Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms

Abstract

3-Nitrotyrosyl adducts in proteins have been detected in a wide range of diseases. The mechanisms by which reactive nitrogen oxide species may impede protein function through nitration were examined by using a unique model system, which exploits a critical tyrosyl residue in the fluorophoric pocket of recombinant green fluorescent protein (GFP). Exposure of purified GFP suspended in phosphate buffer to synthetic peroxynitrite in either 0.5 or 5 μM steps resulted in progressively increased 3-nitrotyrosyl immunoreactivity concomitant with disappearance of intrinsic fluorescence (IC₅₀ ≈ 20 μM). Fluorescence from an equivalent amount of GFP expressed within intact MCF-7 tumor cells was largely resistant to this bolus treatment (IC₅₀ > 250 μM). The more physiologically relevant conditions of either peroxynitrite infusion (1 μM/min) or de novo formation by simultaneous, equimolar generation of nitric oxide (NO) and superoxide (e.g., 3-morpholinosydnonimine; NONOates plus xanthine oxidase/hypoxanthine, menadione, or mitomycin C) were examined. Despite robust oxidation of dihydrorhodamine under each of these conditions, fluorescence decrease of both purified and intracellular GFP was not evident regardless of carbon dioxide presence, suggesting that oxidation and nitration are not necessarily coupled. Alternatively, both extra- and intracellular GFP fluorescence was exquisitely sensitive to nitration produced by heme-peroxidase/hydrogen peroxide-catalyzed oxidation of nitrite. Formation of nitrogen dioxide (NO₂) during the reaction between NO and the nitroxide 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide indicated that NO₂ can enter cells and alter peptide function through tyrosyl nitration. Taken together, these findings exemplified that heme-peroxidase-catalyzed formation of NO₂ may play a pivotal role in inflammatory and chronic disease settings while calling into question the significance of nitration by peroxynitrite.

The prevalence of increased 3-nitrotyrosine in both acute inflammatory events and numerous chronic diseases suggests that protein nitration may be an important component in pathophysiologic processes (1–7). However, there is disagreement regarding the predominant mechanism by which tyrosyl nitration occurs under these conditions (1–16). The reaction between superoxide (O) and nitric oxide (NO) results in formation of peroxynitrite (1–12, 17). Numerous studies have concluded that this nitrogen oxide mediates the majority of nitration, on the basis of 3-nitrotyrosine formation after bolus application of synthetically produced peroxynitrite. Nitration by this route is strongly influenced by buffer composition, in particular, the concentration of protons, transition metals, and carbon dioxide (CO₂; refs. 1, 6, 20–24). Both neutrophils and macrophages have been shown to elicit nitration reactions through myeloperoxidase (MPO) activity (15–18). Catalysis of nitrite oxidation by heme-peroxidase in the presence of hydrogen peroxide provides an alternative mechanism for formation of a species capable of nitrating tyrosine or other susceptible substances.

Proteins may become irrevocably altered by nitration of critical tyrosyl residues, for instance, located in domains containing phosphorylation, dimerizeration, or enzyme active sites. Tyr-66 is a key residue within the centrally located fluorophore of recombinant enhanced green fluorescent protein (GFP) F64L, S65T derived from Aequorea victoria jellyfish (25–27). Because addition of nitro groups to an aromatic ring quenches fluorescence, we considered that nitration of Tyr-66 would impair GFP fluorescence thereby serving as a model for the impact of nitration chemistry on proteins in real-time. Currently, the relationship between 3-nitrotyrosyl formation and protein function is based solely on end-point analysis after extensive sample processing (9). The GFP paradigm permitted a direct assessment of the relevance of either peroxynitrite or heme-peroxidase catalyzed nitration of peptide function while either present in solution or within intact cells.

Materials and Methods

GFP.

