Transmembrane Nitration of Hydrophobic Tyrosyl Peptides

LOCALIZATION, CHARACTERIZATION, MECHANISM OF NITRATION, AND BIOLOGICAL IMPLICATIONS*

Hao Zhang, Kalpana Bhargava, Agnes Keszler, Jimmy Feix, Neil Hogg, Joy Joseph, and B. KalyanaramanDagger

From the Biophysics Research Institute and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Received for publication, November 13, 2002, and in revised form, December 30, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have shown previously that peroxynitrite-induced nitration of a hydrophobic tyrosyl probe is greater than that of tyrosine in the aqueous phase (Zhang, H., Joseph, J., Feix, J., Hogg, N., and Kalyanaraman, B. (2001) Biochemistry 40, 7675-7686). In this study, we have tested the hypothesis that the extent of tyrosine nitration depends on the intramembrane location of tyrosyl probes and on the nitrating species. To this end, we have synthesized membrane spanning 23-mer containing a single tyrosyl residue at positions 4, 8, and 12. The location of the tyrosine residues in the phospholipid membrane was determined by fluorescence and electron spin resonance techniques. Nitration was initiated by slow infusion of peroxynitrite, co-generated superoxide and nitric oxide (·NO), or a myeloperoxidase/hydrogen peroxide/nitrite anion (MPO/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP>) system. Results indicate that with slow infusion of peroxynitrite, nitration of transmembrane tyrosyl peptides was much higher (10-fold or more) than tyrosine nitration in aqueous phase. Peroxynitrite-dependent nitration of tyrosyl-containing peptides increased with increasing depth of the tyrosyl residue in the bilayer. In contrast, MPO/H2O2/ NO<UP><SUB>2</SUB><SUP>−</SUP></UP>-induced tyrosyl nitration decreased with increasing depth of tyrosyl residues in the membrane. Transmembrane nitrations of tyrosyl-containing peptides induced by both peroxynitrite and MPO/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP> were totally inhibited by ·NO that was slowly released from spermine NONOate. Nitration of peptides in both systems was concentration-dependently inhibited by unsaturated fatty acid. Concomitantly, an increase in lipid oxidation was detected. A mechanism involving ·NO2 radical is proposed for peroxynitrite and MPO/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP>-dependent transmembrane nitration reactions.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein nitration has been detected in several inflammatory disease states (1-3). Nitration of tyrosine or tyrosyl residues associated with peptides and proteins is perceived to be a diagnostic marker of reactive nitrogen species (e.g. peroxynitrite, OONO-/ONOOH; nitrogen dioxide radical, ·NO2) (3-5). Nitration of protein tyrosyl has been postulated to modulate protein metabolism, function, and signal transduction pathways (1). Recent reports indicate that hydrophobic protein tyrosyl residues are preferentially nitrated by reactive nitrogen species (1, 6, 7). Thus, in vitro mechanistic studies using "free" tyrosine in the aqueous phase may not adequately reflect the mechanisms of nitration of protein tyrosyl residues in vivo (8-10). In addition, the efficiency of nitration of tyrosine induced by peroxynitrite in the aqueous phase has been reported to be remarkably low (1-4%) (11-13). In contrast, nitrations of membrane-incorporated gamma -tocopherol (a membrane-associated chromanol) and N-t-BOC l-tyrosine tert-butyl ester were increased by 3- to 5-fold (12, 14, 15). A plausible reason for the increased efficiency of lipid-phase nitration is the greater membrane permeability of reactive nitrogen species coupled with decreased lateral diffusion of membrane-bound tyrosyl free radical, making radical-radical dimerization unlikely (15).

These previous studies demonstrated preferential nitration of gamma -tocopherol and tyrosyl analogs that are distributed throughout the hydrophobic phase of the bilayer (12, 15). The objective of this study was to test the hypothesis that the extent of nitration of membrane-incorporated tyrosyl probe is dependent on its intramembrane location. To test this hypothesis, we have synthesized membrane spanning 23-mer peptides containing a single tyrosyl residue at positions 4, 8, and 12 and monitored their nitration reactions in the membrane bilayer.

In this study, we have used HPLC,1 electron spin resonance (ESR), and fluorescence techniques for detection and characterization of transmembrane nitration reactions of tyrosyl probes induced by peroxynitrite and ·NO2. Nitrating agents employed include slow infusion of peroxynitrite, generation of superoxide and nitric oxide (using SIN-1) that rapidly combines to form peroxynitrite in situ, and a myoperoxidase/H2O2/ NO<UP><SUB>2</SUB><SUP>−</SUP></UP> system that slowly generates ·NO2 free radical gas (16-21). The present findings point to an intermediacy of ·NO2 in transmembrane nitration reactions.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The following chemicals and enzymes were purchased from various sources as indicated: tyrosine, hydrogen peroxide, sodium nitrite, sodium bicarbonate, 3-nitrotyrosine, and thiobarbituric acid (Sigma), tetranitromethane and tetramethoxypropane (Aldrich), 3-morpholinosydnonimine HCl (SIN-1; Calbiochem), 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC) and 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocoline (PLPC) (Avanti Polar Lipids), and myeloperoxidase (Calbiochem). Rink amide methylbenzhydrylamine resin and all Fmoc-protected amino acids were purchased from Calbiochem-Novabiochem (La Jolla, CA). Diisopropylcarbodiimide (DIC), 1-hydroxybezotriazole (HOBt), triisopropylsilane, piperidine, and N-methylpyrrolidione (NMP) were purchased from Fisher Scientific and used without further purification. Spin-labeled fatty acids (5-doxyl)-stearic acid and (12-doxyl)-stearic acid are from Avanti Chemicals. Peroxynitrite was synthesized according to the published procedure (18, 19), and its concentration in 0.01 M NaOH was determined using the extinction coefficient (epsilon  = 1,670 M-1 cm-1 at 302 nm). All reagents were prepared in double-distilled deionized water.

