Reactivity of Superoxide Anion Radical with a Perchlorotriphenylmethyl

Dec 15, 2007 - S.I.C., Campus de la UAB, 08193 Bellaterra, Spain. ReceiVed: August 18 ... The reaction of superoxide radical with a tricarboxylate der...
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J. Phys. Chem. B 2008, 112, 158-167

Reactivity of Superoxide Anion Radical with a Perchlorotriphenylmethyl (Trityl) Radical Vijay Kumar Kutala,† Frederick A. Villamena,† Govindasamy Ilangovan,† Daniel Maspoch,‡ Nans Roques,‡ Jaume Veciana,‡ Concepcio´ Rovira,‡ and Periannan Kuppusamy*,† Center for Biomedical EPR Spectroscopy and Imaging, DaVis Heart and Lung Research Institute, The Ohio State UniVersity, Columbus, Ohio 43210, and Institut de Ciencia de Materials de BarcelonasC.S.I.C., Campus de la UAB, 08193 Bellaterra, Spain ReceiVed: August 18, 2007; In Final Form: October 22, 2007

The reaction of superoxide radical with a tricarboxylate derivative of perchlorotriphenylmethyl radical (PTMTC) is studied. PTM-TC is a stable (“inert”) free radical, which gives a single sharp electron paramagnetic resonance (EPR) peak in aqueous solutions. PTM-TC also gives a characteristic optical absorption at 380 nm. Superoxide, on reaction with PTM-TC, induced a decrease in the intensity of the EPR signal and optical absorption of PTM-TC at 380 nm. The signal loss was specific to superoxide and linearly dependent on the superoxide flux in the system. Competitive kinetics experiments revealed that PTM-TC reacts with superoxide with an apparent second-order rate constant of 8.3 × 108 M-1 s-1. Electrochemical and mass spectrometric analyses of the reaction suggested the formation of perchlorotriphenylmethane and molecular oxygen as products. The high sensitivity of detection of PTM-TC combined with the high rate constant of the reaction of superoxide with PTM-TC may offer a potential opportunity for measurement of superoxide in biological systems. In conclusion, the PTM-TC molecule has high sensitivity and specificity for superoxide radicals and thus may enable quantitative detection of superoxide generation in biological systems using EPR and/or spectrophotometric methods.

Introduction Superoxide, a one-electron reduction product of molecular oxygen, has been implicated in several pathophysiological processes such as ischemia reperfusion and acute lung injury,1,2 neurodegenerative diseases,3,4 hypertension,5 aging,6,7 cell cycle,8 apoptosis,9,10 and cancer.8,9,11 Increasing evidence suggests that enhanced generation of O2-• decreases the bioavailability of nitric oxide, leading to endothelial dysfunction.12,13 The increasing knowledge of the role of superoxide in the development and progression of oxidative stress-related diseases and physiological events requires improved techniques for the detection, characterization, and quantification of this species. During recent years, efforts have been directed toward improving existing techniques for measuring this ubiquitous reactive oxygen species.7,14-16 A variety of analytical techniques have been described for the quantification of O2-•, some of which have been successfully applied to give an estimate of it in biological systems.10,14,15 However, several of these methods present drawbacks that limit their sensitivity and specificity and have difficulties in quantifying the amount or rate of O2-• production.17 Hence, it is important to identify new probes (reagents) improving the efficacy of the assays. Our previous studies have demonstrated a triarylmethyl free radical (TAM Ox063) as an efficient probe for the EPR and spectrophotometric detection of superoxide in biological systems.18,19 Furthermore, this method is not influenced by the presence of other ROS (H2O2, HO•, ROO•), and biological oxidoreductants such as hydrogen peroxide, ascorbate, glutathione, and iron. The rate constant of the reaction of TAM * Corresponding author. E-mail: [email protected]. Phone: 614292-8998. Fax: 614-292-8454. † The Ohio State University. ‡ C.S.I.C.

with superoxide is 3.1 × 103 M-1 s-1, which is twice that of commonly used nitrone spin-traps.20 In the current study, we studied a new class of trityl probe based on the perchlorotriphenylmethyl (PTM, Figure 1) radical. The PTM radical and its derivatives are highly stable against a variety of reactive chemical agents, and hence are called “inert free radicals”.21 They can withstand temperatures as high as 250 °C. Their chemical inertness and thermal stability are due to the full steric blockage of the central carbon, where most of the spin density resides.21 The probe used in the current study is a water-soluble tricarboxylic acid derivative of PTM (PTM-TC, Figure 1). We observed that superoxide reacts with PTM-TC at near diffusioncontrolled rates in aqueous solution. The measurements can be conveniently performed using EPR spectroscopy or UV-visible spectrophotometry with comparable sensitivity and reliability. PTM-TC revealed a higher sensitivity and specificity for superoxide when compared with many of the existing probes used in methods for superoxide detection. Materials and Methods Chemicals. PTM-TC was synthesized following the procedure described in the literature.22 PTM was a kindly provided by Dr. Ballester (Barcelona, Spain). TAM (Ox063) was a gift from Nycomed Innovation (Sweden). Glutathione, hydrogen peroxide, xanthine, ferricytochrome c, superoxide dismutase (SOD), catalase, diethylenetriaminepentaacetic acid (DTPA), and xanthine oxidase were purchased from Sigma (St. Louis, MO). All solutions were prepared in phosphate buffer (0.1 M, pH 7.4) in the presence of DTPA (0.1 mM). The buffers were pretreated with a Chelex-100 resin (40 g/l) to remove adventitial metal ions. Fresh stock solutions of PTM-TC were prepared in phosphate or tris(hydroxymethyl)aminomethane (Tris)-HCl buffer and kept in the dark until use.