Recombinant enhanced GFP (F64L, S65T) and pEGFP-N1 vector were purchased from CLONTECH. MCF-7 human breast carcinoma cells (American Type Culture Collection) were washed twice in Hepes-buffered saline containing 6 mM glucose, then electroporated with pEGFP-N1 vector (10 μg of DNA per 5 × 10⁶ cells in 0.5 ml). Cells were plated in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% FBS (HyClone), with the addition of 400 μg/ml G418 (Life Technologies) after 48 h. Clones expressing GFP were identified by fluorescence microscopy (Zeiss axiovert 110) and isolated with cloning cylinders by using trypsin-EDTA. Stable GFP transfectants were maintained in selection media at 37°C, 5% CO₂, and 95% air. To control for possible induction of GFP expression during experimentation (<2 h), cells were pretreated with cycloheximide (10 μM for 1 h; Roche Molecular Biochemicals) and found to exhibit no differences in fluorescence relative to non-cyclohexamide-treated cells.

Peroxynitrite.

Synthetic peroxynitrite was prepared by simultaneously mixing solutions of 0.5 M NaNO₂ in 0.5 M HCl and 0.5 M H₂O₂, followed by rapid quenching in 1 M NaOH (28). The resulting basic solution was exposed to MnO₂ to remove excess H₂O₂, which was reduced to <1% per mol of peroxynitrite. After filtering, aliquots were stored at −20°C for <2 wk. Directly before use, the concentration of synthetic peroxynitrite was determined from the A at 302 nm (ɛ = 1,670 M⁻¹⋅cm⁻¹; ref. 28). Reactions commenced with dilution of 2–10 mM synthetic peroxynitrite into 0.1 M phosphate buffer (2-ml fluorometric cuvette, Spectrocell, Oreland, PA; stirring, pH 7.4, 37°C) containing diethylenetriaminepentaacetic acid (DTPA, 50 μM; Sigma) to give a final concentration of either 0.5 or 5 μM/application. Alternatively, synthetic peroxynitrite (2 mM in 1 M NaOH, 4°C) and 1 M HCl were infused from separate syringes (CMA 102 pump, North Chelmsford, MA) at a constant flow rate of 1 μl/min into buffer as above. For CO₂ experiments, 25 mM NaHCO₃ was added to sample buffer contained in a septum-sealed cuvette in an atmosphere of 5% CO₂ and 95% air. Maintenance of pH 7.4 throughout the experiment was verified.

Peroxynitrite was formed de novo by reacting O and NO at equimolar ratios. The rate of O formation during xanthine oxidase (XO; Roche)-catalyzed degradation of hypoxanthine (500 μM; Sigma) was assessed by reduction of cytochrome c (570 nm, ɛ = 21,000 M⁻¹⋅cm⁻¹; ref. 29). In the absence of XO, the steady-state concentration of NO produced during either spermine/NO or PAPA/NO degradation (generous gifts from J. A. Hrabie, National Cancer Institute, Frederick, MD; ref. 30) was determined from the electrochemical signal of an NO probe (World Precision Instruments, Sarasota, FL) controlled by a DUO18 amplifier and suspended into the cuvette under identical conditions. Signals were calibrated by using argon-purged PBS solutions of saturated NO gas (Matheson) after determination of NO concentration with 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS; 660 nm, ɛ = 12,000 M⁻¹⋅cm⁻¹; ref. 31). Rhodamine formation from dihydrorhodamine 123 (DHR, 50 μM; λ_ex/em 500/570 nm, 2.5-mm slit widths; Molecular Probes) was determined with varied concentrations of XO (0–10 μM O/min) and spermine/NO (0–200 μM). Urate formation under these conditions was monitored at 305 nm (ɛ = 8030 M⁻¹⋅cm⁻¹) rather than 295 nm to avoid interference from high levels of xanthine formed from hypoxanthine. Formation of peroxynitrite de novo within parental and transfected cells was assessed by peak intracellular DHR oxidation during coexposure to either menadione (0–100 μM; Sigma) or mitomycin-C (0–100 μM; Sigma) and spermine/NO (0–100 μM) while maintained at 37°C in a 5% CO₂ and 95% air mixture. The 3-morpholinosydnonimine (SIN-1) was purchased from Alexis (San Diego, CA).

NO₂.