Peptide Synthesis and Purification-- Sequences of the peptides used in this study are given in Table I. The peptides were acetylated and amidated to stabilize their alpha -helical secondary structure. The peptides were chemically synthesized using standard Fmoc solid phase peptide synthesis chemistry on an Advanced Chemtech Model 90 synthesizer (Louisville, KY). Rink amide methylbenzhydrylamine resin (loading 0.72 mmol/g) was used as a solid support. Fmoc-protected amino acids were coupled as HOBt esters. All amino acids were double coupled using HOBt/DIC. The following steps were performed in the reaction vessel for each double coupling: deprotection of the Fmoc group with 20% piperidine in NMP for 30 min (2 times), three NMP washes, two dichloromethane washes, first coupling for 1 h with 5-fold excess of Fmoc amino acid in 0.5 M HOBt and 0.5 M DIC, second coupling using a fresh addition of the same reagent for another hour, three NMP washes, and two dichloromethane washes. Final acetylation was performed using acetic anhydride/HOBt/DIC for 30 min (twice). The resin was washed twice with dichloromethane and three times with methanol and then dried under vacuum prior to cleavage. The peptide was deprotected and cleaved from the resin with 90% trifluoroacetic acid containing triisopropylsilane for 3 h at room temperature. The resin was removed by filtration and washed with trifluoroacetic acid, and the combined trifluoroacetic acid filtrates were evaporated to dryness under a steam of dry N2 gas. The oily residue was washed three times with cold ether to remove scavengers, and the dry crude peptide was dissolved in acetonitrile/H2O (1:1) and lyophilized. The crude peptides were purified by semi-preparative reverse-phase HPLC on a Vydac RP-C8 (10 × 250 mm) column (Hesperia, CA) using a CH3CN/water gradient (20 to 100% CH3CN over 50 min) containing 0.1% trifluoroacetic acid at a flow rate of 3 ml/min with detection at 220 nm. Elution of the peptides generally occurred between 50 and 70% CH3CN. Purity and homogeneity of the peptides were cross-checked by analytical reverse-phase HPLC, and the purified peptides (~95% pure) were characterized by liquid chromatography/mass spectrometry on an Agilent 1100 series liquid chromatography/mass spectrometer.

Synthesis of Nitro Peptides-- 10 mg/ml peptide dissolved in MeOH/1% trifluoroacetic acid was mixed with NO2BF4 (5 mg/ml). After 20 min, the product was purified by a semi-preparative HPLC (C-18; 250 × 10 mm), pre-equilibrated with 50% CH3CN in 0.1% trifluoroacetic acid. The nitro peptide was eluted by a linear CH3CN gradient (0.025%/min). Collected nitro peptide peak was dried under a stream of nitrogen and redissolved in MeOH as standard for analysis. Nitro peptides show a characteristic UV-visible spectrum. After adding NaOH, the 350-nm absorption peak in MeOH was pH-sensitive-shifted to 430 nm with an extinction coefficient of 4100 M-1 cm-1.

Spin Labeling Depth Measurements-- HPLC-purified peptides in methanol were mixed with a 5-fold molar excess of (1-oxyl-2,2,5,5-tetramethylpyrroline)-3-methylmethanethiosulfonate (MTSL) (Toronto Research Chemicals) and incubated overnight at 4 °C. Unreacted spin labeled and unlabeled peptides were removed by semi-preparative HPLC as described above, and the MTSL-labeled peptides were collected and lyophilized. To prepared liposomes, spin-labeled peptides were dissolved in methanol and added to a thin film of DLPC to give a lipid:peptide molar ratio of 200:1. The samples were dried under a stream of N2 and placed under vacuum for at least 1 h. The dried lipid:peptide films were then hydrated at room temperature in 50 mM Tris, 100 mM NaCl, pH 7.5, with or without 200 mM nickel (II) ethylenediaminediacetate (NiEDDA). ESR spectra were obtained on a Varian E-9 spectrometer equipped with a loop-gap resonator using a field modulation amplitude of 1.0 G. Samples were contained in a TPX capillary to facilitate equilibration with either air or high purity N2.

Incorporation of Peptides into DLPC Liposome-- Liposomes were prepared according to a previous report (15). A methanolic solution of peptide was added to DLPC dissolved in methanol. The mixture was then dried under a stream of N2 gas and kept in a vacuum dessicator overnight. Multilamellar liposomes were formed by thoroughly mixing the dried lipid in phosphate buffer (100 mM, pH 7.4) containing DTPA (100 µM). Unilamellar liposomes were prepared by five cycles of freeze-thawing using liquid nitrogen followed by five cycles of extrusion through a 0.2-µm polycarbonate filter (Nucleopore, Pleasanton, CA) in an extrusion apparatus (Lipex Biomembranes, Inc., Vancouver, BC). The nitration of peptides in liposomes prepared by both methods showed no significant difference.

Nitration of Tyrosyl Peptides in Liposomes-- A typical reaction was initiated by adding 1 µl of various concentration of peroxynitrite diluted in 0.05 M NaOH into 29 µl of liposome (30 mM DLPC)-containing peptide (0.3 mM peptide). Incubation mixtures were in 0.1 M phosphate buffer containing 100 µM DTPA. After 20 min at room temperature, the reaction mixture was diluted with 600 µl of distilled water and centrifuged at 12800 rpm for 120 min, and the pelleted liposome was dried by a speedvac. The dried liposome was dissolved in MeOH containing 2.5% trifluoroacetic acid for HPLC analysis. Compared with liposomes dried directly, the recovery of peptide was greater than 95%. Neither decomposed peroxynitrite nor addition of NO<UP><SUB>2</SUB><SUP>−</SUP></UP>/NO<UP><SUB>3</SUB><SUP>−</SUP></UP> (200 µM) gave any detectable nitration products.

Peroxynitrite Infusion-- The slow infusion of peroxynitrite was performed using an infusion/withdraw pump from Harvard Apparatus (model 966) under constant stirring of liposomes or aqueous solution containing tyrosine probes. Peroxynitrite (15 mM diluted in 0.25 M NaOH) was infused at a constant rate (0.82 ± 0.05 µl per 10 min) into 0.25 ml of liposomes (30 mM DLPC containing 300 µM peptide) until the final concentration of peroxynitrite was measured to be 50 µM. The infusion rates for peroxynitrite were 5.0 µM/min. The maximum pH shift was less than 0.1 units after infusion. Peroxynitrite decomposition under these conditions was evaluated by following the absorption change at 302 nm and its nitration efficiency before and after infusion. After 45 min, the changes of optical absorption and nitration efficiency after infusion were less than 5% of the starting peroxynitrite stock solution.

Myeloperoxidase/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP> System-- Peptides (0.3 mM) in 30 mM DLPC liposomes were incubated with 0.5 mM NaNO2, 0.1 or 0.2 mM H2O2, and 50 nM MPO in 0.1 M phosphate buffer containing 100 µM DTPA at 37 °C for 1 h. Samples were extracted and analyzed by HPLC as described below.

HPLC Analysis of Nitration Products-- Typically, 2 µl of sample was injected into a capHPLC system (HP1100) with a cap C-18 column (150 × 2 mm) equilibrated with 50% CH3CN in 0.5% trifluoroacetic acid. The peptide and its nitration product were separated by a linear increase of CH3CN concentration (0.1%/min) at a flow rate of 15 µl/min. The elution was monitored by a variable UV detector at 280 and 350 nm. Peptide and nitrated peptide were eluted at 12 or 17.5 min, respectively.