10.1021/jp076656x CCC: $40.75 © 2008 American Chemical Society Published on Web 12/15/2007

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Figure 1. Chemical structure and spectroscopic properties of PTM-TC. (A) PTM-TC is a tricarboxylic acid derivative of perchlorotriphenylmethyl (PTM) radical. Both PTM and PTM-TC are stable (“inert”) free radicals. PTM is insoluble in aqueous solutions. (B) A geometry optimized rendering of PTM molecule showing the central carbon shielded by the bulky perchlorophenyl groups. (C) The EPR spectrum was measured from an aerated solution of PTM-TC (10 µL of 1 µM) in Tris-HCl buffer (pH 7.4). The peak-to-peak width was 55 µT. The inset shows the EPR spectrum of a 10 nM solution (10 µL) with a signal-to-noise ratio of 2.5. Instrumental settings were frequency, 9.78 GHz; microwave power, 10 mW; modulation amplitude, 20 µT; scan, 10 × 30 s. (D) UV-visible absorption spectrum of PTM-TC (50 µM) in Tris-HCl buffer (pH 7.4). The spectrum shows a peak absorption at 380 nm ( ) 18 600 M-1 cm-1).

EPR Measurements. Superoxide radicals were generated by a xanthine/xanthine oxidase (X/XO) system. The reaction was initiated by adding XO (0.02 U/mL) to a mixture of PTM-TC (50 µM) and X (0.5 mM) containing DTPA (0.1 mM) in TrisHCl buffer (50 mM, pH 7.4). EPR spectra were recorded at room temperature using X-band Bruker ER300 EPR spectrometer with a TM110 cavity. Solutions were taken in a quartz EPR flat cell or in 50 µL borosilicate glass capillary tubes (Fisher Scientific, Pittsburgh, PA). Some measurements were performed under constantly aerated conditions using a gas-permeable Teflon tubing (0.8 mm diameter, Zeus Industrial Products, Orangeburg, SC) as reported.23 The spectra were acquired continuously for up to 20 min. Each experiment was conducted at least three times. The following acquisition parameters were used: modulation amplitude, 0.2 µT; time constant, 40 ms; scan time, 10 s; microwave power, 10 mW; microwave frequency, 9.79 GHz; and modulation frequency, 100 kHz. Data acquisition and processing were performed using custom-developed software. Spectrophotometric Measurements. Superoxide radicals were generated by X/XO system as in the case of EPR measurements. The decrease in the absorbance at 380 nm was monitored spectrophotometrically (CARY 50 BIO UV-visible spectrophotometer, Varian) for 20 min at room temperature. For validation of the X/XO superoxide-generating system, a cytochrome c assay was also performed. Superoxide was generated by the action of XO (0.02 U/mL) on X (0.5 mM) in the presence

of catalase (500 U/mL), EDTA (0.1 mM) in Tris-HCl buffer (0.05 M) at pH 7.4. The initial rates of superoxide generation were measured spectrophotometrically by following the SODinhibitable reduction of ferricytochrome c (50 µM) at 550 nm. Determination of Rate Constant. The apparent second-order rate constant of the reaction of PTM-TC and superoxide was determined by using SOD as a competitive inhibitor of the reaction of PTM-TC with superoxide radicals.24,25 The kinetic experiments were conducted to determine (i) the rate of decrease in the EPR signal intensity of PTM-TC and (ii) the rate of decrease in absorbance of PTM-TC (OD at 380 nm) induced by a constant flux of superoxide generated using X/XO. The competition kinetics was performed using various concentrations of SOD. The initial rates of reaction in the absence and presence of SOD were used to calculate the rate constant, as reported in ref 25. Isolation of Neutrophils. Freshly isolated polymorphonuclear neutrophils (PMNs) were used. The PMNs were isolated from blood collected from human volunteers using a Percoll gradient centrifugation method.26 The cells were suspended in PBS containing glucose (1 mg/mL) and albumin (1 mg/mL) and counted using a hemocytometer. Cell viability was >95% as assessed by the trypan blue exclusion method. PMNs (2 × 106 cells/mL) were activated with PMA (400 ng/mL) and used for superoxide generation. The assay mixture also consisted of PTM-TC (50 µM) and 100 µM diethylenetriaminepentaacetic acid (DTPA) in 50 mM Tris-HCl buffer at pH 7.4. The EPR