Formation of NO₂ from nitrite was catalyzed by either horseradish peroxidase (HRP, 2–8 units/ml; Sigma) or MPO (0.5 unit/ml; Sigma) plus hydrogen peroxide as indicated in either PBS solution (pH 7.4) or 0.1 M phosphate buffer (pH 7.4), both containing DTPA (50 μM). The 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO, Calbiochem) was reacted with PAPA/NO as indicated to produce various amounts of either NO₂ or N₂O₃ (32).

Protein Analysis.

Extracts were made by suspending washed MCF-7 cells (10 × 10⁶) into PBS buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor mixture (Calbiochem), followed by brief sonication and centrifugation at 10,000 × g. Protein concentration of supernatant was determined by bicinchoninic acid method (Pierce). Immunoblots of GFP and 3-nitrotyrosine required 2 and 25 ng of purified GFP, respectively, or 10 μg of cell extract. After electrophoresis on either 16% (GFP) or 4–20% gradient (extract) Tris-glycine acrylamide gels (Novex), samples were transferred to poly(vinylidene difluoride) membranes (Immunolon P) and probed with either rabbit polyclonal GFP (1:10,000; CLONTECH) or 3-nitrotyrosine (0.5 μg/ml, Upstate Biotechnology, Lake Placid, NY) Abs. Bands were visualized with HRP-conjugated secondary Abs (Sigma), chemiluminescent substrate (Pierce), and x-ray film.

Spectroscopy Instrumentation.

UV-visible spectroscopy was performed with a Hewlett–Packard 8452A diode-array spectrophotometer. Fluorescence measurements were obtained on a Perkin–Elmer LS50B fluorometer equipped with water-jacketed cuvette holder that stirred and maintained the reaction solution (2 ml) at 37°C. Additional fluorescence measurements were obtained on a Perkin–Elmer HTS 7100 fluorescent plate reader (0.2 or 1 ml, nonstirring, 37°C).

Results

Nitration of GFP Decreases Fluorescence.

Purified recombinant GFP (0.5 μg/ml) suspended in 0.1 M phosphate buffer (pH 7.4) was exposed to stepwise additions of synthetic peroxynitrite as a bolus of either 0.5 or 5 μM/application. Aliquots were removed and subjected to gel electrophoresis. Western blotting with Abs directed against either 3-nitrotyrosine or GFP consistently showed a progressive increase in immunoreactivity for 3-nitrotyrosyl adducts (Fig. 1A, 0.5 μM). Concomitantly, immediate drops in GFP fluorescence (Fig. 1 B and C) were observed, which were stable between each successive application and were essentially nonreversible (<5%) with mercaptoethanol. Excitation and emission scans (200–750 nm) indicated that fluorescence decreases were not due to spectral peak shifts. Decomposed peroxynitrite, nitrite, or NaOH at equivalent exposure levels were without effect.

Figure 1.

Effect of bolus synthetic peroxynitrite application. (A) Purified recombinant GFP (1 μg) in 2 ml of 0.1 M phosphate buffer (pH 7.4, stirring, 37°C) containing DTPA (50 μM) was exposed to synthetic peroxynitrite in 0.5-μM steps. Aliquots (25 μl) were removed from the reaction vessel and were subjected to PAGE and Western blotting with Abs to either 3-nitrotyrosine or GFP. Data are representative of three trials. (B and C) Fluorescence (λ = 488_em/512_ex nm, 5-mm slit widths) of purified recombinant GFP (■, 1 μg) or an equivalent concentration of GFP expressed within MCF-7 cells (▴, 3 × 10⁵) suspended in buffer (as in A) was monitored during exposure to synthetic peroxynitrite in 0.5- (B) or 50- (C) μM steps. Shown are representative data normalized to percentage of initial values (≈200 arbitrary units, AU). Fluorescent intensity was stable between bolus additions. Changes were not observed on exposure to equivalent concentrations of synthetic peroxynitrite vehicle (data not shown).