Nitration of tyrosine in aqueous solution was performed under the same conditions, and the product analysis of tyrosine reaction in aqueous solution was according to previous reports (12, 19, 22, 23). Briefly, 2 µl of sample was injected into a capHPLC system (HP1100) with a cap C-18 column (150 × 2 mm) equilibrated with 4% MeOH in 50 mM phosphate buffer, pH 3.0, and the tyrosine and nitrotyrosine were eluted at 5 and 12 min at a flow rate of 15 µl/min. The elution was monitored by a variable UV detector at 280 and 350 nm.

Fluorescence Quenching Measurements-- Fluorescence spectra of peptides incorporated into liposomes (0.3 mM peptide in 30 mM DLPC in 0.1 M phosphate buffer containing 100 µM DTPA) were monitored using an excitation wavelength of 278 nm (slit width, 5 nm) and monitoring emission from 300-350 nm (slit width, 10 nm). The fluorescence quenching experiments were performed as follows: methanolic solutions of spin labels, 12-doxyl SA, or 5-doxyl SA in glass vials were dried under a stream of nitrogen, and liposomes containing peptides (Y-4, Y-8, or Y-12) were pipetted into these vials and vortexed for 5 min, allowing incorporation of the doxyl SA into the liposomes.

TBA-MDA Assay-- We used a modified HPLC method to measure TBA-MDA adduct (24). A mixture of 25 µl of sample (or standard), 75 µl of 0.15 M H3PO4, 25 µl of 0.44 mM TBA, and 25 µl of distilled H2O was heated in boiling water for 1 h and then cooled down on ice. An aliquot of 50 µl was neutralized with 50 µl of methanol-sodium hydroxide solution and analyzed by HPLC. Reverse-phase isocratic separation was applied on a Kromasil C-18 column (250 × 4.6 mm, 5 µm) with a methanol/water mobile phase (70/30%) (v/v) at 0.5 ml/min flow rate. The TBA-MDA adduct eluting at 4.7 min was detected at 532 and 553 nm extinction and emission, respectively. The injected amount was 10 µl.

ESR Measurements-- ESR spectra were recorded at room temperature on a Bruker ER 200 D-SRC spectrometer operating at 9.8 GHz and a cavity equipped with a Bruker Aquax liquid sample cell. Typical spectrometer parameters were as follows: scan range, 100 G; field set, 3510 G; time constant, 0.64 ms; scan time, 20 s; modulation amplitude, 5.0 G; modulation frequency, 100 kHz; receiver gain, 2 × 105; and microwave power, 20 mW. Reaction mixtures consisting of Y-4 (0.4 mM) and 20 mM 2-methyl-2-nitrosopropane (MNP) spin trap incorporated into 40 mM DLPC liposome in a phosphate buffer (0.1 M, pH 7.4) containing DTPA (0.1 mM) were rapidly mixed with 2 mM peroxynitrite. Samples were subsequently transferred to a 100-µl capillary tube for ESR measurements.

Statistic Analysis-- The data are presented as average ± S.D. The significance was calculated using a Student's t test.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of Transmembrane Peptides in Liposomes-- Table I shows the amino acid sequences and mass spectral data for the various tyrosyl and cysteinyl peptides used in the present study. These peptides were synthesized and purified as described under "Experimental Procedures." To assess the localization of the tyrosine residues in these peptides with respect to the membrane bilayer, we examined their intrinsic fluorescence properties and susceptibility to quenching by nitroxide-labeled fatty acids. As shown in Fig. 1A, fluorescence intensity for this series of peptides increases in the order Y-12 > Y-8 > Y-4, indicating a progressively increasing hydrophobic environment (membrane depth) for the tyrosine residues. A similar order of sensitivity to fluorescence quenching by nitroxides attached at the 5-position of stearic acid (5-doxyl SA) and at the 12-position of stearic acid (12-doxyl SA) was observed (Fig. 1B). Fluorescence quenching by 5-doxyl SA, which localizes near the glycerol backbone of phospholipid bilayers, was greater for Y-4 than for Y-8 or Y-12 (Fig. 1B), further indicating that the tyrosine residue of Y-4 resides just below the membrane surface. On the other hand, fluorescence quenching by 12-doxyl SA was greater for Y-12 than for Y-4 indicating that Y-8 and Y-12 are located deeper in the bilayer. A schematic representation of the intramembrane position of tyrosyl peptides and nitroxide-labeled fatty acids is shown in Fig. 1C. These data are all consistent with the expected transmembrane orientation of these peptides (25-29) and indicate that incorporation into a repeating leucine-alanine motif anchored at each end by lysine residues is an effective strategy for positioning tyrosine residues across the lipid bilayer.

                              
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Table I
Characteristics of transmembrane peptides


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Fig. 1.   Fluorescence spectra of membrane-incorporated tyrosyl peptides. A, fluorescence spectra of tyrosyl peptides (0.3 mM) incorporated into liposomes (30 mM DLPC in 0.1 M phosphate buffer containing 100 µM DTPA) were obtained using an excitation wavelength of 278 nm. B, the effect of 5-doxyl and 12-doxyl stearic acid nitroxides (1 mM) on the ratio of the fluorescence intensity of tyrosyl peptides in the absence (F0) and in the presence of nitroxide (F). C, schematic representation of relative intramembrane position of tyrosyl peptides and doxyl probes.

To further characterize bilayer depth we synthesized the analogous peptides with cysteine replacing tyrosine (i.e. C-4, C-8, and C-12), modified the single cysteine in each peptide with a sulfhydryl-specific nitroxide spin label MTSL, and measured bilayer depth by established methods using O2 and NiEDDA as paramagnetic relaxation agents (30, 31). The ESR spectra of MTSL-labeled peptides in DLPC liposomes are shown in Fig. 2A. The ESR spectra of spin-labeled peptides in Fig. 2 are significant in that they demonstrate that all of the peptide has been incorporated into the membrane, giving homogeneous, single component spectra. Peptides with a similar sequence may distribute between a transmembrane orientation and alignment along the surface of the bilayer, which would give rise to a two-component ESR spectrum. The spectra in Fig. 2 show that, at least for our methods of liposome preparation, this is not the case. MTSL-C-12 shows a slightly greater degree of motional restriction than the other two peptides. Isotropic hyperfine coupling constants for the three peptides ranged between 13.45 and 13.9 G, consistent with their hydrophobic location.