160 J. Phys. Chem. B, Vol. 112, No. 1, 2008 spectrum of PTM-TC was acquired continuously for 15 min, and the concentration of superoxide was determined. Each experiment was conducted at least three times. Effect of Free Radicals and Oxido-Reductants. The effect of various biological oxidizing and reducing agents such as hydrogen peroxide, glutathione, ascorbate, and free radicals such as hydroxyl and peroxyl radicals, and nitric oxide on the assay of superoxide with PTM-TC was examined. Hydrogen peroxide (500 µM), glutathione (1 mM), and ascorbate (100 µM) was used. Ferrous ammonium sulfate (0.1 mM) and hydrogen peroxide (1 mM) were used to generate hydroxyl radicals. Alkylperoxyl radicals were generated by thermal decomposition of 2,2-azobis-(2-amidinopropane)dihydrochloride (5 mM) at 37 °C. S-nitroso-N-acetylpenicillamine (SNAP, 1 mM) in phosphate buffer (pH 7.4, 0.1 M) at room temperature was used to generate nitric oxide (NO). Cyclic Voltammetry. Cyclic voltammetry measurements were performed using 5 mM solution of TAM or PTM-TC in PBS (pH 7.4) at room temperature using AutoLab, PGSTAT30 (Brinkmann Instruments, Westbury, NY). The redox potentials, reported in this study, were measured using a platinum disc (1 mm2) as a working electrode, Ag/AgCl reversible redox couple as a reference electrode, and platinum gauze (BAS, West Lafayette, IN) as a counter electrode. Electrochemical Measurements of H2O2 and O2. The oxygen and H2O2 content were measured using an Apollo 4000 multichannel analyzer (WPI, Sarasota, FL) with Clark-type oxygen (ISO-OXY-2, WPI) and H2O2 (ISO-HPO-100, WPI) electrodes in a closed-chamber of 2 mL volume with magnetic stirring at 37 °C. The reaction mixture (2 mL) contained X (0.5 mM), DTPA (0.1 mM), and Tris-HCl (50 mM, pH 7.4). The measurements were performed in presence or absence of SOD (400 U/mL) or PTM-TC (50 µM). The reaction was initiated by adding XO (0.02 U/mL) with a Hamilton syringe through an aperture, and the amounts of O2 and H2O2 were continuously measured and then quantified using a standard curve. Gas Chromatography-Mass Spectrometry (GC-MS). GCMS analyses were carried out using a Flennigan TraceGC Ultra and Trace DSQ system equipped with positive ion electron impact ionization (EI). A 2 µL solution of 230 µM PTM in DMSO was injected into the column. In another experiment, a 200 µL solution of KO2 (5 mg/500 µL) in DMSO was added to a 200 µL solution of PTM (230 µM in DMSO) and then acidified with 10 µL of 1 mM HCl. Two microliters of the DMSO-acid mixture was injected into the column at an initial temperature of 40 °C using a ramping of 20 °C/min with a maximum temperature of 350 °C. MS detection was carried out using a 200 °C ion source temperature, an electron energy of 70 eV, and a scan speed of 1.6584 s-1. Data analysis. All experiments were performed in triplicate with 3 or 4 independent measurements. The data were analyzed and compared using Student’s t-test for statistical significance. Results EPR Spectrum of PTM-TC in Solution. PTM-TC shows a single-peak EPR spectrum in aerated Tris-HCl buffer (pH 7.4, 50 mM) at 37 °C with a peak-to-peak width of 55 µT (Figure 1C). The width under anoxic conditions was 51 µT suggesting that the spectral width is sensitive to the concentration of dissolved molecular oxygen (paramagnetic broadening) in the solution. Under nonsaturating conditions, the intensity (obtained by double integration of the signal) is directly proportional to the concentration of the PTM-TC and is independent of oxygen concentration. Figure 1C shows that a

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Figure 2. Effect of superoxide on the EPR signal intensity of PTMTC in solution. The EPR spectrum of PTM-TC was measured as a function of time after mixing in a continuously aerated solution (see Methods for details) consisting of X (0.5 mM), XO (0.02 U/mL), DTPA (100 µM), PTM-TC (50 µM) in 50 mM Tris-HCl buffer (pH 7.4) at 22 °C. The reaction was initiated by the addition of XO. In the absence of SOD, superoxide induced a decrease in the signal intensity of PTMTC. Addition of SOD (2000 U/mL) completely inhibited the signal loss.