Human breast carcinoma MCF-7 cells were stably transfected with GFP to determine the effect of intracellular localization on protein nitration. Microscopic examination revealed GFP fluorescence was nearly uniform in its distribution throughout the cell (data not shown). The fluorescence intensity from 3 × 10⁵ GFP expressing MCF-7 cells suspended in 0.1 M phosphate buffer (pH 7.4) was equivalent to that from 0.5 μg/ml purified GFP. Addition of synthetic peroxynitrite in bolus aliquots (5 μM/application) had only a marginal effect on GFP fluorescence (Fig. 1C). The concentrations of bolus synthetic peroxynitrite required to achieve a 50% decrease in fluorescence (IC₅₀) were in excess of 250 μM, conditions that resulted in substantial levels of instantaneous cell lysis. From these data, it was evident that synthetic peroxynitrite does not readily cross the intact plasma membrane to affect GFP fluorescence.

Effect of Synthetic Peroxynitrite Infusion on GFP.

The effect of a stable stream of synthetic peroxynitrite delivered into the reaction vessel by syringe pump at a flow rate of 1 μM/min was examined. This procedure resulted in a steady increase in DHR oxidation achieving an increase in product formation of ≈20-fold after 1 h (Fig. 2A). After 20 min of infusion, providing a concentration of synthetic peroxynitrite equal to the IC₅₀ from bolus (Fig. 1C), the fluorescence intensity of purified GFP suspended 0.1 M phosphate buffer (pH 7.4) was decreased only ≈5% (Fig. 2B). After 1 h of infusion (three times the bolus IC₅₀), GFP fluorescence was diminished 7 ± 5% (n = 5). The signal from control (vehicle) infusion run in parallel was decreased 4 ± 3% (n = 5). A similar decrease (5%) in GFP fluorescence was observed when synthetic peroxynitrite was delivered into buffer containing bicarbonate (25 mM, pH 7.4) while maintaining a 5% CO₂ and 95% air mixture in the vessel head space.

Figure 2.

Effect of synthetic peroxynitrite infusion. (A) Rhodamine formation from DHR (50 μM) in 2 ml of 0.1 M phosphate buffer (pH 7.4, stirring, 37°C) containing DTPA (50 μM) was monitored (λ = 500_em/570_ex nm, 2.5-mm slit widths) during exposure to either vehicle (◊) or ONOO⁻ (■) delivered at a rate of 1 μM/min. (B) Fluorescence from purified recombinant GFP (1 μg) suspended in buffer as A was monitored (λ = 488_em/512_ex nm, 5-mm slit widths) during exposure to either vehicle (◊) or synthetic peroxynitrite (ONOO⁻) delivered at a rate of 1 μM/min either with (●) or without (■) the presence of CO₂ (25 mM NaHCO₃ with 5% CO₂ and 95% air). Shown are representative data of at least five experiments, which in B are normalized to percentage of initial values (≈200 AU).

Changes in fluorescence were not observed with MCF-7 cells expressing an equivalent level of GFP during exposure to synthetic peroxynitrite in this manner regardless of CO₂ presence. To determine whether the protonation status of peroxynitrite (pK_a 6.8, ref. 24) would affect its ability to enter cells and react with GFP (9, 20, 21), intracellular GFP fluorescence was evaluated by suspending cells in either pH 6 or 8 phosphate buffer. Under these conditions, fluorescence baseline equilibrated at ≈55% of the intensity from cells suspended in pH 7.4 buffer. Subsequent infusion of synthetic peroxynitrite resulted in no further change in fluorescence (data not shown). The decrease in intensity was fully reversible on return of pH to ≈7.4 after respective addition of NaOH or HCl to the cell suspensions.

GFP Fluorescence Was Not Affected by NO/O Reaction.

Several paradigms were used to test the effect of de novo peroxynitrite formation on GFP. Over the concentration ranges used, a maximum for DHR oxidation was observed with XO- catalyzed formation of superoxide from hypoxanthine (500 μM) at a rate of 1.5 μM O/min (based on cytochrome c reduction assay) in combination with 100 μM spermine/NO (Fig. 3A). This concentration of spermine/NO produces a 7 ± 1 μM steady-state of NO during decomposition in the reaction vessel in the absence of XO. Under these conditions, urate formation was quantified to be 0.25 nM/min. Significant quenching of peroxynitrite chemistry generally requires >100-fold excess of urate indicating experimental accumulation was inconsequential. This de novo peroxynitrite preparation did not elicit changes in the fluorescence intensity of either purified (Fig. 3B) or intracellular GFP (data not shown).