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Fig. 2.   ESR spectrum of MTSL-labeled cysteinyl peptides in DLPC liposomes. A, the ESR spectrum of MTSL-labeled C-4 peptide (top), C-8 peptide (middle), and C-12 peptide (bottom). The lipid:peptide ratio was 200:1. The scan width was 100 G. B, continuous wave power saturation for MTSL-C8 in DLPC liposomes under N2 (circles), air (squares), and in the presence of 200 mM NiEDDA (triangles). The greater shift to higher powers in the presence of O2 relative to NiEDDA is characteristic of a hydrophobic environment for the spin label.

Bilayer depths of the nitroxide side chains were determined by power saturation ESR measurements (Fig. 2B) based on relative collision rates with O2 and NiEDDA (30, 31). In agreement with the fluorescence studies described above, the C-4 position was nearest to the membrane surface at an estimated depth of 9 Å below the lipid phosphates, whereas nitroxide side chains attached at C-8 and C-12 were located further below the lipid phosphates (~ 12.5 Å).

Peroxynitrite-dependent Oxidation and Nitration of Tyrosyl Peptides in Membranes: Comparison with Tyrosine in Aqueous Solution-- Previous studies implicated that peroxynitrite-induced nitration and oxidation reactions are favored in the hydrophobic phase of lipid membranes (12, 15). In this study, we sought to demonstrate this phenomenon with tyrosyl residues that are anchored at specific positions in the lipid bilayer. Peroxynitrite-dependent nitration of tyrosyl-containing peptides in DLPC liposomes was monitored by HPLC with UV-visible detection at both 280 and 350 nm (Fig. 3, A and B). The Y-8 peptide eluted with a retention time of 12 min, and pre-synthesized NO2-Y-8 eluted after 17.5 min. A bolus addition of peroxynitrite (100 µM) to DLPC liposomes containing Y-8 peptide (300 µM) resulted in the formation of a new compound eluting at 17.5 min, along with the parent peptide eluting at 12 min (Fig. 3, middle trace). The peak eluting at 17.5 min was assigned to the nitration product, NO2-Y-8 peptide, by comparison with the authentic standard (Fig. 3, top trace). Decomposed peroxynitrite did not induce any nitration of peptide in DLPC liposomes (Fig. 3, bottom).


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Fig. 3.   HPLC traces of nitrated tyrosyl peptides in liposome. Y-8 peptide (0.3 mM) in 30 mM DLPC liposome was reacted with peroxynitrite in 0.1 M phosphate buffer containing 100 µM DTPA and analyzed by capillary HPLC. A, HPLC traces were monitored at 280 nm. Top indicates the authentic 20 µM NO2-Y-8 peptide, middle shows the nitrated reaction product from the reaction between peroxynitrite and Y-8 peptide and bottom is the same as above but in the presence of decomposed peroxynitrite (left) HPLC traces monitored at 280 nm that showed both original Y-8 peptide (12 min) and nitration product (17.5 min). B, HPLC traces monitored at 350 nm for nitration products. Experimental conditions were the same as in A.

Previous reports suggest that the rate at which peroxynitrite is added to a solution containing tyrosine can drastically alter the nitration yield (8, 9, 15, 22, 23, 32, 33). A bolus addition of increasing concentrations of peroxynitrite showed a significant increase in formation of NO2-Y-8 (Fig. 4A). Under identical conditions, bolus addition of peroxynitrite to a phosphate buffer containing tyrosine (300 µM) also yielded a dose-dependent increase in the formation of nitrotyrosine as monitored by HPLC-UV. At high peroxynitrite concentrations (~300 µM), the yields of NO2-Tyr were about 2-fold higher than NO2-Y-8 peptide (Fig. 4A). However, this difference was diminished as peroxynitrite concentration decreased; a bolus addition of 50 µM peroxynitrite induced greater nitration of Y-8 peptide (6.8 µM NO2-Y-8 peptide) in DLPC liposomes as compared with tyrosine (4.2 µM) in aqueous solution (Fig. 4A). The nitration profile of Y-8 peptide in DLPC membrane was similar to that of N-t-BOC tyrosine tert-butyl ester (BTBE), a hydrophobic tyrosine analog (15). Fig. 4B shows the nitration yields of tyrosyl residues anchored at different depths in DLPC liposomes. HPLC analysis showed that the nitration yields of tyrosyl peptides in membranes were significantly higher than that of tyrosine in aqueous phase. Results showed that the formation of nitrated peptides slightly increased as the depth of tyrosyl residues in the bilayer were increased (NO2-Y-4, 5.8 µM; NO2-Y-8, 6.8 µM; and NO2-Y-12, 7.4 µM).


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Fig. 4.   Nitration of tyrosine in the aqueous phase and tyrosyl peptides in membranes. A, tyrosine (0. 3 mM) or Y-8 (0.3 mM) in DLPC (30 mM) liposomes was incubated with various concentrations of peroxynitrite for 20 min in sodium phosphate buffer (0.1 M, pH 7.4) containing DTPA (100 µM) and analyzed by HPLC (n = 3 ± S.D.). B, tyrosine (0. 3 mM) or Y-4, Y-8, and Y-12 peptides (0. 3 mM each) in DLPC (30 mM) liposomes were incubated with peroxynitrite (50 µM) for 20 min in sodium phosphate buffer (0.1 M, pH 7.4). C, tyrosine and tyrosyl peptides (0.3 mM) in DLPC (30 mM) liposomes were incubated with SIN-1 (1 mM) in sodium phosphate buffer (0.1 M, pH 7.4) containing DTPA (100 µM) overnight. Nitrated products were analyzed by HPLC. D, tyrosine (0.3 mM) or tyrosyl peptides (0.3 mM) in DLPC (30 mM) liposomes were incubated with NaNO2 (0.5 mM), H2O2 (0.2 mM), and myeloperoxidase (50 nM) in a phosphate buffer (0.1 M, pH 7.4) containing DTPA (100 µM) at 37 °C for 1 h. The sample was extracted and analyzed by HPLC as described under "Experimental Procedures."

