The antioxidant mechanism of nitroxide TEMPO: scavenging with glutathionyl radicals

Abstract

A rhodamine-nitroxide probe (R-NO˙), combining rhodamine fluorophore with a 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO) receptor unit was introduced to probe glutathionyl radicals (GS˙) with high sensitivity and selectivity. The R-NO˙ probe could effectively scavenge GS˙ radicals with fluorescence enhancement since the nitroxide group restored the fluorescence properties. In this work, horseradish peroxidase (HRP)-catalyzed and metal-catalyzed oxidation systems were selected as the model of simulating the generation of GS˙, and we found that the metal-catalyzed system had the same experimental results with the HRP-catalyzed system, which provided a new approach to demonstrate the strong oxidant ability of the hydroxyl radical (˙OH) to initiate toxic GS˙. Furthermore, we confirmed that the production of GS˙ abided by a radical-initiated peroxidation mechanism of GSH with the mass spectrometry (MS) analysis and fluorescence spectroscopy. By using combined high-performance liquid chromatography (HPLC) detection and MS analysis, we also demonstrated that the R-NO˙ was converted into fluorescent secondary amine derivative (R-NH). The application of the probe in biological system was explored to monitor GS˙ in HL-60 cells and secondary amine fluorescence was observed upon stimulation by hydrogen peroxide and phenol. Development of fluorescence was prevented via preincubation with the thiol-blocking reagent N-ethylmaleimide (NEM).

---

Introduction

The excess oxygen- and nitrogen-centered radicals generated in biological organisms can lead to the potential damage of vital cellular components such as lipids, proteins, and nucleic acids,^1,2 which can produce secondary reactive species and then induce a series of cellular response or severely endanger cell health and viability, ultimately leading to irreparable cell damage.³

Generally, in vivo, glutathione (GSH) is one of the most abundant antioxidants with a concentration in the millimolar range.⁴ Although GSH is effective to scavenge various deleterious radicals such as carbon centered radicals, hydroxyl radical (˙OH), peroxyl radical (RO₂˙) as well as peroxynitrite (ONOO⁻),^5–7 it can potentially turn into glutathionyl radicals (GS˙),⁸ which are viewed as toxic species for their strong oxidant ability and these radicals can induce cell damage by reacting with protein thiols as well as with unsaturated acyl chains of phospholipids in the membrane.⁹ Therefore, it is of vital importance to block GS˙ from initiating further oxidation reactions and damaging other cellular components.