Figure 3.

Effect of simultaneous exposure to NO and O. (A) Cumulative rhodamine formation from DHR (50 μM) in 200 μl of PBS solution (pH 7.4) containing hypoxanthine (500 μM) and DTPA (50 μM) in the presence of various XO-catalyzed O₂ concentrations either with (■) or without (○) spermine/NO (100 μM) was determined (λ = 500_em/550_ex nm) after 1-h incubation at 37°C. Data with XO alone (○) were subtracted from XO plus spermine/NO (■) to show de novo peroxynitrite formation. (B) Fluorescence from purified recombinant GFP (1 μg) was monitored (λ = 488_em/512_ex nm, 5-mm slit widths) under conditions that resulted in peak DHR oxidation as in A (XO to produce 1.5 μM O/min, 100 μM spermine/NO). Shown are representative data of at least three experiments normalized to percentage of initial values (≈200 AU). Urate formation under these conditions was found to be 15 μg/initial 60 min (data not shown).

GFP fluorescence levels dropped precipitously (70–80% of control) when the concentration of XO was substantially increased (≥50 μM O/min), regardless of the presence and absence of NO (PAPA/NO). GFP fluorescence was restored to near baseline values by brief aeration of the reaction vessel with 100% oxygen (data not shown). Decomposition of the compound SIN-1 results in cogeneration of NO and O that putatively combine to produce peroxynitrite (33). For each SIN-1 concentration examined (0.5, 1, and 5 mM), fluorescence from purified GFP decreased to a plateau of ≈80% of control within 30 min of exposure. These decreases also were largely reversible upon aeration of the solution with 100% oxygen (data not shown). Taken together, these data indicate that severe oxygen depletion affects GFP fluorescence in a reversible manner.

To test whether de novo formation of peroxynitrite within cells may have an impact on GFP function, cells were exposed to NO released by spermine/NO concomitant with either menadione or mitomycin C, which catalyze formation of O through the intermediacy of semiquinone radicals. Peak de novo formation of peroxynitrite within cells was again assessed by oxidation of DHR. A plateau value for intracellular DHR oxidation in the presence of constant spermine/NO (10 μM) was observed with 100 μM quinone (menadione or mitomycin C) within 30–60 min of exposure (data not shown). At this level of menadione or mitomycin C (100 μM), higher concentrations of spermine/NO (>10 μM) decreased intracellular DHR oxidation. These patterns were analogous in parental and GFP-transfected MCF-7 cells, ruling out potential contribution of spectral overlap between GFP and DHR/rhodamine. Under each of these conditions, GFP fluorescence within cells was not decreased regardless of the presence of DHR. Therefore, neither extra (XO/NO) nor intracellular exposure to NO and O results in an appreciable decrease in GFP fluorescence.

Heme-Peroxidase-Catalyzed Oxidation of Nitrite Results in GFP Nitration.

The ability of heme-containing peroxidases to catalyze nitration of GFP was tested by using HRP and MPO. Exposure to hydrogen peroxide (200 μM) caused a small decrease (<20%) in purified GFP fluorescence, which was abated by the metal chelator DTPA regardless of HRP presence (2 units/ml). Addition of nitrite resulted in a progressive decrease in GFP fluorescence (IC₅₀ ≈ 25 μM nitrite) that inversely correlated with increased 3-nitrotyrosine immunoreactivity (data not shown). A substantially greater IC₅₀ (400 μM nitrite) for GFP fluorescence within MCF-7 cells was observed. Further addition of either hydrogen peroxide or nitrite at this point had no effect; however, subsequent addition of HRP (2 units/ml) resulted in complete loss of fluorescence over a second 1-h period (data not shown). When hydrogen peroxide was lowered to levels anticipated during inflammation (2 μM), the maximum fluorescence decrease of purified GFP was ≈50% at ≥100 μM nitrite and HRP (2 units/ml).

