Determination of Superoxide in Seawater Using 2-Methyl-6-(4

Anal. Chem. 2008, 80, 1215-1227

Determination of Superoxide in Seawater Using 2-Methyl-6-(4-methoxyphenyl)-3,7dihydroimidazo[1,2-a]pyrazin-3(7H)-one Chemiluminescence Andrew L. Rose,*,†,‡ James W. Moffett,‡,§ and T. David Waite†

Centre for Water and Waste Technology, School of Civil and Environmental Engineering, The University of New South Wales, Sydney, Australia, and Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts

Superoxide, the one-electron reduced form of dioxygen, is known to be generated in marine environments by photochemical and biological processes. Because of its selective reaction with only a few commonly occurring compounds, superoxide is expected to approach concentrations in the high picomolar or low nanomolar range in seawater. Most currently existing methods do not have both the necessary sensitivity and selectivity to measure naturally occurring concentrations. In contrast, we demonstrate here that the chemiluminescence reagent 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[l,2-a]pyrazin3(7H)-one (MCLA) is selective for superoxide in seawater and can be used with a detection limit of around 50 pM. Although a wide range of potential interferences were shown not to react with MCLA directly, some care must be taken when analyzing samples containing nanomolar concentrations of Fe(II), Cu(I), Mo(V), V(III), or V(IV), since these compounds can react with oxygen to produce superoxide during analysis that is subsequently detected. We describe two methods for calibrating the system, one employing photochemically generated superoxide standards and the other employing the superoxide-generating xanthine/xanthine oxidase system and discuss limitations on the use of each. The method was successfully used in the field to determine steady-state superoxide concentrations in the water column in the eastern equatorial Pacific Ocean. Superoxide (O2 ) was first identified by Linus Pauling in 1933 as somewhat of a chemical curiosity.1 Since then, it has been established as a critical molecule in biological systems as a mediator of both beneficial reactions (e.g., as a defense mechanism by mammalian immune systems2 and an intercellular * Corresponding author. E-mail: [email protected]. Phone: +61 2 9385 5214. Fax: +61 2 9313 8624. † The University of New South Wales. ‡ Woods Hole Oceanographic Institution. § Present address: Department of Biology, University of Southern California, Los Angeles, CA. (1) Neuman, E. W. J. Chem. Phys. 1934, 2, 31-33. (2) Segal, A. W. J. Clin. Invest. 1989, 83, 1785-1793. 10.1021/ac7018975 CCC: $40.75 Published on Web 01/18/2008

© 2008 American Chemical Society

signaling molecule3) and detrimental reactions (e.g., “oxidative stress”4) for cells, the latter being responsible for the widespread distribution of the enzyme superoxide dismutase (SOD) across all types of living organisms.5 In environmental systems the importance of superoxide is not well understood, but there is increasing evidence that it may play a pivotal role in many abiotic and biologically mediated redox reactions in natural waters. In particular, superoxide is able to reduce a range of biologically important redox-active trace metals (including iron6 and copper7) and may also react with some organic molecules8 under typical environmental conditions. However superoxide is relatively selective in its reactions (due to its unusually stable bonding structure1), conferring it with the potential for considerable longevity in natural waters. This property, in combination with its rapid photochemical and/or biological production, suggests that it may persist at concentrations in the picomolar range, or possibly even higher, in marine waters.8-10 Several methods currently exist for determination of superoxide concentrations. The simplest of these is direct observation of superoxide by UV spectrophotometry; however, the molar extinction coefficient at 240 nm, the wavelength of maximum absorption in the neutral to alkaline pH range, is too small to permit determination of environmentally relevant concentrations (240 ) 2350 M-1 cm-1).11 Furthermore, a variety of organic (and possibly inorganic) molecules present in natural waters will also absorb significantly at this wavelength. The most popular alternative methods involve reaction of superoxide with a chemical probe to form products that can be quantified spectrophotometrically (such (3) Aguirre, J.; Rios-Momberg, M.; Hewitt, D.; Hansberg, W. Trends Microbiol. 2005, 13, 111-118. (4) Lesser, M. P. Annu. Rev. Physiol. 2006, 68, 253-278. (5) Wolfe-Simon, F.; Grzebyk, D.; Schofield, O.; Falkowski, P. G. J. Phycol. 2005, 41, 453-465. (6) Rose, A. L.; Waite, T. D. Environ. Sci. Technol. 2005, 39, 2645-2650. (7) Voelker, B. M.; Sedlak, D. L.; Zafiriou, O. C. Environ. Sci. Technol. 2000, 34, 1036-1042. (8) Goldstone, J. V.; Voelker, B. M. Environ. Sci. Technol. 2000, 34, 10431048. (9) Petasne, R. G.; Zika, R. G. Nature 1987, 325, 516-518. (10) Zafiriou, O. C. Mar. Chem. 1990, 30, 31-43. (11) Bielski, B. H. J.; Cabelli, D. E.; Arudi, R. L.; Ross, A. B. J. Phys. Chem. Ref. Data 1985, 14, 1041-1100.