Stable nitroxide radicals, such as 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO), possess important antioxidant activity due to their ability of scavenging reactive radicals.^10–14 To date, most of the attentions have been paid to the abilities of nitroxides to degrade superoxide and peroxide,¹⁵ inhibit Fenton reactions,¹⁶ and undergo radical–radical recombination,¹⁷ especially for carbon centered radicals. However, few studies have focused on the antioxidant effect of nitroxides for secondary radicals such as GS˙. Borisenko et al.¹⁸ employed a fluorescent probe containing nitroxide and acridine moieties, 4-((9-acridinecarbonyl) amino)-2,2,6,6-tetramethylpi-peridin-1-oxyl (Ac-TEMPO) to scavenge GS˙, and they confirmed that the main product of the reaction between nitroxides and GS˙ was the corresponding secondary amine.^18,19 Previous work also demonstrated that the reaction between GS˙ and nitroxides had low energy barriers, being radical–radical reactions, and had rate constants near the diffusion limit (i.e., >10⁸ dm³ mol⁻¹ s⁻¹).²⁰ Since the TEMPO derivative has been used in the interception of GS˙, it is meaningful to make clear the interactive effect of TEMPO derivative with GS˙. Based on this point, we introduced a rhodamine-nitroxide probe, combining rhodamine fluorophore with TEMPO receptor unit covalently (R-NO˙, Fig. 1A), to probe GS˙ with high sensitivity and selectivity. In addition, the rhodamine-nitroxide probe has excellent photophysical and photochemical properties in aqueous solution and can be excitable at a long wavelength (556 nm), so it does not cause severe cell damage and is also free from interference by autofluorescence of biological molecules.²¹ Furthermore, this method can indirectly indicate the lesion of biomolecules by quantifying the product of R-NO˙ derivative as well. Herein, horseradish peroxidase (HRP) was used as peroxidase in peroxidase-catalyzed reactions in vitro. The oxidation of phenolic compounds by HRP/H₂O₂ generated phenoxyl radicals, and then phenoxyl radicals recycled back to phenol through the oxidation of GSH to GS˙.^18,22 Compared with the previous work, metal-catalyzed oxidation of GSH as a model of yielding GS˙ was also introduced in the present work. Since ˙OH was capable of abstracting hydrogen atom from GSH,²³ we inferred the proposed mechanism of the interaction between R-NO˙ with GS˙ in metal-catalyzed system (Fig. 1B). Additionally, we also found that the metal-catalyzed system had the same experimental results with HRP-catalyzed system, which further confirmed the generation of GS˙ by ˙OH, providing a new approach to demonstrate the strong oxidant ability of ˙OH to initiate toxic GS˙. Furthermore, by using combined high-performance liquid chromatography (HPLC) detection and MS analysis, we confirmed that the production of GS˙ abided by a radical-initiated peroxidation mechanism, and we also demonstrated that the R-NO˙ was converted into fluorescent secondary amine derivative (R-NH) as reported in the previous work (Fig. 1A).^18,24 Finally, this probe was applied in biological system to track the GS˙ radicals in HL-60 cells and fluorescence enhancement was observed upon stimulation by H₂O₂ and phenol.

Fig. 1 (A) The structures of rhodamine-nitroxide probe (a) and secondary amine derivative (b) and the interaction between R-NO˙ with GS˙ radicals; (B) the inferred reaction processes among GSH, R-NO˙ and ˙OH.

Experimental section

2.1. Reagents and chemicals

The probe was synthesized following a previous route established by our laboratory and the detailed procedure of R-NO˙ was described in our previous published paper.²⁵ 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (4-OH-TEMPO), rhodamine B, cysteine and xanthine oxidase were purchased from J&K Scientific Ltd (Beijing, China). Xanthine was purchased from Sangon Biotech Co. Ltd (Shanghai, China). Glutathione reduced was purchased from Roche. Horseradish peroxidase (HRP) and N-ethylmaleimide (NEM) were purchased from Aladdin and TCI. H₂O₂ (30%), sodium hypochlorite, ferrous sulfate, ethylenediamine tetraacetic acid disodium salt (EDTA) and phenol were purchased from Xilong Chemical Co. Ltd (Sichuan, China). HL-60 cells were purchased from the tumor cell bank of Chinese academy of Medical Sciences (Beijing, China). Cell culture media were purchased from Thermo Scientific HyClone. All chemicals used were of analytical grade or chromatographical purity without further purification. Sodium phosphate buffers were prepared using ultra-pure water from a Millipore Direct-Q purification system (18.2 MΩ). All experiments were carried out in 10 mM sodium phosphate buffer solution at pH 7.4 and at room temperature.

2.2. Fluorescence spectrometry analysis

Fluorescence spectra were measured on a Perkin-Elmer LS 55 scanning spectrofluorometer equipped with a Xenon flash lamp. The fluorescence intensities were recorded with excitation and emission wavelengths of 556 nm and 590 nm, and excitation and emission slit widths were 15 nm and 3 nm, respectively.

2.3. UV-visible spectrometry analysis

The concentration of H₂O₂ was determined by its absorbance at 240 nm (ε = 43.6 M⁻¹ cm⁻¹) on a Perkin-Elmer Lambda-35 UV-visible double beam scanning spectrophotometer, and the NaClO stock solution was determined by its absorbance at 292 nm (ε = 350 M⁻¹ cm⁻¹).