To investigate the nitrating ability of peroxynitrite that is generated in situ (Fig. 4C), DLPC liposomes containing tyrosyl peptides (300 µM) or tyrosine (300 µM) in phosphate buffer were incubated with SIN-1 (1 mM) (Fig. 4C). Nitration of tyrosine in aqueous phase by SIN-1, which generates equal amounts of nitric oxide and superoxide, gave very low yields of nitrotyrosine (~ 0.5 µM) (Fig. 4C) (9, 12). Under similar conditions, the nitration yield of Y-12 peptide in membrane was about 18 times higher (9 µM) than that of tyrosine in aqueous solution (Fig. 4C). Slow infusion of peroxynitrite (5 µM/min; final concentration, 50 µM) into DLPC liposome containing Y-8 peptide also induced a 5-fold increase in nitration (6.2 µM) compared with the nitration of tyrosine (1.2 µM) in aqueous phase (Fig. 5C). Incubation of SIN-1 (1 mM) with DLPC liposome containing these peptides (Y-4, Y-8, and Y-12) revealed that nitration of those peptides was enhanced with increasing membrane depth for the tyrosine residues (Fig. 4C). Investigation of the nitration profile of Y-4, Y-8, and Y-12 obtained in MPO/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP> system produced an interesting yet unexpected result (Fig. 4D). In contrast to SIN-1-catalyzed nitration profile, incubation of DLPC liposome containing Y-4, Y-8, and Y-12 in the presence of MPO, H2O2, and NO<UP><SUB>2</SUB><SUP>−</SUP></UP> caused a decrease in the nitration of peptide with increasing membrane depth (Fig. 4D).


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Fig. 5.   The effect of bicarbonate on peroxynitrite-mediated nitration of tyrosine in aqueous solution and tyrosyl peptides in membranes. A, tyrosine (0. 3 mM) or Y-4, Y-8, and Y-12 peptides (0. 3 mM each) in DLPC (30 mM) liposomes were incubated with peroxynitrite (50 µM) for 20 min in sodium phosphate buffer (0.1 M, pH 7.4) containing DTPA (100 µM) with or without 25 mM bicarbonate. B, tyrosine and tyrosyl peptides (0.3 mM) in DLPC (30 mM) liposomes were incubated with SIN-1 (1 mM) and bicarbonate (25 mM) in sodium phosphate buffer (0.1 M, pH 7.4) containing DTPA (100 µM) overnight. Nitrated products were analyzed by HPLC. C, tyrosine (0.3 mM) and Y-8 peptide (0.3 mM) in DLPC (30 mM) liposomes were infused with peroxynitrite at 5 µM/min (accumulating to a final concentration of 50 µM) with and without bicarbonate (25 mM) in phosphate buffer (0. 1 M, pH 7.4) containing 100 µM DTPA and analyzed by HPLC.

To investigate the effect of tyrosine on the relative product yields, we incubated SIN-1 or MPO in the reaction mixtures containing free tyrosine at the same concentration as Y-8 peptide in DLPC liposomes. Table II shows the relative yields of nitrated peptide, nitrotyrosine, and dityrosine. Even in the presence of free tyrosine, the nitration yield in the membrane is higher than that of free tyrosine in the aqueous phase. However, the overall yield of peroxynitrite from SIN-1 is considerably lower in the presence of tyrosine. The effect of tyrosine on the mechanism of decomposition of SIN-1 is not known. With SIN-1, there was no detectable change in the dityrosine concentration in the presence or absence of DLPC liposome containing Y-8 (Table II). With MPO/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP>-induced nitration, the relative nitration yield in the membrane was significantly enhanced in the presence of tyrosine. Under these conditions, the dityrosine yield in the aqueous phase was increased by more than 2-fold (Table II). Clearly, the hydrophobic membranes significantly affect the MPO/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP>-induced dityrosine formation in the aqueous phase. We are actively investigating the mechanism of this reaction in detail.

                              
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Table II
The effect of tyrosine on the relative product yields of free and peptide-bound tyrosine

Effect of Bicarbonate on Peroxynitrite-mediated Nitration of Tyrosyl Peptides-- Next we compared the effect of bicarbonate anion (HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) on nitration of tyrosyl peptides in membrane with that of aqueous tyrosine, as HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> had been shown to enhance peroxynitrite-dependent oxidation and nitration of tyrosine (34-39). Peroxynitrite reacts with CO2 to generate the nitrosoperoxycarbonate (ONOOCO<UP><SUB>2</SUB><SUP>−</SUP></UP>) intermediate that is responsible for increased nitration and oxidation of tyrosine in the aqueous phase (15). Fig. 5 shows the effect of increasing concentrations of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> on the nitration of tyrosine caused by a bolus and the slow addition of peroxynitrite. Bicarbonate considerably enhanced the nitration of tyrosine in aqueous solution as expected (15, 33, 39), whereas nitration of peptides in DLPC liposomes by a bolus addition of peroxynitrite was inhibited by bicarbonate (Fig. 5A). As shown in Fig. 5A, the inhibitory effect of bicarbonate on the nitration of peptides in membrane increased in the order Y-12 (60%) > Y-8 (30%) > Y-4 (20%). This indicates a lack of diffusion or slow diffusion of the intermediate radical pair (·NO2  ... CO<UP><SUB>3</SUB><SUP>&cjs1138;</SUP></UP>) into membranes (36). Although the degree to which bicarbonate inhibited membrane nitration varied to some extent, the overall effect of bicarbonate on membrane nitration was minimal (Fig. 5, A-C). Based on these findings, we conclude that bicarbonate does not affect nitration of tyrosyl peptides in membrane exposed to co-generated O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> and ·NO at the same flux or to slowly infused peroxynitrite.

Effect of Unsaturated Fatty Acid on Tyrosine Nitration and Lipid Oxidation-- Biological membranes are composed of unsaturated lipids. Recently, several papers reported that peroxynitrite can diffuse into membranes (15, 38, 40, 41) and oxidize unsaturated fatty acids. To investigate the effect of unsaturated fatty acids on transmembrane nitration of tyrosyl peptides, we used PLPC as a model liposomal system. Peroxynitrite (50 µM) was added as a bolus or generated in situ from SIN-1 in incubations containing Y-8 peptide (0.3 mM) incorporated into liposomes (30 mM PC), composed of differing percentages of PLPC. Nitration of tyrosyl-containing peptides was inhibited by unsaturated fatty acid in a concentration-dependent manner (Fig. 6A). The nitration yield of Y-8 peptide was reduced from 6.5 µM in 100% DLPC to about 1.6 µM in 100% PLPC liposome under otherwise identical conditions. Concomitantly, an increase in MDA, an oxidation product of polyunsaturated fatty acids, was observed (Fig. 6B). These results indicate that unsaturated fatty acid effectively scavenges peroxynitrite or its reactive intermediate and inhibits tyrosine nitration in membranes containing PLPC.


