect of azide on the MB photooxidation of HTAU is accompanied by the production of nitrite and nitrate and that nitrite is the nitrogen species responsible of the production of 3-nitrotyrosine observed 1 when tyrosine is added to the HTAU/N 3 = O2 system. We previously suggested that azide could stimulate the singlet oxygen dependent oxidation of HTAU by a mechanism involving the strong one-electron oxidant azidyl radical, N3 , which dissociates from the chargetransfer complex generated during the quenching of 1 O2 by azide [7]: 1 N 3 þ O2 ! ðN3 . . . :O2 Þ ! N3 þ O2
It was proposed that the azidyl radical could act as HTAU oxidant N3 þ HTAU ! HTAU þ N 3 generating the HTAU radical to be converted into TAU by further reactions. This interpretation, however, does not account for the production of NO 2 =NO3 (Fig. 5). Therefore, consistent with the present results, it is more likely that HTAU could interact, via one-electron transfer, with the charge-transfer complex in such a way as to cause production of an azide-derived intermediate nitrogen species (NOx ) which subsequently generates nitrite and nitrate: HTAU þ ðN3 . . . :O 2 Þ ! HTAU þ NOx # #
TAU
NO 2 þ NO3
L. Pecci et al. / Biochemical and Biophysical Research Communications 301 (2003) 411–416
The finding that the yield of nitrate is higher than that of nitrite (Fig. 5) suggests the formation of peroxynitrite as intermediate nitrogen species. The spontaneous decay of peroxynitrite produces nitrite and nitrate in yields which depends on pH: at pH 5.7 peroxynitrite undergoes protonation to give peroxynitrous acid (ONOOH, pka ¼ 6:8) which isomerizes almost completely to nitrate [25]. In the presence of compounds which react with ONOOH, nitrite is also formed and the NO 2 =NO3 yields appear to be dependent on the mechanism of oxidation [26,27]. Since HTAU can be oxidized to TAU by peroxynitrite in a reaction accompanied by a fast oxygen consumption (Fontana et al., work in progress), the possible occurrence of this reaction in the HTAU/ 1 N 3 = O2 system could account for the product distribution of nitrite and nitrate and for the increase of the oxidation rate of HTAU observed in the presence of azide. In this interpretation HTAU acts either as the reactive compound able to generate peroxynitrite by its interaction with the azide/singlet oxygen complex, or the target molecule oxidizable by ONOOH. Therefore, although we do not know at present the mechanism of the oxidation of HTAU by peroxynitrous acid, the following reactions can be proposed: ONOOH þ HTAU ! HTAU þ NO 2 þ NO3
HTAU þ O2 !! TAU In addition to oxidant properties peroxynitrite is also a nitrating species able to nitrate tyrosine [28]. However, under our experimental conditions, the competitive reaction of ONOOH with HTAU (1 mM) should prevent the nitration of tyrosine (100 lM). Accordingly, our results indicate that the formation of 3-nitrotyrosine, observed when tyrosine was added to the HTAU/ 1 N 3 = O2 system, involves only nitrite through the previously reported pathway [21]: 1 NO 2 þ Tyr þ O2 ! NO2 Tyr þ O2 1 The nitration of tyrosine by the HTAU/N 3 = O2 system indicates the occurrence of another mechanism of production of azide-derived nitrogen reactive species to be added to that involving peroxidase-catalyzed oxidation of azide in the presence of H2 O2 [12]. Since azide is widely used either as quencher of singlet oxygen or inhibitor of metalloenzymes, its possible contribution to generate nitrating species deserves further studies which will be undertaken in the future.
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