Analytical Chemistry, Vol. 80, No. 4, February 15, 2008 1215

as cytochrome c, tetrazolium salts, and hydroethidine12). The major disadvantage of these methods is that the probe molecules are generally not specific in their reaction with superoxide, and thus the magnitude of non-superoxide mediated reaction must also be determined. In addition, naturally occurring concentrations of superoxide will produce only small quantities of probe products that are likely to be below the detection limit of even the most sensitive spectrophotometric equipment. Other methods that have been employed include the use of 15N-labeled 15NO as a probe,13 which reacts with superoxide to form 15NO3 (ref 14) whose concentration can be determined by mass spectrometry, and conversion of superoxide to hydrogen peroxide,9 which can be more easily determined by a variety of methods. While these techniques are more specific for superoxide, they still result in detection limits that are too high to determine in situ superoxide concentrations in natural waters. The most sensitive methods available are those that employ probes that form chemiluminescent products on reaction with superoxide. With the availability of photon-counting photomultiplier tubes, the technology required to employ such methods is relatively inexpensive and robust, and the sensitivity of the method is limited primarily by the quantum efficiency of the chemiluminescence (CL) reaction. The major types of such probe compounds are (i) luminol (and its derivatives); (ii) lucigenin; and (iii) coelenterazine and its derivatives Cypridina luciferin analogue (2-methyl-6-phenyl-3,7-dihydroimidazo[l,2-a]pyrazin-3-one; CLA) and methyl Cypridina luciferin analogue (2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[l,2-a]pyrazin-3(7H)-one; MCLA). Members of the first class of compounds typically undergo univalent oxidation to produce a radical anion, which is then further oxidized by superoxide to produce the light emitting species.15 Since the first oxidation step is nonspecific, luminol and its derivatives are generally poor probes for superoxide due to their low specificity and prevalence of competing side-reactions. CL of the second type of compound, lucigenin, involves univalent reduction of the parent compound to produce a radical cation which then reacts with superoxide to yield a light emitting precursor and is thus also not entirely specific for superoxide.16 In addition, as the cation radical may reoxidize by univalent reduction of oxygen (thus producing superoxide), reaction conditions must be carefully controlled to precisely determine how much CL is due to superoxide that is present in the sample compared to that which is produced by other reactions of lucigenin.16 In contrast, the third class of compounds have a high specificity for superoxide and singlet oxygen (O2 in the1∆g excited state), as the primary mechanism of CL involves either hydrogen atom extraction by hydroperoxyl (the conjugate acid of superoxide) followed by addition of a second superoxide molecule to yield a dioxetane intermediate as a precursor to the light-emitting species, or addition of singlet oxygen to the parent compound to yield the dioxetane intermediate directly (see Kambayashi and Ogino17 and (12) Zhao, H.; Kalivendi, S.; Zhang, H.; Joseph, J.; Nithipatikom, K.; VasquezVivar, J.; Kalyanaraman, B. Free Radical Biol. Med. 2003, 34, 1359-1368. (13) Zafiriou, O. C.; Blough, N. V.; Micinski, E.; Dister, B.; Kieber, D.; Moffett, J. Mar. Chem. 1990, 30, 45-70. (14) Blough, N. V.; Zafiriou, O. C. Inorg. Chem. 1985, 24, 3502-3504. (15) Mere´nyi, G.; Lind, J.; Eriksen, T. E. J. Biolumin. Chemilumin. 1990, 5, 53-56. (16) Lu, C.; Song, G.; Lin, J.-M. Trends Anal. Chem. 2006, 25, 985-995. (17) Kambayashi, Y.; Ogino, K. J. Toxicol. Sci. 2003, 28, 139-148.