detection sensitivity of better than 10 µL of 10 nM PTM-TC can be achieved at typical X-band frequencies. EPR and Spectrophotometric Detection of Superoxide Reaction with PTM-TC. The effect of superoxide, generated by a mixture of xanthine (0.5 mM) and xanthine oxidase (0.02 U/mL), on PTM-TC (50 µM) was studied in a 50 mM TrisHCl buffer. Superoxide induced a decrease in the EPR signal intensity of PTM-TC in a time-dependent manner (Figure 2). SOD, at a concentration of 2000 U/mL (∼17 µM), completely inhibited the decrease in EPR signal intensity of PTM-TC by superoxide. PTM-TC has a characteristic peak at 380 nm in the UV-visible absorption spectrum with an extinction coefficient of 18 600 M-1 cm-1 (Figure 2B). The reaction between PTM-TC (50 µM) and superoxide radicals, generated by X/XO, caused a time-dependent decrease in the absorbance at 380 nm (Figure 3). The initial rate of decrease in the EPR signal intensity of PTM-TC, obtained during 5 min after mixing, was linearly dependent on the superoxide flux (Figure 4A). In separate experiments, it was established that the observed rate of decay was not due to either xanthine or xanthine oxidase alone (data not shown). In order to determine whether hydrogen peroxide, which is a product of superoxide disproportionation, has any role in the observed decay of the PTM-TC signal, we performed additional experiments in the presence of catalase (500 U/mL). The results revealed that catalase did not have any significant effect on the decay of PTM-TC signal. Similarly, the rate of decrease in absorbance of PTM-TC at 380 nm was linearly dependent on the superoxide flux (Figure 4B). Detection of Superoxide in Polymorphonuclear Leukocytes (PMNs). To evaluate the possible application of PTMTC as a probe for detection of superoxide in cellular systems in vitro, we used a cellular system consisting of stimulated PMNs, which produce superoxide radicals through the NADPH oxidase system. Unstimulated (resting) neutrophils had no significant effect on the EPR signal intensity of PTM-TC (Figure 5). But, when the PMNs were stimulated with PMA (400 ng/mL), a continuous decay of the signal was observed which was inhibited by SOD (2000 U/mL). The stimulated cells showed a superoxide production of 2.76 ( 0.25 µmol/106 cells/ 15 min.

Reactivity of Superoxide with a Trityl Radical

Figure 3. Effect of superoxide on the UV-visible absorption spectrum of PTM-TC. PTM-TC (50 µM) was incubated with X (0.5 mM), XO (0.02 U/mL), and DTPA (100 µM) in 50 mM Tris-HCl buffer (pH 7.4) at 22 °C. The reaction was initiated by the addition of XO. (A) PTM-TC shows a characteristic absorption at 380 nm ( ) 18 600 M-1 cm-1), which decreased with time (as indicated in the inset) on reaction with superoxide. (B) Change of absorbance at 380 nm as function of time. In the absence of SOD, superoxide induced a decrease in the absorbance. Addition of SOD (2000 U/mL) completely inhibited the decrease in absorption.

Rate Constant for the Reaction of Superoxide with PTMTC. The apparent second-order rate constant for the reaction of PTM-TC and superoxide was determined using SOD as a competitive inhibitor of the reaction of PTM-TC with superoxide radicals. Competition kinetics was studied using various concentrations of SOD (Figure 6). The initial rates of reaction in the absence and in the presence of SOD were measured, and their ratio was plotted against the concentration of SOD. As can be seen, a linear response was observed, in both EPR (Figure 6A) and spectrophotometry (Figure 6B), suggesting that SOD competes with PTM-TC for superoxide. By using the reported rate constant of the SOD reaction with superoxide (3.8 × 109 M-1 s-1),25 the rate constant for the reaction of superoxide with PTM-TC was calculated as 6.5 ( 1.9 × 108 M-1 s-1 from EPR and as 8.3 ( 1.5 × 108 M-1 s-1 from spectrophotometry. PTM-TC Versus Cytochrome c Assay of Superoxide. Cytochrome c assay is a well-established method for superoxide determination in biological systems. The method is based on the reduction of ferricytochrome c by superoxide with a bimolecular rate constant of 5 × 105 M-1 s-1.27 This rate constant is comparable to that of self-dismutation by disproportionation of superoxide (7.6 × 105 M-1 s-1). Thus, cytochrome c needs to compete with the self-dismutation reaction of superoxide. Since the rate constant of the reaction of PTM-

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Figure 4. Determination of superoxide by EPR and spectrophotometry. (A) Effect of superoxide flux on the EPR detection of superoxide. The incubation mixture contained X (0.5 mM), DTPA (100 µM), PTMTC (50 µM) in 50 mM Tris-HCl buffer (pH 7.4) at 22 °C. Various concentrations of XO were used to initiate the reaction. The rate of decay of PTM-TC (µM/min) was measured during the first 5 min after initiation of the reaction. The rate of decay was linear with XO (255 ( 12 µM/min/U). (B) Spectrophotometric determination of the rate of superoxide generation under conditions as in A. The plot B shows that the rate was linear with XO (246 ( 15 µM/min/U).

TC with superoxide is about 3 orders of magnitude greater than that of cytochrome c or self-dismutation of superoxide, we compared the efficiency of PTM-TC against cytochrome c in the determination of superoxide by spectrophotometry under identical conditions. The results, as shown in Figure 7, revealed that the sensitivity of PTM-TC for the detection of superoxide was 1.42-fold higher compared with cytochrome c. The higher sensitivity is attributed to the 3 orders of magnitude higher reactivity of superoxide with PTM-TC compared with cytochrome c. Effect of Free Radicals and Biological Oxido-Reductants. The effect of free radicals such as hydroxyl, peroxyl, and nitric oxide and various biological oxidizing and reducing agents on the assay of superoxide with PTM-TC was examined. Alkyl peroxyl radicals, hydroxyl radicals, nitric oxide, hydrogen peroxide (500 µM), glutathione (1 mM), or ascorbate (100 µM) did not show any significant effect on the EPR signal intensity of PTM-TC (Figure 8) implying that the paramagnetic property of PTM-TC is not affected. The results, however, do not rule out the possibility of nonspecific reactions at sites other than

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Figure 5. Determination of PMN-mediated superoxide flux with PTMTC. The assay mixture consisted of PMNs (2 × 106 cells/mL), PMA (400 ng/mL), PTM-TC (50 µM), DTPA (100 µM) in 50 mM Tris-HCl buffer (pH 7.4). The measurements were performed by X-band EPR for 15 min at room temperature. All experiments were carried out with and without SOD (2000 U/mL).