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Fig. 6.   The effect of unsaturation on peroxynitrite- and MPO/H2O2/nitrite-induced nitration of tyrosyl peptides. A, Y-8 peptide (0.3 mM) in liposomes composed of various amounts of DLPC and PLPC (total, 30 mM) were incubated with 50 µM peroxynitrite in sodium phosphate buffer (0.1 M, pH 7.4) containing 100 µM DTPA. Nitrated Y-8 was analyzed by HPLC (n = 3 ± S.D.). The effect of unsaturation on MPO/nitrite/H2O2-induced nitration of tyrosyl peptide was determined. Conditions were essentially the same except that incubations were performed with 0. 5 mM NaNO2, 0.2 mM H2O2, and 50 nM MPO. Nitrated Y-8 was analyzed by HPLC (n = 3 ± S.D.). B, formation of MDA was measured by HPLC under the reaction conditions that were the same as A.

The effect of different proportions of PLPC on MPO/H2O2/ NO<UP><SUB>2</SUB><SUP>−</SUP></UP>-dependent transmembrane tyrosyl nitration was then investigated. As shown in Fig. 6B, nitropeptide formation was inhibited by unsaturated fatty acid in a dose-dependent manner. With increasing amount of unsaturated fatty acid, MPO/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP> caused an increase in lipid oxidation (Fig. 6B) while decreasing tyrosine nitration. These data suggest that ·NO2 generated from MPO/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP> is scavenged by PLPC resulting in increased MDA formation and decreased nitration.

The Effect of Nitric Oxide on Peroxynitrite- and MPO/ H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP>-induced Nitration of Tyrosyl Peptides-- Nitrogen dioxide radical is rapidly scavenged by ·NO to form N2O3 that is hydrolyzed to form NO<UP><SUB>2</SUB><SUP>−</SUP></UP> (42). Peroxynitrite also reacts with ·NO to form N2O3 (43). As both ·NO and ·NO2 can readily diffuse into the membrane (44), this radical-radical recombination reaction should rapidly occur in the hydrophobic domain of membranes. Fig. 7 shows that ·NO dramatically inhibits transmembrane nitration. We investigated the effect of ·NO released slowly from spermine NONOate (SNN) (5 µM/min) on transmembrane nitration of tyrosyl peptides induced by MPO/ H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP> and by ONOO- generated in situ. As shown in Fig. 7A, ·NO generated by SNN inhibited MPO/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP>- dependent tyrosine nitration in the aqueous phase by 80% and Y-8 nitration in the membrane by more than 90%. Under the same conditions, SNN inhibited SIN-1-mediated aqueous phase nitration by 33% and Y-8 nitration in the membrane by over 80% (Fig. 7B). Decomposed SNN did not inhibit nitration (not shown). These results indicated that ·NO inhibits membrane nitration much more effectively than tyrosine nitration in the aqueous phase. This is attributed to the rapid reaction between ·NO and ·NO2 or ·NO and peroxynitrite.


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Fig. 7.   The effect of spermine NONOate on peroxynitrite- and MPO/H2O2/NO<UP><SUB><B>2</B></SUB><SUP><B>−</B></SUP></UP>-induced nitration of tyrosyl peptides in the membrane. A, Y-8 peptide (0.3 mM) in DLPC liposome (30 mM) or tyrosine (0.3 mM) was incubated with NaNO2 (0.5 mM), H2O2 (0.2 mM), and 50 nM MPO in phosphate buffer (0.1 M, pH 7.4) containing DTPA (0.1 mM) at 37 °C for 1 h. B, Y-8 peptide (0.3 mM) in DLPC liposome (30 mM) or tyrosine (0.3 mM) was incubated with SIN-1 (1 mM) at room temperature overnight. Spermine NONOate (1 mM) was added to incubation mixtures in A and B as indicated. The samples were extracted and analyzed by HPLC as described under "Experimental Procedures."

Spin Trapping of Tyrosyl Radical in Membrane-- To investigate whether peroxynitrite induces nitration of tyrosyl peptides via a radical mechanism, we used MNP, a lipophilic spin trap, to detect tyrosyl peptide-derived radicals. Previously, MNP has been used to detect and characterize tyrosyl radical formed during MPO/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP>-catalyzed oxidation of tyrosine in solution (45). As shown in Fig. 8, the addition of peroxynitrite (2 mM) to DLPC liposome (40 mM) incorporated with Y-4 (0.4 mM) and MNP (20 mM) in a sodium phosphate buffer (0.1 M, pH 7.4) containing DTPA (0.1 mM) gave rise to an ESR spectrum of a spin adduct that is motionally restricted. Spectral features show similarity to MTSL-C-4 (see Fig. 2). No ESR spectrum of a spin adduct was obtained with decomposed peroxynitrite (Fig. 8). The spin adduct was extracted into methanol, which gave an isotropic ESR (alpha N = 15.6 G) spectrum. Because of the poor signal-to-noise ratio and oxygen broadening, the spectrum could not be resolved further even in the presence of fully deuterated spin trap (data not shown). It is also noteworthy that the spectra in Fig. 2 are quite similar to those obtained by spin trapping during peroxynitrite-induced tyrosyl peptide nitration (Fig. 8). Thus, the ESR data provide further support for radical formation from tyrosyl peptides in the presence of active peroxynitrtie.


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Fig. 8.   Spin trapping of tyrosyl radical formed during peroxynitrite-induced nitration of tyrosyl peptides in membrane. Y-4 (0.4 mM) in DLPC (40 mM) was reacted with peroxynitrite (2 mM) in the presence of MNP (20 mM) in a phosphate buffer (0.1 M, pH 7.4) containing DTPA (0.1 mM). The ESR spectrum was recorded using a modulation amplitude of 5 G, scan time, 20 s; time constant, 0.64 ms; microwave power, 20 mW. A modulation of 2G was used to obtained the spectrum of spin adduct in methanol.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of Tyrosyl Peptides-- The rationale for this study was to examine whether protein tyrosyl residues exposed to the hydrophobic interior of biological membranes are more prone to nitration than their aqueous counterparts. Biological nitration of tyrosine residues has been the subject of much interest. At present two major candidate mechanisms for nitration have been identified that involve either the formation of peroxynitrite from the diffusion-limited reaction between nitric oxide and superoxide or the peroxidatic activity of peroxidase enzymes, using nitrite as the electron donating substrate. It should be noted that these mechanisms are not mutually exclusive, nor is nitration the only modification that results from these agents. This study also attempts to clarify the mechanisms of hydrophobic tyrosine nitration and to examine how the source of nitration affects the relative product yields in the hydrophobic interior of membranes. To examine these issues we have synthesized model tyrosyl-containing peptides designed to position the tyrosyl group at varying depths within the membrane. The peptides consisted of an alanine-leucine repeat that has been shown previously to form transmembrane helices, anchored at both ends by two lysine residues. Tyrosyl residues were inserted at positions 4, 8, and 12 (termed Y-4, Y-8, and Y-12) to place the nitration target at the surface of the bilayer, at approximately one-quarter of the way into the bilayer and at approximately the center of the bilayer. The position of the residues within the bilayer was confirmed by two approaches. The first used the intrinsic fluorescence of the tyrosyl residue and the ability of fatty acid nitroxides to quench this fluorescence. The second method used a complimentary set of peptides containing cysteine in place of tyrosine. The cysteine residues were spin-labeled, and the depth of the label was determined using the ESR/power saturation technique. In both cases, the data were consistent with the predicted depth of the tyrosyl residue. The present data indicate that the peptide was properly inserted into the bilayer, most likely as a transmembrane alpha -helix.