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Analytical Chemistry, Vol. 80, No. 4, February 15, 2008

Akutsu et al.18 for a detailed reaction scheme). The contribution of superoxide alone can be obtained by addition of a singlet oxygen scavenger such as azide or by eliminating the source of any singlet oxygen production (e.g., by removing samples from the light) immediately prior to analysis, since its lifetime (4 µs in water19) is much shorter than that of superoxide. In the case of CLA and MCLA, autoxidation of the anionic form of the parent compound by ground state dioxygen also occurs to some extent, producing background chemiluminescence whose magnitude must be determined in order to accurately quantify the amount of superoxide-mediated CL. However the autoxidation product is a caged CLA or MCLA radical such that only a very small amount of free superoxide is released into the reaction matrix, even though the overall background CL may be quite high (see Fujimori et al.20 for a detailed reaction scheme). Thus, under appropriate conditions it is relatively simple to correct for CL produced by autoxidation and to obtain an accurate determination of superoxide in the sample. MCLA CL has previously been used with flow injection analysis to determine concentrations of hydroperoxyl in atmospheric water droplets after trapping it in an alkaline solution.21 While this previous study demonstrated the effectiveness of MCLA CL for determination of hydroperoxyl (and thus superoxide) at concentrations as low as 120 pM, the calibration technique employed 60Co and is unsuitable for field studies. In addition, while the technique was shown to be insensitive to interference from low concentrations of H2O2 and O3, seawater can contain many more potential interferences (e.g., reduced metals). In this work, we use MCLA CL as the basis for determination of superoxide concentrations and decay rates in seawater. The major aims of the work are to establish simple and accurate protocols for superoxide determination in seawater and to verify the efficacy of the method for use in the field. EXPERIMENTAL SECTION Chemiluminescence Analysis. Samples were continuously drawn via peristaltic pump into a FeLume chemiluminescence system (Waterville Analytical) where they were mixed with MCLA reagent in a spiral glass flow cell positioned in front of a photomultiplier tube. The delay between the sample and the flow cell was 15-60 s (depending on the pump speed and tubing size) and was minimized by pumping the reagent and waste streams such that the sample stream was drawn directly into the instrument without passing via the pump. The pump was operated at 5-10 rpm, corresponding to flow rates of 0.5-1.0 mL min-1 that were equal for sample and reagent streams. MCLA reagent consisted of 5 µM 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[l,2-a]pyrazin-3(7H)-one hydrochloride (MCLA; TCI America, >95.0% purity) in 50 mM sodium acetate (Sigma) with 50 µM diethylenetriaminepentaacetate (DTPA; Fluka) adjusted to pH 6.0 with analytical grade HCl (Sigma). Upon receipt, MCLA (10 mg) was dissolved in 14 mL of 18.2 MΩ cm resistivity Milli-Q water, (18) Akutsu, K.; Nakajima, H.; Katoh, T.; Kino, S.; Fujimori, K. J. Chem. Soc., Perkin Trans. 2 1995, 1699-1706. (19) Wilkinson, F.; Helman, W. P.; Ross, A. B. J. Phys. Chem. Ref. Data 1995, 24, 663-677. (20) Fujimori, K.; Nakajima, H.; Akutsu, K.; Mitani, M.; Sawada, H.; Nakayama, M. J. Chem. Soc., Perkin Trans. 2 1993, 2405-2409. (21) Zheng, J.; Springston, S. R.; Weinstein-Lloyd, J. Anal. Chem. 2003, 75, 46964700.