2.4. HPLC and MS analysis

HPLC analysis was performed on an Agilent 1100 series equipped with UV-Vis and fluorescence detectors. HPLC conditions were as follows: LICHROM C18 column (5 μm, 4.6 × 250 mm), the mobile phase consisted of a solvent A (75 mM ammonium acetate, 50 mM citric acid, 5% acetonitrile, pH 3.5)²⁶ and a solvent B (acetonitrile) (V/V 20/80) was run in the isocratic mode. The flow rate was 1.0 mL min⁻¹, and the injection volume was 20 μL. The eluent was monitored by UV-Vis detector at 547 nm and by fluorescence detector (FLD) using an excitation wavelength of 556 nm and emission wavelength of 590 nm.

The MS analysis was carried out on an Agilent 1100 series LC/MSD Trap. The mass spectrometer was operated in positive ion electrospray mode.

2.5. Fluorescence imaging

HL-60 cells were cultured in the Roswell Park Memorial Institute (RPMI) medium supplemented with 10% (v/v) fetal calf serum and incubated at 37 °C under 5% CO₂ atmosphere. Cells were seeded into culture dishes with appropriate density and cultured for 24 h, and then the well-grown cells were selected and treated accordingly. Cells were preincubated with the probe (2 μM) for 10 min. Phenol (10 μM) and H₂O₂ (2 μM) were then added for another 10 min incubation. To block reactions of low molecular weight thiols, cells were preincubated with NEM for 10 min, which preferentially depletes GSH and free cysteine. After all the treatments, cells were centrifuged at 1000 g for 5 min and resuspended in PBS. Fluorescence and bright field images were acquired using an Olympus BX51 fluorescence microscope with 10× eye lens and 20× objective lens.

Results and discussion

3.1. Glutathionyl radicals formed by HRP-catalyzed oxidation of GSH

GS˙ can be generated in the presence of H₂O₂, HRP, GSH and phenol. In this system, phenol was oxidized by HRP/H₂O₂, and transformed to the corresponding phenoxyl radicals, which were then quenched by GSH, resulting in the formation of GS˙.^18,22 The R-NO˙ probe can be changed to its diamagnetic derivative via reacting with GS˙ and the fluorescence intensity was significantly enhanced (Fig. 2a). In addition, the R-NO˙ probe showed a fast response toward GS˙ based on the kinetic study. After the generation of GS˙, a marked fluorescence enhancement was observed within 2 min, and leveled off in about 6 min thereafter (ESI Fig. S1,† black line), which indicated that the probe had a fast response to GS˙. Meanwhile, the fluorescence intensity of the R-NO˙ probe remained unchanged for up to 12 min (ESI Fig. S1,† red line), which indicated that the probe had good photostability. For control, negligible changes in fluorescence were observed in the absence of either GSH, HRP, H₂O₂ or phenol (Fig. 2a), which demonstrated that each of them was essential to the catalytic recycling to produce GS˙, so we can draw the conclusion that the fluorescence enhancement was exactly due to the reaction between GS˙ and R-NO˙, and the probe hardly reacted directly with the phenoxyl radical. In order to confirm the role of GS˙, the thiol-blocking reagent NEM was added into the reaction mixture before initiating the reaction. Development of fluorescence intensity was not observed at that time (Fig. 2a), which also fitted well with our conjecture.

Fig. 2 Effects of GSH, H₂O₂, HRP and phenol on the fluorescence production with incubation conditions: (a) 5 μM R-NO˙, 20 μM phenol, 5 μM GSH, 5 μM H₂O₂ and 0.0625 U mL⁻¹ HRP; same as above system, but minus GSH; minus HRP; minus H₂O₂; minus phenol; fluorescence intensity were measured in the presence of combinations of HRP/phenol/GSH/H₂O₂ and 50 μM NEM as well; (b) 20 μM phenol, 5 μM H₂O₂, final [GSH]: 0–5 μM; (c) 5 μM GSH, 20 μM phenol, final [H₂O₂]: 0–5 μM; (d) 5 μM GSH, 5 μM H₂O₂, final [phenol]: 0–20 μM. R-NO˙ (5 μM) and HRP (0.0625 U mL⁻¹) were used in all experiments. The F₀ value was the intensity of R-NO˙. All experiments were carried out in 10 mM sodium phosphate buffer solution at pH 7.4 and at room temperature for 10 min. Every data point was the mean of three measurements.