Figure 6. Determination of the rate constant for the reaction of PTMTC with superoxide radical using a competitive inhibition method. SOD was used as a competitive inhibitor. A constant flux of superoxide was generated by 0.5 mM X and 0.02 U/mL of XO in 50 mM Tris-HCl buffer (pH 7.4) containing 100 µM DTPA and 10 µM PTM-TC. The initial rates of the reaction were measured by (A) EPR, (B) spectrophotometry, in the absence (V) and the presence (v) of various concentrations of SOD. The rate constant of the reaction of PTM-TC and superoxide was calculated as 6.5 ( 1.9 × 108 M-1 s-1 from EPR (A) and 8.3 ( 1.5 × 108 M-1 s-1 from spectrophotometry (B) using the reported rate constant of the SOD reaction with superoxide (3.8 × 109 M-1 s-1).25

the central carbon atom of PTM-TC. This is particularly important in the case of highly reactive hydroxyl radicals. Stability of PTM-TC in Blood and Plasma. Experiments were also performed to study the stability of PTM-TC in the

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Figure 7. Comparison of superoxide determination by PTM-TC and cytochrome c (cyt c) assays. The assay mixture for PTM-TC consisted of Tris-HCl buffer (50 mM, pH 7.4), DTPA (100 µM), X (0.5 mM), PTM-TC (50 µM), and varying concentrations of XO (0.005, 0.01, 0.02, 0.04 U/mL). The assay mixture for cytochrome c assay consisted of Tris-HCl buffer (50 mM, pH 7.4), DTPA (100 µM), X (0.5 mM), catalase (200 U/mL), cytochrome c (50 µM), and varying concentrations of XO (0.005, 0.01, 0.02, 0.04 U/mL). The reaction was started after the addition of XO and monitored for 10 min at room temperature. The rate of decrease in absorbance was measured at 380 nm for PTMTC and increase in absorbance at 550 nm for cytochrome c. The concentration of superoxide with PTM-TC or cytochrome c was measured using molar extinction coefficient of PTM-TC ( ) 18 600 M-1cm-1) and cytochrome c ( ) 21 000 M-1 cm-1). Results show that the assays are linearly correlated (R2 ) 0.99) with a slope of 1.42 ( 0.05 indicating that the PTM-TC assay is more sensitive than the cytochrome c assay.

Figure 8. Effect of biological oxido-reductants on the detection of superoxide by PTM-TC. Hydrogen peroxide (500 µM), GSH (1 mM), and ascorbate (100 µM) were used. Ferrous ammonium sulfate (0.1 mM) and hydrogen peroxide (1 mM) were used to generate hydroxyl radicals. Alkylperoxyl radicals were generated by thermal decomposition of 2,2-azobis-(2-amidinopropane)dihydrochloride (5 mM) at 37 °C. S-nitroso-N-acetylpenicillamine (SNAP, 1 mM) in phosphate buffer (100 mM, pH 7.4) at room temperature was used to generate nitric oxide (NO). Values are expressed as mean ( SD (n ) 3).

presence of biological fluids such as blood and plasma in vitro using EPR spectroscopy. For this study, PTM-TC (50 µM) was added to human blood or plasma (1:9 volume) which was collected from healthy volunteers, and EPR measurements were performed for 30 min. The results indicated that the EPR signal intensity of PTM-TC was significantly decreased (40%) in the presence of blood or plasma (data not shown), which could be due to the presence of oxido-reductants present in higher concentrations. This suggests that PTM-TC may not be suitable for in vivo applications, though the studies with common

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Figure 9. Cyclic voltammogram (CV) of PTM-TC and TAM. The CV measurements were performed on (A) PTM-TC and (B) TAM using an Ag/AgCl reversible redox couple as a reference electrode. PTM-TC shows only a reduction occurring at -0.320 V (-0.123 V versus NHE). TAM shows a reduction at -0.535 V (-0.338 V versus NHE) and an oxidation at +0.510 V (+0.707 V versus NHE).