MPO is a principal heme-peroxidase in humans (16, 18). Interestingly, MPO catalyzed a substantial decrease in GFP fluorescence in PBS solution in the absence of either substrate. This decrease is putatively due to chlorination (155 mM Cl⁻). In chloride-free phosphate buffer, addition of nitrite was required to abate fluorescence (Fig. 4). Relative to HRP, the intracellular GFP IC₅₀ concentrations of MPO, hydrogen peroxide, and nitrite were lower (Fig. 4B).

Figure 4.

Effect of nitrite oxidation by MPO/peroxide. Fluorescence from either purified recombinant GFP (1 μg) (A), or an equivalent concentration of GFP expressed within MCF-7 cells (▴, 3 × 10⁵) (B) suspended in 2 ml of 0.1 M phosphate buffer (pH 7.4, stirring, 37°C) containing DTPA (50 μM) was monitored (λ = 488_em/512_ex nm, 5-mm slit widths). Shown are representative data normalized to percentage of initial values (≈200 AU) in the presence of MPO (0.5 unit/ml) and either H₂O₂ (2 μM) and nitrite as shown (μM) (A) or H₂O₂ and nitrite as shown (μM) (B).

GFP Nitration Can Be Ascribed to NO₂ Formation.

To test the hypothesis that NO₂ was the salient species mediating nitration of GFP, nitrogen dioxide was generated by oxygen transfer from PTIO to NO. Although PTIO absorbs light in the 500 nm region, 25 μM was found to only minimally interfere with GFP fluorescence (78% of control). An IC₅₀ for GFP fluorescence was observed with 5–50 μM PAPA/NO (which releases two NO molecules per molecule) in the presence of 25 μM PTIO (Fig. 5A). The effect of this reaction on GFP fluorescence was progressively diminished as the concentration of PAPA/NO was further increased. A similar U-shaped pattern was evident in experiments conducted with GFP within MCF-7 transfectants (Fig. 5B). Because of the enhanced resistance of intracellular GFP, a larger quantity of PTIO (800 μM) was required to achieve an IC₅₀ for fluorescence, which obscured analysis of GFP in real-time. Therefore, PTIO was removed by centrifugation and washing before end-point analysis.

Figure 5.

Effect of NO₂. (A) Fluorescence from of purified GFP (1 μg) in 2 ml of PBS solution (pH 7.4, stirring, 37°C) containing DTPA (50 μM) and PTIO (25 μM) was monitored (λ = 488_em/512_ex nm, 5-mm slit widths) 30 min after addition of PAPA/NO (μM) as indicated. Changes were not observed with PAPA/NO in the absence of PTIO (data not shown). Shown are representative data normalized to percentage of initial values (≈150 AU). (B) MCF-7 cells (3 × 10⁵) expressing GFP at equivalent levels were suspended in 2 ml of PBS solution (stirring, pH 7.4, 37°C) containing DTPA (50 μM), PTIO (800 μM), and PAPA/NO as indicated. After 30 min, cells were centrifuged and washed, and fluorescence was read (λ = 488_em/512_ex nm, 5-mm slit widths).