The same phenomenon occurred for other phenolic compounds such as L-tyrosine. While no fluorescence enhancement was observed for L-tyrosine in the absence of GSH (ESI Fig. S2†), which further confirmed the essential role of phenolic compounds associated with GSH oxidation. Another thiol compound—cysteine, was also investigated and research for cysteine had the same results as GSH (ESI Fig. S3†).

The effects of GSH, H₂O₂ and phenol concentration on fluorescence intensity were also investigated. As can be seen from Fig. 2b, the fluorescence intensity of the probe enhanced with the increase of the GSH concentration, and there was a good linearity between the relative fluorescence intensity (F − F₀) and GSH concentrations in the range of 0-3.75 μM (F − F₀ = −2.811 + 100.64 × [GSH] μM, R² = 0.9983, ESI Fig. S4†). The detection limit, which is calculated to be three standard derivations of the background signal, was estimated to be about 9.3 nM. The concentration of H₂O₂ influenced the total yield of fluorescence as well (Fig. 2c). However, unlike H₂O₂ and GSH, changes in phenol concentration did not affect total reaction yield (Fig. 2d). The reaction was sufficient to convert R-NO˙ to its fluorescent derivative even with low phenol concentrations (1 μM), but with a long reaction time (ESI Fig. S5†), which demonstrated the fact that phenol was recycled in this system and peroxidase activity was essential.

3.2. Glutathionyl radicals formed by metal-catalyzed oxidation of GSH

˙OH that generated via Fenton reaction, was used as a model of yielding GS˙ in the presence of GSH. When the R-NO˙ probe was added in ˙OH—produced by Fenton reaction, in the presence of GSH, rapid increase in fluorescence was observed (Fig. 3a). While the R-NO˙ was alone in ˙OH or GSH, no fluorescence enhancement or only weak fluorescence increase was observed (Fig. 3a). We also demonstrated that R-NO˙ could not directly react with ˙OH and GSH by mass spectrometry, because the main molecular ion peak was still R-NO˙ (ESI Fig. S6†). Moreover, in the absence of Fe²⁺ or H₂O₂, the molecular ion peak of secondary amine (m/z 582) could hardly be observed (ESI Fig. S7†), which further confirmed the significant role of ˙OH in the production of GS˙.

Fig. 3 Factors affecting the fluorescence intensity in the metal-catalyzed GSH oxidation with incubation conditions: (a) 5 μM R-NO˙, 12.5 μM GSH, 20 μM Fe²⁺ ([Fe²⁺]/[H₂O₂] = 1 : 10); same as above system, but minus GSH; minus ˙OH; minus H₂O₂; minus Fe²⁺; fluorescence intensity were measured in the presence of combinations of GSH/˙OH and 50 μM NEM as well; (b) 5 μM R-NO˙, 12.5 μM GSH, final [Fe²⁺]: 0–25 μM; (c) 5 μM R-NO˙, 20 μM Fe²⁺-EDTA ([Fe²⁺]/[H₂O₂] = 1 : 10), final [GSH]: 0–15 μM. The F₀ value was the intensity of R-NO˙. All experiments were carried out in 10 mM sodium phosphate buffer solution at pH 7.4 and at room temperature for 1 h. Every data point was the mean of three measurements.