biological oxido-reductants, such as glutathione, hydrogen peroxide, and ascorbate showed no interference. Characterization of the Reaction between PTM-TC and Superoxide. Perchlorotriphenylmethyl (PTM) radicals, due to the effective shielding of the central carbon atom, have been shown to be very stable (hence, they are called “inert free radicals”) against many common oxidoreductants. However, under special conditions, these radicals can be oxidized to PTM+ or reduced to the PTM- ion.28,29 Interestingly, both PTM+ and PTM- are stable forms and can be reversibly interconverted by a two-electron redox process through the formation of a neutral PTM radical.30 This observation prompted us to examine the redox potential of PTM-TC using cyclic voltammetry (CV). The CV pattern of PTM-TC showed only a reduction occurring at -0.123 V versus NHE (Figure 9A). The data suggested that in the presence of superoxide, with a redox potential of -0.33 V (O2/O2•-, versus NHE),31 a one-electron reduction of the perchlorotriphenylmethyl radical to the corresponding perchlorotriphenylmethide anion would occur. For comparison, we have also measured the CV pattern of TAM, an open form of trityl which we have previously reported to be a scavenger of superoxide radicals.18,19 As shown in Figure 9B, TAM showed a well-defined reversible oxidation at +0.707 V (versus NHE) indicating that it is oxidized to TAM+ ion by superoxide, rather than getting reduced to TAM- ion. One-electron reduction of PTM-TC by superoxide would result in the conversion of superoxide to molecular oxygen, as opposed to self-dismutation of superoxide to molecular oxygen and hydrogen peroxide (Scheme 1). In order to confirm this hypothesis, we performed electrochemical measurements to find evidence for the generation of oxygen and hydrogen peroxide during the reaction of superoxide with PTM-TC. The amounts of O2 and H2O2 generated during the reaction were measured

Figure 10. Measurement of oxygen and hydrogen peroxide evolution during the reaction of superoxide with PTM-TC. The concentrations of oxygen and hydrogen peroxide in the solution were determined using Clarke-type O2 and H2O2 electrodes. The reaction mixture contained Tris-HCl (50 mM, pH 7.4), DTPA (100 µM), X (0.5 mM), XO (0.02 U/mL) with and without SOD (400 U/mL), or PTM-TC (50 µM) at 37 °C. (A) O2 concentration measured at the end of the first 5 min of the reaction. (B) H2O2 accumulation measured at the end of the first 5 min of the reaction. Values are expressed as mean ( SD (n ) 3). *p < 0.05 vs X + XO; **p < 0.05 vs +SOD.

SCHEME 1

simultaneously by using Clark-type electrodes. As shown in Figure 10, molecular oxygen is consumed by X/XO to produce superoxide; hence, a decrease in O2 concentration was observed, whereas the H2O2 concentration increased because of the spontaneous disproportionation of superoxide into H2O2 and O2. Addition of SOD to the reaction mixture significantly increased the O2 and H2O2 concentrations compared with X + XO (p < 0.05) due to SOD-mediated dismutation of superoxide to H2O2 and O2. The O2 concentration was significantly higher in the

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Figure 11. GC-MS analysis of the reaction of superoxide with PTM. A solution of PTM (230 µM) in DMSO (200 µL) was reacted with KO2 (2 mg) and then subjected to a GC-MS assay. (A) GC-MS of PTM in DMSO, at retention time 13.98 min, showing a molecular ion peak [PTM + H]+ of 761.4 m/z consistent with the molecular weight of PTM (760). Reaction of PTM with KO2 and subsequent acidification, showing a GC peak at 15.09 min corresponding to [PTM + H]+ of 762.2 m/z. The results suggested the superoxide-mediated reduction of PTM to PTM- which on subsequent protonation yielded perchlorotriphenylmethane.

presence of PTM-TC, and H2O2 was significantly decreased, as compared with the SOD (p < 0.05), suggesting the predominant conversion of superoxide to molecular oxygen via reduction of PTM-TC by superoxide, rather than self-dismutation of superoxide to give equimolar amounts of oxygen and hydrogen peroxide. Hence, it was clear that PTM-TC was reduced by superoxide to give molecular oxygen and perchlorotriphenylmethane. In order to obtain evidence for the formation of perchlorotriphenylmethane in the reaction between PTM-TC and superoxide, GC-MS experiments were performed. We used PTM, the parent compound of PTM-TC (see Figure 1 for structure) to take advantage of the solubility of PTM in DMSO so that KO2 can be used to generate large amounts of superoxide without X/XO, which may interfere with GC-MS analyses. We treated PTM with KO2 in DMSO and subjected the solution to a GC-MS assay. Figure 11 shows the GC-MS of pure PTM in DMSO, at a retention time of 13.98 min, showing a molecular ion peak for [PTM + H]+ of 761.4 m/z consistent with the molecular weight of PTM (m/z: 760). The reaction of PTM

with KO2 and subsequent acidification showed a weak GC peak at 15.09 min corresponding to [PTM + H]+ of 762.2 m/z. The results suggested the superoxide-mediated reduction of PTM to PTM-, which on subsequent protonation yielded perchlorotriphenylmethane. Discussion The present study clearly showed that the PTM-TC radical reacted with superoxide with high sensitivity and specificity. The EPR study indicated that the rate of decrease in the signal intensity was linearly correlated with the superoxide flux. The decrease in the signal intensity of PTM-TC was specifically due to superoxide, as confirmed by the complete inhibition of the signal by SOD. One of the most sensitive and standard methods of free radical determination is by spectrophotometry. Therefore, we used this technique to verify the results obtained with the EPR spectroscopy. First, we monitored the reaction of PTM-TC with superoxide at 380 nm using spectrophotometry. The reaction of PTM-TC with superoxide produced a decrease