Table 1. Potential Interferences Tested

redox couplea

compound tested

manufacturer and product no.

primary stock concn (mM)

3+ HCrO4 /Cr 2SeO4 /SeO23 NO3 /NO2 Cu2+/Cu+

Cr(NO3)3‚9H2O Na2SeO3 NaNO2 CuBr

Aldrich 379972 Aldrich 214485 Sigma S2252 Aldrich 254185

1 1 1 2.4

Sb(V)/Sb(III)

SbCl3

Sigma-Aldrich 311375

0.82

3+ VO+ 2 /V + VO2 /VO2+

VCl3 VOSO4‚xH2O

Aldrich 208272 Aldrich 204862

5.9 0.47

MoO4 /Mo(V)

MoCl5

Aldrich 642452

0.49

IO3 /I MnO2/Mn2+

KI MnCl2‚4H2O CoCl2‚6H2O Fe(NH4SO4)2‚6H2O NaAsO2

Ajax Finechem 610136 BDH 10152 Ajax Chemicals 110868 Sigma F2262 Aldrich 40,737-2

1 1 1 1 1.8

CoOOH (s)/Co2+ Fe(OH)3 (s)/Fe2+ H3AsO4/HAsO2 a

From Hopkinson and Barbeau.22

b

secondary stock concn (µM)

10

59

1 1 1

solvent

special conditionsb

0.1 M HCl Milli-Q water Milli-Q water 5 mM HCl (deoxygenated) ethanol (deoxygenated) 0.1 M HCl 0.1 M HCl (deoxygenated) ethanol (deoxygenated) Milli-Q water 0.1 M HCl 0.1 M HCl 0.1 M HCl Milli-Q water

none avoid strong acids none prepare and store under inert gas prepare and store under inert gas none prepare and store under inert gas prepare and store under inert gas none none none none avoid strong acids

Consult MSDS for full details.

divided into 1 mL (≡ 2.5 µmol MCLA) aliquots and frozen at -80 °C. MCLA reagent was prepared in an acid-cleaned amber glass or opaque HDPE bottle from stocks of frozen MCLA, 500 mM sodium acetate, and 5 mM DTPA with Milli-Q water, stored at 4 °C when not in use, and replaced at least weekly. DTPA was added to this and other reagents (as described) to inhibit superoxide decay by forming superoxide-unreactive complexes with any trace metals present in the solution.8 Effect Of Superoxide Dismutase On Background Chemiluminescence. Superoxide-free seawater was prepared by adding 500 Unit L-1 SOD (from bovine erythrocytes; Sigma) to 0.22 µm filtered seawater from Sydney Offshore Reference Station (SORS) and allowing it to stand overnight in an opaque, acid-cleaned HDPE bottle. Subsequently, SOD was added at concentrations of 10, 20, 30, 40, and 50 kUnit L-1 to superoxide-free seawater samples and the FeLume signal recorded for 120 s. A standard addition of 50 nM superoxide generated via the photochemical method (described below) was also performed in a sample of superoxide-free seawater to establish the magnitude of background CL relative to superoxide-induced CL. Interference Studies. Primary stock solutions of potentially interfering compounds (the same as those chosen by Hopkinson and Barbeau22 for similar tests with the luminol method for Fe(II) determination in seawater) were prepared in polypropylene centrifuge tubes by dissolving a preweighed quantity of the solid in 50 mL of the appropriate solvent under the conditions described in Table 1. Secondary stock solutions, where necessary, were prepared by serial dilution of primary stock solutions with the same solvent. Solutions were prepared within 12 h of use (and typically immediately prior to use), as many of the compounds are unstable in solution. Interference tests were conducted by standard additions into 0.22 µm filtered seawater from SORS to give initial concentrations that were at least an order of magnitude greater than the total concentration of the element typically found in seawater.22 FeLume signals were then monitored for 120 s after (22) Hopkinson, B. M.; Barbeau, K. A. Mar. Chem., 2007, 106 (1-2), 2-17.