As expected, when NEM existed, no increase in fluorescence intensity was observed, which further confirmed the fact that enhanced fluorescence was derived from the role of GS˙. Therefore, we deduced that the metal-catalyzed oxidation of GSH was involved in the formation of ˙OH by means of the Fenton reaction, because ˙OH is viewed as the most aggressive one among the various oxygen species,^27,28 and it can oxidize GSH to GS˙. We also inferred that GS˙ was formed via H-abstraction by ˙OH, for it has proved that S–H bond has the lowest single bond energy compared with C–H, N–H, O–H bond energies, thus being the most reactive type of site toward ˙OH via H-abstraction,²³ and then the R-NO˙ can rapidly detoxify GS˙ to generate corresponding derivative with fluorescence enhancement.

In addition, the effects of GSH and ˙OH concentration on the fluorescence production were investigated. The fluorescence intensity enhanced with increasing the amount of ˙OH, which was direct proportional to the concentration of Fe²⁺ (Fig. 3b). The relationship between fluorescence intensity and GSH concentration was shown in Fig. 3c. With the increase of GSH, the generation of GS˙ also increased.

3.3. Test for probe selectivity

The complexity of the intracellular system presents a great challenge for biosensors not only in sensitivity but more importantly in selectivity.²⁹ The selectivity experiments were carried out by evaluating the interference of other reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), hydroxyl radical (˙OH), hypochlorite (ClO⁻), superoxide anion (O₂˙⁻) and singlet oxygen (¹O₂), which may coexist in the living system. As shown in Fig. 4, the changes in fluorescence intensity before and after the addition of other ROS were not increased, indicating that fluorescence enhancement occurred only upon reaction with GS˙. Therefore, we made the conclusion that these potential ROS interferences showed negligible effects on the signal for GS˙ sensing and the probe exhibited excellent selectivity toward GS˙ among various ROS.

Fig. 4 Fluorescence intensity of the probe (5 μM) toward various ROS (0.5 mM), ˙OH was produced by Fenton reaction ([Fe²⁺] 0.5 mM, [Fe²⁺]/[H₂O₂] = 1 : 10), O₂˙⁻ was generated by the enzymatic reaction of xanthine/xanthine oxidase, and ¹O₂ was prepared by the reaction of NaClO with H₂O₂ ([ClO⁻]/[H₂O₂] = 10 : 1), GS˙ was formed in the HRP-catalyzed oxidation of GSH. Every data point was the mean of three measurements.

3.4. Identification of the R-NO˙ metabolites by HPLC and MS

R-NO˙ and its metabolites were subjected to the HPLC analysis with FLD (Ex 556 nm, Em 590 nm) and UV-Vis detector (absorbance wavelength at 547 nm), respectively. Fig. 5 showed that R-NO˙ was eluted with a retention time (t_R) of 7.7 min (peak 1) and t_R for the major fluorescent product of both HRP-catalyzed and metal-catalyzed oxidation of GSH was 8.7 min (peak 2). In addition, compared with R-NO˙ chromatogram, the total fluorescence response of reaction mixture increased significantly. The structure of the R-NO˙ metabolite was confirmed by MS analysis (ESI Fig. S8†). It demonstrated that R-NO˙ (m/z 597) was predominantly converted to the compound with m/z 582, which is consistent with [M − O + H]⁺ of corresponding rhodamine-nitroxide secondary amine derivative.

Fig. 5 HPLC detection of R-NO˙ and its derivative (a and c were HPLC-UV/VIS detection; b and d were HPLC-FLD detection). Sample preparation: (a and b) reaction mixture, 5 μM R-NO˙, 20 μM phenol, 5 μM GSH, 5 μM H₂O₂ and 0.0625 U mL⁻¹ HRP, incubation time 10 min; (c and d) R-NO˙ standard (5 μM) only. Peak 1 retention time, 7.7 min; peak 2 retention time, 8.7 min (the metal-catalyzed oxidation of GSH had the same result); FLD, Ex 556 nm, Em 590 nm; UV-Vis, absorbance wavelength at 547 nm.