Reactivity of Superoxide with a Trityl Radical in the intensity of absorbance. The rate of decrease in absorbance was linearly correlated with superoxide flux. During recent years, efforts have been directed toward improving existing techniques for measuring this ubiquitous reactive oxygen species.7,14-16 A variety of analytical techniques have been described for the quantification of O2-•, some of which have been successfully applied to give an estimate of it in biological systems.10,14,15 Current methods used for determining O2-• include cytochrome c and nitrosoblue tetrazolium (NBT) reduction assays, dihydroethidium oxidation to ethidium, aconitase inhibition, chemiluminescence, and electrochemical and electron paramagnetic resonance (EPR) spin-trapping techniques.32 The most popular assays for the detection of O2-• in cells and tissues involve the use of fluorescence14,15,33 and EPR techniques.34-36 However, several of these methods present drawbacks that limit their sensitivity and specificity and have difficulties in quantifying the amount or rate of O2-• production.17 For instance, the commonly used nitrone spin traps have a very low efficiency for the trapping of superoxide radicals, and the nitroxide radical formed is not stable in the presence of biological oxidoreductants.37-40 Recently, the sensitivity and specificity of the dihydroethidium fluorescence assay for superoxide detection was further improved by identifying a new oxidation product of dihydroethidium with superoxide, using fluorescence and HPLC methods.14,15,41,42 The rate constant for the reaction of superoxide with PTMTC was obtained by competitive kinetic analysis using SOD as a competitive inhibitor of superoxide. As the rate of reaction of superoxide with PTM-TC was very high, comparable with that with SOD, we could not use other inhibitors such as cytochrome c for the determination of the rate constant. Unlike the catalytic nature of the reaction of superoxide with SOD, the reaction between superoxide and PTM-TC was stoichiometric as evidenced from the decrease of the EPR intensity, as well as, the spectrophotometric OD observed during the reaction of PTM-TC with superoxide. The decrease in OD is also indicative of modification(s) in the electronic absorption spectrum of the PTM-TC molecule. Under the conditions used to generate superoxide by X (0.5 mM) and XO (0.02 U/mL), a superoxide flux of 3-5 µM/min is expected.19,41 The EPR, as well as, spectrophotometric data shown in Figure 4 revealed that the rate of reduction of PTM-TC was about 4 µM/min under similar conditions. This further suggested that the reaction between PTM-TC and superoxide is 1:1 stoichiometric; that is, one molecule of superoxide is involved in the reaction with one molecule of PTM-TC (Scheme 1), which is once again consistent with the GC-MS and CV results. An important feature of PTM-TC is its reaction rate with superoxide under biological conditions. The rate constant (8.3 × 108 M-1 s-1) obtained using competitive inhibition studies with SOD is several orders of magnitude higher than those of commonly used nitrone traps such as DMPO (3.0 × 101 M-1 s-1)20 or DEPMPO (5.8 × 101 M-1 s-1),43 hydroxylamine oxidation (8.4 × 102 M-1 s-1),44,45 or cytochrome c reduction (5 × 105 M-1 s-1).27 It should be noted that the rate constant of PTM-TC with superoxide is also 3 orders of magnitude higher than that of self-disproportionation of superoxide (7.6 × 105 M-1 s-1)46 and close to that of dismutation by SOD (3.8 × 109 M-1 s-1).25 This is evident from the observation that a very high concentration of SOD (2000 U/mL) was required to completely scavenge the superoxide generated by X/XO in the presence of PTM-TC. Previously, we reported the use of TAM (Ox063), which was specifically designed and developed as a contrast agent for