the standard addition. Standard additions of superoxide that was generated via the photochemical method23 (described in the following section) were also performed for comparison. In the case that a signal that was significantly different from the baseline was observed, 50 kUnit L-1 SOD (from bovine erythrocytes; Sigma) was also added in the presence of the potential interference and further standard additions were conducted (in the absence of SOD) with lower concentrations. Calibration Using Photochemically-Generated Superoxide Standards. Superoxide stock solutions were generated by UV photolysis of an alkaline aqueous solution of acetone and ethanol according to the method of McDowell et al.,23 with modifications as described previously.6 Superoxide was generated by irradiating a 1 cm path length quartz fluorescence cuvette containing the photolysis solution using a low-pressure mercury pen-lamp (PenRay) attached to a cuvette holder (Waterville Analytical). The superoxide concentration was quantified continuously during photolysis by UV spectrophotometry performed at a 90° angle to irradiation from the pen-lamp using a USB4000 spectrometer coupled to a USB-DT lamp (Ocean Optics Inc.). When the superoxide concentration in the photolyzed solution had reached 50 µM, an aliquot of appropriate volume was quickly withdrawn by a pipettor and a standard addition made to a seawater sample for immediate analysis in the FeLume. The FeLume signal (corresponding to decay of superoxide from the standard addition) was recorded for 5-10 min, then 50 kUnit L-1 SOD (from bovine erythrocytes; Sigma) was added to remove any remaining superoxide from the solution and thereby establish the magnitude of background CL. The FeLume signal at the time of the standard addition was determined by extrapolation of the signal to time zero by either a log-linear transform of the signal data (when superoxide decay was determined to follow first-order kinetics) or by a plot of the reciprocal of the signal data versus time (when superoxide decay was determined to follow second-order kinetics). (23) McDowell, M. S.; Bakacˇ, A.; Espenson, J. H. Inorg. Chem. 1983, 22, 847848.


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A calibration curve was subsequently constructed by regression of time zero signals resulting from standard additions of various concentrations of photochemically generated superoxide. Calibration Using Xanthine And Xanthine Oxidase. As an alternative calibration method, the xanthine/xanthine oxidase (X/ XO) system was used by exploiting the fact that XO enzymatically mediates the reduction of oxygen by xanthine to produce superoxide. The superoxide concentration in a solution to which X/XO has been added increases until the rate of superoxide decay equals the rate of its production by X/XO. The resulting FeLume signal at steady-state can then be related to the steady-state superoxide concentration, provided this quantity can be calculated. Calibration using the X/XO method was performed as follows. Xanthine (Sigma) was added at 50 µM to seawater samples that had been allowed to stand for at least 30 min to allow any superoxide to decay to near the instrument detection limit. Xanthine oxidase (Sigma X4376) was then added into 10-20 mL of a seawater-xanthine solution at nominal concentrations between 0.1 and 5 Unit L-1 (based on the manufacturer’s specification) in duplicate and the solution analyzed using the FeLume. The FeLume signal was monitored until steady-state had been reached and the resulting steady-state superoxide concentration determined by analysis of the kinetics of the FeLume signal over time. Xanthine Oxidase Activity. The enzymatic activity of XO was determined according to the manufacturer’s assay, based on the definition of 1 Unit as the amount of XO that converts 1.0 µmol of xanthine to urate per minute at pH 7.5 and 25 °C. Briefly, 10 µL of XO solution (with approximate activity of 100-200 Unit L-1) was added to a 1 cm path length quartz cuvette containing 2.99 mL of 50 mM KH2PO4 and 50 µM xanthine that had been adjusted to pH 7.5 with 1.0 M HCl (Fluka) and warmed to 25 °C in a water bath. The cuvette was placed in a Cary 50 Bio spectrophotometer and the absorbance monitored at 292 nm for 5 min. The rate of urate production was then calculated by linear regression of the data and converted to concentration based on a molar absorptivity at 292 nm of 11 000 M-1 cm-1.24 The activity of XO in seawater solutions as a function of pH was also determined by UV spectrophotometry. Solutions of 0.22 µm filtered SORS seawater containing 50 µM DTPA and 50 µM xanthine were prepared and left to equilibrate in the dark overnight. Subsequently, the pH of the solution was adjusted to approximately pH 9 and 10 mL added into an acid-cleaned polypropylene tube along with a nominal concentration of 2 Unit L-1 XO based on the manufacturer’s specification. A 2 mL aliquot of the solution was then transferred to a 1 cm path length quartz cuvette and positioned in a cuvette holder with optical fiber connections to an Ocean Optics DT-1000 lamp and USB4000 spectrometer. The spectrometer was zeroed and the absorbance of the solution monitored at 292 nm for 5 min. The pH of the solution was then adjusted by approximately 0.2 units by addition of 1.0 M HCl, and the procedure was repeated over the pH range 7-9. The pH of the solution was measured using a Hanna pH meter (previously calibrated with NIST-traceable buffers) before and after each addition of HCl, and the entire solution was maintained at 25 ( 0.2 °C by immersion in a water bath. The rate of urate production was determined in the same way as described (24) Bergmann, F.; Dikstein, S. J. Am. Chem. Soc. 1955, 77, 691-696.