That is to say, secondary amine was observed since R-NO˙ scavenged GS˙. It was suggested that the GS˙ radicals formed via HRP-catalyzed or metal-catalyzed oxidation of GSH were immediately scavenged by R-NO˙ to give an unstable intermediate adduct, which would be decomposed owing to its weak N–O–S bond, thus leading to the formation of rhodamine-nitroxide secondary amine derivative (Fig. 1A).²⁴

3.5. Detection of GS˙ in HL-60 cells

To develop a selective and sensitive fluorescent probe is of great demanded for the quantitative analysis of intracellular species generation in complicated biological systems, especially, for developing in situ intracellular species fluorescent probe, which could provide real-time physiological information within live cells responding to various external stimuli.³⁰ As demonstrated above, the rhodamine-nitroxide probe showed high selectivity and sensitivity toward GS˙, which provided a reliable platform for bioimaging and biosening of GS˙ in living cells. The application of the rhodamine-nitroxide probe in biological system was explored to track the production of GS˙ in HL-60 cells using fluorescence microscope. HL-60 cells preincubated with probe displayed sufficiently weak background fluorescence (Fig. 6a). Addition of phenol or H₂O₂ to probe-loaded cells induced fluorescence slightly (Fig. 6b and c); in contrast, addition of phenol and H₂O₂ together to HL-60 cells caused a striking production of fluorescence (Fig. 6d). To confirm that the fluorescent recovery of the cells was caused by GS˙, the probe-loaded cells were first preincubated with NEM for 10 min, and then with phenol and H₂O₂ for another 10 min, as a result the fluorescence intensity was obviously suppressed (Fig. 6e). Bright-field measurements indicated that the cells were viable throughout the imaging experiments (Fig. 6f–j).

Fig. 6 Fluorescence imaging of HL-60 cells under different conditions with R-NO˙: (a) HL-60 cells incubated with 2 μM R-NO˙ for 10 min; (b) and (c) HL-60 cells incubated with 2 μM probe for 10 min and then treated with phenol (10 μM) and H₂O₂ (2 μM) for another 10 min, respectively; (d) probe-loaded cells incubated with phenol and H₂O₂ together for 10 min; (e) probe-loaded cells preincubated with NEM for 10 min and then addition of phenol and H₂O₂ together for 10 min; (f)–(j) were the corresponding bright field images.

Conclusions

The rhodamine-nitroxide probe was employed to probe GS˙ in model systems and in living cells. In both the HRP-catalyzed and metal-catalyzed oxidation of GSH, R-NO˙ significantly scavenged GS˙ and generated derivative with strong fluorescence. Consequently, we can make a conclusion that nitroxide TEMPO combined with GSH can act as efficient scavengers against free radical-initiated peroxidation. Moreover, this probe would help for elucidating the reaction mechanisms of nitroxides with GS˙ and giving a pathway to detoxify GS˙. The method can also be applied to monitor quantitatively the lesion of free radicals on GSH by detecting the product of secondary amine, which is under active research in our laboratory.

Acknowledgements

This work was supported by the Around five top priorities program of “One-Three-Five” Strategic Planning of Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, and the National Science & Technology Major Project of China (no. 2011ZX05011).