J. Phys. Chem. B, Vol. 112, No. 1, 2008 165 Overhauser-enhanced MRI,47 for the detection of superoxide by EPR18 and spectrophotometry.19 TAM, on reaction with superoxide, leads to the loss of EPR signal and also to the appearance of a new electronic absorption peak at 546 nm, possibly due to a product of the reaction. The assay is independent of the interference of common biological oxidoreductants. However, the redox reaction mechanism of TAM appears to be different from that of PTM-TC. As shown in Figure 9, TAM shows a well-defined reversible oxidation at +0.707 V (versus NHE) indicating that it is oxidized to the TAM+ ion by superoxide, rather than getting reduced to the TAM- ion. But, in the case of PTM-TC, there is no oxidation peak for up to 1.0 V (Ag/AgCl), thus making the reduction of PTM-TC to PTM-TC- anion (E0 ) 0.123 V) by superoxide as the favorable reaction. Superoxide reacts with TAM with an apparent second-order rate constant of 3.1 × 103 M-1 s-1, suggesting that the reaction of superoxide with PTM-TC is about 6 orders of magnitude higher than TAM. In addition, the sensitivity of the measurement of superoxide by PTM-TC using spectrophotometry is higher compared with TAM, because PTM-TC gives a sharp and strong ( ) 18 600 M-1 cm-1) optical absorption peak at 380 nm, which decreases on reaction with superoxide. On the contrary, the assay of superoxide using TAM was suggested on the basis of the measurement of a possible product of the reaction which gave a broad optical absorption band at 546 nm. Furthermore, the synthesis of TAM requires a multistep procedure,48 whereas PTM-TC can be synthesized more easily.22 The nature of the reaction between PTM-TC and superoxide was clearly established by the electrochemical studies. Superoxide can react with substrates either by oxidation or by reduction processes. Superoxide, itself, can be reduced at 0.94 V or oxidized at -0.33 V (vs NHE).31,49,50 The reduction potential of PTM-TC in PBS at pH 7.4, measured by cyclic voltammetry, was -0.123 V (vs NHE). Because of its lower reduction potential as compared with superoxide, PTM-TC can be reduced by superoxide resulting in the formation of oxygen. The observed increase in the oxygen concentration during the reaction of PTM-TC with superoxide (Figure 10) revealed a reductive conversion of PTM-TC. The evolution of oxygen during the reaction of superoxide with PTM was also confirmed by EPR oximetry (data not shown). The GC-MS verification of the reduced product of PTM-TC further provided support to the above conclusion. Previously, it was reported that a PTM radical on one-electron oxidation yields a PTM cation (PTM+), which showed a new intense absorption peak at 690 nm as observed by UV-visible spectroscopy.30 In our current study, we did not observe the presence of such a peak at 690 nm, during the reaction between PTM-TC and superoxide, indicating that the reaction was not an oxidation process. On the basis of the above observations, the reaction between PTM-TC and superoxide is clearly established as a reduction process, and perchlorotriphenylmethane appears to be a product of the reaction. The classical EPR spin-trap method has certain advantages over the “spin-loss” method: the appearance and growth of an EPR signal in the spin-trap method positively indicates the presence and generation of radicals, and the method could enable the identification of the radical species trapped. The spin-trap method, however, requires the use of a large concentration of the trapping agents because of their slow reactivity to superoxide and instability of the superoxide adduct. On the other hand, PTM-TC undergoes redox reaction with superoxide with high sensitivity and specificity. The probe gives a single sharp line

166 J. Phys. Chem. B, Vol. 112, No. 1, 2008 with a width of 51 µT, which is much smaller when compared with about 100-150 µT obtained with the spin adducts. This means that the working concentration of the probe can be decreased to 3 orders of magnitude, for example, 10 µM compared with >10 mM concentration of spin traps. This low concentration combined with the fact that the probe is inert to biological oxido-reductants should enable measurements of radicals at virtually unperturbed conditions. In addition, the present method of superoxide determination may also have certain advantages over the use of hydroxylamines, which are shown to be oxidized by superoxide to paramagnetic nitroxides. However, unlike the hydroxylamine/nitroxide couple, the PTMTC/PTM-TCH couple seems to be very stable against biological oxido-reductants. Further, because of its instability in blood or plasma, PTM-TC may not be suitable for in vivo applications. Although the proposed method appears to be very promising for in vitro determination of superoxide in biological systems, there are several important questions that remain to be addressed. The most important questions pertaining to this new class of spin probes are as follows: (i) Can we internalize these probes in cells to measure intracellular generation of superoxide? (ii) Can these probes be targeted to specific cellular compartments, such as mitochondria? (iii) Can we enhance the stability of the probe for in vivo applications? (iv) Can we enhance the functionality of these probes by derivatizing them with fluorescence conjugates? The preliminary results of the ongoing research in our laboratory seem to indicate a potential opportunity ahead of us. Summary and Conclusions In the present study, we described the biochemical characterization of the reaction of superoxide with a new trityl radical, PTM-TC, with high sensitivity and specificity. The reaction of superoxide with PTM-TC resulted in the loss of EPR signal intensity or spectrophotometric OD at 380 nm. The signal loss, which was specific to superoxide, was linearly dependent on the superoxide flux in the system. The high sensitivity of detection of PTM-TC by the two methods and the high rate constant of the reaction (approaching the diffusion-controlled rate) may have potential advantages to enable reliable measurements of superoxide in biological systems. Acknowledgment. We are grateful to Dr. Xiaoping Liu for his help with the electrochemical measurement of oxygen and hydrogen peroxide and to Ms. Nancy Trigg for her critical reading of the manuscript. Dr. Kutala was on sabbatical from Nizam’s Institute of Medical Sciences, Hyderabad, India. The synthesis of the trityl compounds was supported by DGI, Spain (Project No. MAT2003-04699). Abbreviations DMPO - 5,5-dimethyl-1-pyrroline-N-oxide DEPMPO - 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline Noxide DTPA - diethylenetriaminepentaacetic acid EPR - electron paramagnetic resonance PBS - phosphate-buffered saline PMN - polymorphonuclear leukocytes PTM - perchlorotriphenylmethyl radical PTM-H - perchlorotriphenylmethane PTM-TC - perchlorotriphenylmethyl tricarboxylic acid ROS - reactive oxygen species SOD - superoxide dismutase

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