Discussion

These data have demonstrated that GFP can be a sensitive tool to study nitration, which gives an unprecedented real time dosimeter of nitration mechanisms within intact cells. Analogous to an enzyme containing a key tyrosyl residue in a protected active site, Tyr-66 of the chromophore is symmetrically located within an 11-stranded β-barrel GFP structure (26). After spontaneous cyclization of residues 65 and 67, Tyr-66 undergoes autoxidation yielding an intrinsically fluorescent peptide (25). We predicted that introduction of an electron withdrawing 3-nitrotyrosyl adduct at this site may quench fluorescence by providing a nonradiative decay mechanism through vibronic coupling. Multiple GFP residues may be susceptible to nitration. The side chains of Tyr-145 and His-148 in particular are situated ≈4 Å from Tyr-66 (26). However, nitration often occurs at selective tyrosyl residues possibly biased by proximity to glycyl turns and charged residues (e.g., glutamyl; ref. 34), which are qualities manifest in Tyr-66 of GFP (26). Previous studies have shown that fluorescence of GFP-related peptides can be modified by anions including chloride (35). The fluorescence decrease catalyzed by MPO in PBS solution without nitrite is consistent with a chlorination process, suggesting that attack from electron deficient species on Tyr-66 (or other aromatic residues) may be a generalized pathway for quenching emission. In contrast to the collisional quenching effect of oxygen on most fluorophores, fluorescence from mature GFP inexplicably requires oxygen (25). This was evident in assays with high concentrations of XO or SIN-1 where oxygen was depleted by reduction to O. Similar to pH effects, this behavior was fully reversible. In contrast, fluorescence decreases induced by nitration and putatively by chlorination were not recoverable. These data indicate that redox status and oxygen tension within cells or tissue may strongly influence GFP fluorescence, which is an important consideration for reporter, trafficking and biosensor applications.

Nitration mechanisms are often examined by reacting samples with a bolus quantity of synthetic peroxynitrite. This procedure was effective at nitrating purified GFP with an IC₅₀ for fluorescence at ≈25 μM when applied in 5-μM steps. In contrast, after exposure to an equimolar amount of synthetic peroxynitrite infused at a rate of 1 μM/min, GFP fluorescence was still 98% of control values. Previous studies have shown that the poor efficiency of tyrosine nitration by synthetic peroxynitrite by using the bolus format was enhanced by CO₂ through formation of a nitosoperoxocarboxylate (ONOOCO) intermediate (20, 21). However, the presence of CO₂ had a negligible effect on GFP fluorescence during peroxynitrite infusion (98% of control values at 60 min, Fig. 2B).

These findings show that the ability of peroxynitrite to affect a change in peptide function, fluorescence in the case of GFP, through nitration was dependent on en mass reaction at sufficient concentration (bolus) as opposed to an accrual of reactions over time with infusion or de novo formation. In addition to nitration, numerous processes exist for peroxynitrite consumption within the intracellular milieu. Fluorescence from GFP inside MCF-7 cells was largely resistant to synthetic peroxynitrite until substantial cell lysis occurred (>250 μM bolus). Under biological conditions, peroxynitrite would be formed de novo from the reaction between NO and O. Accumulation and release of peroxynitrite as a bolus cannot occur because of its extremely reactive nature at physiologic pH. Therefore, sufficient quantities of both NO and O would have to be present for significant nitration from peroxynitrite to occur in vivo. Moreover, these reactants would have to be maintained at near 1:1 stoichiometry to avoid secondary reactions between peroxynitrite intermediates and either NO or O (32, 36–41), which form species incapable of nitration (e.g., dinitrogen trioxide, N₂O₃; peroxynitrate, respectively). Even under optimal experimental conditions, fluorescence from both purified and intracellular GFP was unaffected by balanced exposure to NO and O produced simultaneously by a variety of means (SIN-1; XO/HX + NO, menadione + NO, mitomycin C + NO) at both physiologic and supraphysiologic levels. These findings are consistent with studies that have shown nitration of free tyrosine by bolus synthetic peroxynitrite differs sharply from that elicited by NO + O systems (10, 11).

Nitration reactions differ from oxidation in that a covalent bond must be formed. Two-electron oxidation of DHR by peroxynitrite was used to determine 1:1 stoichiometry for peak de novo peroxynitrite formation in NO + O systems in addition to serving as a positive control in syringe pump experiments. This study clearly shows that oxidation of DHR cannot be used as evidence for either 3-nitrotyrosine formation or origin. Indeed, these data suggest that the major role for peroxynitrite, provided that ideal conditions for de novo formation exist, may be to oxidize rather than nitrate biological targets. In addition, peroxynitrite formation also can contribute intermediates that subsequently react with NO to form the nitrosating species N₂O₃ (refs. 3 and 37 and unpublished observations).