Notes and references

1. M. Valko, D. Leibfritz, J. Moncol, M. T. D. Cronin, M. Mazur and J. Telser, Int. J. Biochem. Cell Biol., 2007, 39, 44–84.
2. O. M. Lardinois, D. A. Maltby, K. F. Medzihradszky, P. R. O. de Montellano, K. B. Tomer, R. P. Mason and L. J. Deterding, Chem. Res. Toxicol., 2009, 22, 1034–1049.
3. I. Dalle-Donne, R. Rossi, R. Colombo, D. Giustarini and A. Milzani, Clin. Chem., 2006, 52, 601–623.
4. F. Q. Schafer and G. R. Buettner, Free Radical Biol. Med., 2001, 30, 1191–1212.
5. B. Halliwell, in eLS, John Wiley & Sons, Ltd, 2001,  DOI:10.1038/npg.els.0003913.
6. Y.-Z. Fang, S. Yang and G. Wu, Nutrition, 2002, 18, 872–879.
7. H. Sies, Free Radical Biol. Med., 1999, 27, 916–921.
8. C. Schöneich, Chem. Res. Toxicol., 2008, 21, 1175–1179.
9. C. Schöneich, U. Dillinger, F. von Bruchhausen and K.-D. Asmus, Arch. Biochem. Biophys., 1992, 292, 456–467.
10. S. Goldstein and A. Samuni, J. Phys. Chem. A, 2007, 111, 1066–1072.
11. D. I. Pattison, M. Lam, S. S. Shinde, R. F. Anderson and M. J. Davies, Free Radical Biol. Med., 2012, 53, 1664–1674.
12. P. J. Wright and A. M. English, J. Am. Chem. Soc., 2003, 125, 8655–8665.
13. O. Augusto, M. G. Bonini and D. Trindade, Free Radical Biol. Med., 2004, 36, 1224–1232.
14. Y. Miura, H. Utsumi and A. Hamada, Arch. Biochem. Biophys., 1993, 300, 148–156.
15. M. C. Krishna, A. Russo, J. B. Mitchell, S. Goldstein, H. Dafni and A. Samuni, J. Biol. Chem., 1996, 271, 26026–26031.
16. J. B. Mitchell, A. Samuni, M. C. Krishna, W. G. DeGraff, M. S. Ahn, U. Samuni and A. Russo, Biochemistry, 1990, 29, 2802–2807.
17. B. P. Soule, F. Hyodo, K.-i. Matsumoto, N. L. Simone, J. A. Cook, M. C. Krishna and J. B. Mitchell, Free Radical Biol. Med., 2007, 42, 1632–1650.
18. G. G. Borisenko, I. Martin, Q. Zhao, A. A. Amoscato and V. E. Kagan, J. Am. Chem. Soc., 2004, 126, 9221–9232.
19. S. Goldstein, A. Samuni and G. Merenyi, J. Phys. Chem. A, 2008, 112, 8600–8605.
20. M. A. Lam, D. I. Pattison, S. E. Bottle, D. J. Keddie and M. J. Davies, Chem. Res. Toxicol., 2008, 21, 2111–2119.
21. K. Tanaka, T. Miura, N. Umezawa, Y. Urano, K. Kikuchi, T. Higuchi and T. Nagano, J. Am. Chem. Soc., 2001, 123, 2530–2536.
22. X. Ji, J. Hong and X. Guo, Analyst, 2012, 137, 710–715.
23. G. Xu and M. R. Chance, Chem. Rev., 2007, 107, 3514–3543.
24. D. A. Stoyanovsky, A. Maeda, J. L. Atkins and V. E. Kagan, Anal. Chem., 2011, 83, 6432–6438.
25. L. Cao, Q. Wu, Q. Li, S. Shao and Y. Guo, New J. Chem., 2013, 37, 2991–2994.
26. G. G. Borisenko, I. Martin, Q. Zhao, A. A. Amoscato, Y. Y. Tyurina and V. E. Kagan, J. Biol. Chem., 2004, 279, 23453–23462.
27. H. Wiseman and B. Halliwell, Biochem. J., 1996, 313, 17–29.
28. J. Cadet, T. Delatour, T. Douki, D. Gasparutto, J.-P. Pouget, J.-L. Ravanat and S. Sauvaigo, Mutat. Res., Fundam. Mol. Mech. Mutagen., 1999, 424, 9–21.
29. M. Zhuang, C. Ding, A. Zhu and Y. Tian, Anal. Chem., 2014, 86, 1829–1836.
30. H. Lee, K. Lee, I.-K. Kim and T. G. Park, Adv. Funct. Mater., 2009, 19, 1884–1890.

---

Footnote

† Electronic supplementary information (ESI) available: The spectra of R-NO˙ and its secondary amine derivative. See DOI: 10.1039/c5ra06129f

---