The Effect of the Nitration Modality on Tyrosyl Peptide Nitration-- Nitration of the Y-8 peptide with bolus addition of chemically synthesized peroxynitrite gave a concentration-dependence similar to our previous observations using BTBE, a non-anchored hydrophobic tyrosine analog (15). In both cases, nitration of the membrane-localized probe was significantly reduced, as compared with tyrosine in the aqueous phase, at high levels of peroxynitrite. However, this difference was lost at lower levels of peroxynitrite, and nitration of membrane-anchored Y-8 exceeded the aqueous phase tyrosine nitration at the lowest concentration tested (50 µM). This illustrates the complex relationship between product yield and the nitration modality that should be taken into consideration when extrapolating the relevance of in vitro nitration studies to the biological arena.

We previously observed that BTBE had significantly reduced dimer formation than tyrosine. In the present study, no dimer product from peroxynitrite-mediated oxidation of the peptides was observed. The most likely explanation for this is that the hindered diffusion substantially inhibits radical-radical interactions necessary to form the tyrosyl dimer. This restriction would be more severe with membrane-anchored peptides than with BTBE.

The extent of peptide nitration by bolus addition of peroxynitrite did not show a strong depth dependence, although a trend toward higher nitration yield at greater depth was apparent. However, in all cases the yield of nitration product was greater than that observed for tyrosine. This indicates that the preferential nitration of membrane-associated tyrosine at low bolus amounts of peroxynitrite had little depth dependence. In contrast, the simultaneous formation of nitric oxide and superoxide from SIN-1 gave a significantly higher yield of peptide nitration compared with tyrosine and exhibited a marked depth dependence. Nitration from SIN-1 increased as a function of depth in the membrane. Most significantly, this pattern was reversed when myeloperoxidase/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP> was used as the source of the nitrating species. In this case the nitration of tyrosyl residues was negatively correlated with tyrosyl depth in the membrane. This not only highlights significant differences in the mechanism of nitration by these two agents but also gives a potential way to discriminate between them in a biological milieu.

Mechanism of Peroxynitrite- and MPO/NO<UP><SUB>2</SUB><SUP>−</SUP></UP>/H2O2-induced Transmembrane Nitration: Intermediacy of Nitrogen Dioxide Radical-- Two major pathways have been suggested to be responsible for tyrosine nitration in vivo (2, 5, 16). These involve either the nitrative chemistry of peroxynitrite or the catalytic action of heme peroxidases using H2O2 and nitrite anion as substrates. Scheme 1 illustrates the reactions involved in both the oxidation and nitration by either a peroxynitrite-dependent route (left hand side) or a myeloperoxidase-dependent pathway (right hand side).


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Scheme 1.   Postulated reaction pathways for transmembrane nitration of tyrosyl peptides anchored in membranes catalyzed by peroxynitrite and myeloperoxidase/nitrite anion/H2O2. The relevant rate constants of peroxynitrite and ·NO2 with ·NO, tyrosine, CO2, and thiols are taken from Ref. 46.

Peroxynitrite exists in equilibrium with peroxynitrous acid as shown in Reaction 1. 

<UP>R<SC>eaction</SC> 1</UP>
Peroxynitrous acid (ONOOH) has been reported to diffuse into membranes at rates similar to that of water molecules (36, 38, 40, 41). Homolytic decomposition of ONOOH in the membrane will form HO· and ·NO2 (47). Although the involvement of HO· in tyrosine nitration in the aqueous phase has been shown to be negligible based on hydroxyl radical scavenger experiments, its role in transmembrane nitration of tyrosyl peptides remains unclear. Transmembrane nitration mediated by peroxynitrite is more likely to be a free radical-dependent mechanism.

In the presence of H2O2, MPO forms higher oxidants (compounds I and II) that will oxidize nitrite into ·NO2 radical and tyrosine to tyrosyl radical. Nitration of tyrosine occurs following a rapid recombination of ·NO2 and the tyrosyl radical (Scheme 1). The oxidation of tyrosine by MPO-compound I to tyrosyl radical is deemed to be essential for tyrosine nitration, as the hydrogen abstraction from the tyrosyl hydroxyl group by ·NO2 in the aqueous phase is relatively slow (46). It is conceivable that tyrosyl peptides incorporated into liposomes are not readily accessible to MPO-compound I. However, the reaction between ·NO2 and the tyrosyl hydroxyl group in the hydrophobic interior of the phospholipid membrane may be more effective.

Nitrogen dioxide radical undergoes hydrolysis very readily in the aqueous phase forming nitrite and nitrate anions. However, ·NO2 is persistent in the hydrophobic phase and will participate in one-electron oxidation reactions with tyrosyl peptides forming the tyrosyl radical that can rapidly combine with ·NO2 to form the nitrated tyrosyl peptide (Scheme 1) (4, 49-51). Recently, it was shown that ·NO2 generated extracellularly by peroxidatic oxidation of nitrite anion diffuses into cells and nitrates a tyrosyl residue present within a green fluorescent protein (52). Nitration of fluorophore quenches the intrinsic green fluorescent protein fluorescence because of an electron-withdrawing effect of the nitro group. In the present work, we show that ·NO2 generated from MPO/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP> diffuses into the membrane and efficiently nitrates tyrosyl peptide. Nitration yield, however, decreased with increasing depth of tyrosyl location (i.e. NO2-Y-4 > NO2-Y-8 > NO2-Y-12). These findings suggest that reactive nitrogen species generated in biological systems can now be quantitated more accurately by monitoring the nitration of tyrosyl peptides incorporated into membranes. This trend is opposite the nitration profile observed with peroxynitrite. Previously, it has been shown that nitration of tyrosyl peptides by ·NO2 is enhanced in systems generating tyrosyl radicals (17). Thus, we tested the hypotheses that (i) Y-4 peptide can be oxidized by compound-I formed by MPO/H2O2 to generate the tyrosyl radical that subsequently reacts with ·NO2 to form NO2-Y-4, and (ii) Y-8 and Y-12 that are buried deeper in the bilayer are less prone to undergo oxidation by compound-I to form the tyrosyl radical. However, results indicate that there were no detectable changes in transmembrane tyrosyl peptides levels (Y-4, Y-8, and Y-12) in liposome treated with MPO and H2O2 (data not shown). These findings suggest that ·NO2 alone is able to cause nitration in membrane by forming tyrosyl radicals through an electron-transfer or a hydrogen abstraction mechanism (51).