Our findings support a predominant role for heme-peroxidase catalyzed oxidation of nitrite in formation of 3-nitrotyrosyl adducts relative to peroxynitrite (15–18). Nitration-induced decreases of both extra- and intracellular GFP fluorescence catalyzed by either HRP or MPO were evident with hydrogen peroxide and nitrite at concentrations relevant to pathophysiological conditions. Hydrogen peroxide is required to convert the resting-state, ferric heme of peroxidases to the hypervalent compound I, which can directly oxidize nitrite by one electron to give NO₂ and compound II. Subsequently, an additional nitrite molecule will reduce compound II by one electron to regenerate the resting state and produce a second NO₂. Nitration may occur under these circumstances either by direct addition of NO₂ to the aromatic ring or by hydrogen atom abstraction followed by addition of a second NO₂.

Nitration of biological compounds by NO₂ has been viewed as unlikely (3, 9, 12–14). In the peroxidase-catalyzed mechanism, it is often presumed that hydrogen peroxide must also oxidize tyrosine to a radical intermediate, which then can combine with NO₂ (e.g., ref. 14). We tested whether exogenous hydrogen peroxide was obligatory for nitration by NO₂ by exposing GFP to incremental concentrations of NO₂ derived via oxygen transfer from PTIO to NO. Decrease of fluorescence and formation of 3-nitrotrysyl GFP both in solution and within cells under these conditions support a mechanism for direct electrophilic addition of NO₂. Tyr-66 is a site of particular interest because it putatively exists as an anionic phenoxide (26), which would be highly susceptible to NO₂ attack (39). The additional π^+⋅ electron may potentially either reduce the dehydrotyrosyl Cα–Cβ bond or reduce oxygen, generating superoxide and ultimately hydrogen peroxide. The U-shaped curve for decline in GFP fluorescence elicited by NO and PTIO is noteworthy (Fig. 5). Nitration chemistry steadily waned as the concentration of NO exceeded that of PTIO. This was indicative of a transition from nitration to nitrosation chemistry through formation of N₂O₃ because of the reaction of NO₂ with NO (32) rather than GFP or other cellular constituents. Although tyrosyl residues can be O-nitrosated at the hydroxyl position, GFP fluorescence was not affected by N₂O₃ illustrative of the contrast between targets and functional outcomes under nitrative vs. nitrosative conditions (32, 40, 41). Similar to NO, both N₂O₃ and NO₂ should be viewed as diffusible species, which are capable of entering cells to selectively modify susceptible targets, whether in host cells or toward combating pathogenic microbes. Hydrolysis of N₂O₃ results in formation of nitrite, which can potentially participate in nitration chemistry if a suitable environment of heme-peroxidase and hydrogen peroxide exists. In general, nitrosation may precede nitration after leukocytic expression of inducible NO synthase in vivo (42, 43).

A principal role for NO₂ in the chemistry of NO after its production in vivo emerges. We have shown that NO₂ is an intermediate of NO autoxidation in cells only within hydrophobic domains, which results in generation of more potent nitrosating species relative to the aqueous process (32). Herein, the impact of peroxynitrite on GFP was evident only when delivered under circumstances that are highly unlikely in vivo (synthetic, bolus). De novo formation and decomposition of peroxynitrite likely generates insufficient NO₂ to conduct nitration chemistry when balanced with competing reactions. In contrast, these findings strongly support the viewpoint that heme-peroxidases such as MPO may manifest 3-nitrotyrosyl modifications during inflammation and chronic disease by catalyzing relatively copious formation of NO₂ from nitrite in the presence of hydrogen peroxide. Moreover, GFP fluorescence provides an unique means to monitor the ability of NO₂ to enter cells and affect protein function via nitration.

Abbreviations

MPO

myeloperoxidase

GFP

green fluorescent protein

XO

xanthine oxidase

HRP

horseradish peroxidase

PTIO

2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide

DHR

dihydrorhodamine 123

DTPA

diethylenetriaminepentaacetic acid

SIN-1

3-morpholinosydnonimine

AU

arbitrary units