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for the standard assay of XO activity, since the molar absorptivity of urate is constant over the pH range 7-9.25 Superoxide Production Rate By Xanthine/Xanthine Oxidase. To correctly analyze the kinetics of the X/XO calibration method, it was necessary to know the rate of superoxide production by the X/XO system. This was determined in the presence of 50 µM xanthine as a function of pH as follows. Xanthine was dissolved in 0.22 µm filtered SORS seawater (pH 8.05) at a final concentration of 50 µM and DTPA added at a final concentration of 50 µM. The seawater was left to equilibrate with the DTPA overnight. XO was then added into 20 mL of a seawater-xanthine solution at nominal concentrations (based on the manufacturer’s specification) of 1 Unit L-1, 2 Unit L-1, and 5 Unit L-1 in duplicate and the superoxide concentration monitored for 10 min using the FeLume. The FeLume was then calibrated using photochemically generated superoxide standards as described. The superoxide generation rate of XO at each added concentration was determined from analysis of the kinetic data as described in Results and Discussion. Actual XO activity was determined using the standard assay described previously. Superoxide generation rates were then normalized to actual XO activity and averaged. This procedure was repeated in seawater that had been adjusted to pH 7.02, 7.54, and 8.51 using high purity 32% w/v HCl (Fluka) and 10 M NaOH (Fluka). Calculated rates of superoxide production by the X/XO system were also verified using an independent method involving reduction of nitro blue tetrazolium (NBT; Sigma) as follows. DTPA was added to 0.22 µm filtered SORS seawater at a final concentration of 50 µM along with 50 µM xanthine in an acid-cleaned opaque plastic bottle and left to equilibrate overnight. XO was added at a nominal concentration (based on the manufacturer’s specification) of 2 Unit L-1 to 1 mL of the solution along with 10 µL of 10 mM NBT stock solution (previously prepared in Milli-Q water and stored at 4 °C when not in use), and 1 mL drawn via syringe into a 1 m path length liquid waveguide capillary cell (World Precision Instruments) connected via an optical fiber to a USB4000 spectrometer and LS-1 tungsten halogen light source (Ocean Optics). The instrument was then zeroed and the absorbance of the solution monitored at 530 nm where the monoformazan that is formed from superoxide-mediated reduction of NBT absorbs most strongly.26 The absorbance of the solution at 720 nm was also monitored and subtracted from the signal at 530 nm to account for both variations in the incident light over time and possible absorbance by the NBT radical that is formed as an intermediate during the process (the NBT radical absorbs equally at 720 and 530 nm, while the monoformazan absorbs negligibly at 720 nm).26 After 5 min, the solution was pushed back out of the waveguide, 10 µL of 5000 kUnit L-1 SOD (from bovine erythrocytes; Sigma) was added, and the solution was drawn back into the waveguide and monitored for a further 5 min. The rate of monoformazan formation in the presence of SOD was subtracted from the rate of monoformazan formation in the absence of SOD to determine the rate of superoxide-induced monoformazan formation. A small amount of 1.0 M HCl was then added to the original seawater-xanthine-NBT solution to lower the pH by approximately 0.2 units, the pH was measured using a Hanna (25) Rubbo, H.; Radi, R.; Prodanov, E. Biochim. Biophys. Acta 1991, 1074, 386391. (26) Bielski, B. H. J.; Shiue, G. G.; Bajuk, S. J. Phys. Chem. 1980, 84, 830-833.