The transmembrane nitration induced by ONOO- is opposite that observed with MPO/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP>. The electron-transfer reaction rate between ·NO2 and tyrosine is dependent on the protonated or unprotonated state of the phenolic group. ·NO2 is able to oxidize the phenoxide ion much more readily than the phenolic hydroxyl group. It is likely that the phenolic hydroxyl group in Y-4 that is anchored closer to the membrane surface is more ionized than that of Y-12. Thus, the formation of Y-4 tyrosyl radical in the presence of ·NO2 is probably more efficient, which will, in turn, accelerate the formation of NO2-Y-4. In the hydrophobic phase, the protonated form of peroxynitrite (ONOOH) is likely to be the dominant species. Homolytic decomposition of ONOOH to ·NO2 and ·OH has recently been reported (47). Tyrosyl peptides in the membrane can be oxidized by ·OH to tyrosyl radical that will rapidly react with ·NO2 to form NO2-Y. Homolytic decomposition of peroxynitrite is more likely to occur deeper in the membrane, which may explain the increased nitration of Y-12 with peroxynitrite. It has been reported recently (16, 53) that heme peroxidases (e.g. eosinophil peroxidase) could form peroxynitrite at lower pH levels and ·NO2 at physiological pH levels. It is conceivable that this marked difference in transmembrane tyrosyl nitration profile may be used to identify the nitrating species in these systems.

Bicarbonate Effect on Peroxynitrite-dependent Transmembrane Nitration-- Previously, several investigators have demonstrated that peroxynitrite-dependent nitration and oxidation product profiles are altered in the presence of CO2 (22, 23, 33, 34). Peroxynitrite anion reacts with CO2 to form a transient adduct, nitrosoperoxycarbonate (ONOOCO<UP><SUB>2</SUB><SUP>−</SUP></UP>), which decomposes in part to form CO<UP><SUB>3</SUB><SUP>&cjs1138;</SUP></UP> and ·NO2 radicals (Scheme 1). Both species can oxidize tyrosine to the tyrosyl radical, which will rapidly react with ·NO2 or another tyrosyl radical to form nitrotyrosine and dityrosine. Typically, nitration of tyrosine or tyrosine analogs is enhanced in the aqueous phase in the presence of peroxynitrite and CO2 (33-35). However, the situation is different in membranes. The transmembrane diffusion of OONOCO<UP><SUB>2</SUB><SUP>−</SUP></UP> across the bilayer is probably limited because of its increased rate of decay as compared with ONOO- (36). In addition, the anionic CO<UP><SUB>3</SUB><SUP>&cjs1138;</SUP></UP> is not sufficiently membrane-permeable. This is consistent with the previous findings that bicarbonate inhibits nitration of hydrophobic phenolic probes, gamma -tocopherol and BTBE (13-15).

Implications in Protein Nitration and Nitrosation-- Emerging literature (6, 7) provides evidence for selective nitration of tyrosines present in the transmembrane domains of proteins (e.g. sarcoplasmic reticulum Ca2+-ATPase, mitochondrial complex-1 protein). These findings support the notion that hydrophobic tyrosine residues are potential targets for nitrating species. Tyrosine nitration in MPO/H2O2/NO<UP><SUB>2</SUB><SUP>−</SUP></UP> system in aqueous phase is mediated by MPO/H2O2 oxidant-generated tyrosyl radical. With the hydrophobic transmembrane tyrosyl nitration, the nitrating species ·NO2 can clearly facilitate nitration of hydrophobic tyrosines. It has been suggested that protein tyrosine nitration is enhanced when tyrosine is in close proximity to negatively charged amino acids (glutamate, aspartate) and is decreased when tyrosine is located adjacent to easily oxidizable amino acids such as cysteine or methionine (48, 54). It is now possible to experimentally verify such effects using specifically custom-synthesized transmembrane peptides. The biophysical approaches outlined in the present study should serve as potentially useful tools to investigate the effect of membrane composition and other factors controlling transmembrane protein tyrosyl nitration. We have proposed in Scheme 1 that N2O3 is a likely intermediate in these reactions. With the availability of hydrophobic transmembrane cysteinyl peptides, it is now possible to investigate transmembrane nitrosation reactions initiated by N2O3 (Scheme 1). As shown in the present study, nitric oxide donors inhibit both myeloperoxidase- and peroxynitrite-induced transmembrane and aqueous phase nitration reactions. It is conceivable that nitric oxide donors could be used as anti-nitration and anti-inflammatory therapeutic agents.

    ACKNOWLEDGEMENTS

We sincerely thank Dr. Kasem Nithipatikom for obtaining the mass spectra of compounds.

    FOOTNOTES

* This work was supported by National Institute of Health Grants RR01008, IPO1HL68769-01, and HL63119.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Biophysics Research Inst., Medical College of Wisconsin, 8701 Watertown Plank Rd., P. O. Box 26509, Milwaukee, WI 53226. Tel.: 414-456-4035; Fax: 414-456-6512; E-mail: balarama@mcw.edu.

Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M211561200

    ABBREVIATIONS

The abbreviations used are: HPLC, high pressure liquid chromatography; CAPHPLC, capillary HPLC; BTBE, N-BOC l-tyrosine tert-butyl ester; DLPC, 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine; ESR, electron spin resonance; PLPC, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocoline; DTPA, diethylenetriaminepentaacetic acid; SA, stearic acid; MTSL, (1-oxyl-2,2,5,5-tetramethylpyrroline)-3-methylmethanethiosulfonate; MPO, myeloperoxidase; MNP, 2-methyl-2 nitroso propane; NiEDDA, nickel (II) ethylenediaminediacetate; SNN, spermine NONOate; TBA, thiobarbituric acid; MDA, malondialdehyde; Fmoc, N-(9-fluorenyl)methoxycarbonyl; DIC, diisopropylcarbodiimide; HOBt, 1-hydroxybezotriazole; NMP, N-methylpyrrolidinone.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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