pH meter (previously calibrated with NIST-traceable buffers), and the procedure was repeated. This was performed repeatedly to obtain superoxide production and xanthine oxidation rates over the pH range 7-9. Field Sampling And Analysis. Field samples were obtained on a cruise on the R/V Knorr in the Costa Rica Dome (CRD) upwelling region of the equatorial Pacific Ocean in August 2005. Samples were collected from various depths using acid-cleaned Go-Flo bottles deployed on a Kevlar line. The bottles were subsequently pressurized with high purity nitrogen and samples rapidly filtered through 0.45 µm polycarbonate membranes into a 50 mL polypropylene tube containing 50 µM final concentration of DTPA (Fluka). The sample was immediately drawn into a FeLume system where it mixed with MCLA reagent. The chemiluminescence signal was monitored for 10-15 min to observe superoxide decay in the filtered sample and background chemiluminescence, due to MCLA autoxidation, was subtracted. The initial superoxide concentration in the sample was determined by linear extrapolation of ln(signal) vs time (based on the observation of first-order superoxide decay kinetics) back to the time at which the sample was filtered, which was typically 30-60 s prior to obtaining a stable FeLume signal. The instrument was calibrated independently for each sample using the X/XO method by adding 50 µM xanthine to a 10 mL aliquot of the sample and allowing it to sit for at least 30 min with periodic shaking (to ensure it was fully reoxygenated) prior to addition of a known concentration of XO and monitoring of the resulting FeLume signal for 5-10 min. Safety Considerations. Most of the compounds used in the interference studies are hazardous and must be prepared under appropriate conditions. In particular, SbCl3 and MoCl5 are air and water sensitive and must be prepared in nonaqueous solvent under an inert gas. V(III) must be handled in a fume hood due to generation of corrosive vapors on exposure to air. Na2SeO3 and Na2AsO2 must be prepared in Milli-Q water as contact with strong acids liberates toxic gases. Relevant Material Safety Data Sheets (MSDS) should be consulted for full details. RESULTS AND DISCUSSION System Performance. As with most CL based methods, optimal performance of the system depends primarily on reaction pH, reagent concentration, and flow rate. On the basis of the previous work of Zheng et al.21 and knowledge of the reaction kinetics of superoxide with CLA,18 which we expect to be very similar to those of MCLA, we have employed conditions that should theoretically be near optimal for the performance of our system. Because of the high specificity of MCLA for superoxide in our system (verified by interference studies described later), optimal analytical performance on the basis of signal-to-noise ratio (SNR) should correspond to the maximum ratio of superoxideinduced CL to autoxidation-induced CL, given that the latter will typically be well above the instrument noise signal. Superoxideinduced CL will be maximal when superoxide reacts essentially quantitatively with MCLA. To ensure that this occurs, reaction of superoxide with MCLA must out-compete possible side-reactions of superoxide, whose rate will (in general) increase with decreasing pH since hydroperoxyl is generally more reactive than the superoxide anion.11 In contrast, autoxidation-induced CL of MCLA decreases with decreasing pH, similarly to that observed for CLA.20 We have previously established that a reaction pH of 6.0 is optimal

when using 5 µM MCLA. It may be possible to further improve the SNR by employing a higher MCLA concentration at lower pH, since the higher MCLA concentration should counteract the higher apparent rate constants for side-reactions yet the rate of autoxidation will decrease. However CL typically declines at MCLA concentrations above about 15 µM,21 likely due to strong absorption of CL at 430 nm by MCLA.27 Additionally, lower MCLA concentrations are more economical as MCLA is quite expensive. Considering a fixed reaction pH of 6.0, the primary requirement for optimizing the reagent concentration and flow rate is that superoxide should react quantitatively with MCLA to produce photons inside the flow cell. At pH 6.1, CLA reacts with superoxide with an apparent rate constant of 4.5 × 105 M-1 s-1.18 Assuming that MCLA reacts with superoxide at pH 6.0 with a similar rate constant, and given that the rate constant for superoxide disproportionation at this pH is 6 × 105 M-1 s-1,11 then a MCLA concentration of >1 µM will ensure that >99.9% of superoxide reacts with MCLA provided that there are no other side-reactions for superoxide. In reality, superoxide will typically also undergo side-reaction with other compounds in the sample such as Fe and Cu. Since this will vary from sample to sample, it may be necessary to increase the MCLA concentration to minimize such sidereactions. In this study, 5 µM MCLA was chosen as a suitable concentration but was not necessarily optimal for all samples. The final parameter that is expected to affect system performance is the flow rate. To ensure that CL occurs entirely inside the flow cell, the flow rate must be sufficiently slow to ensure the reaction of superoxide with MCLA is complete within the residence time of the sample in the flow cell. Again assuming that MCLA reacts with superoxide at a similar rate to CLA, and provided that MCLA is in considerable excess of superoxide, the pseudo-first-order rate constant for the reaction of superoxide with 5 µM MCLA is 2.3 s-1. On the basis of a flow cell volume of 320 µL and a likely detection limit of no better than 10 pM superoxide21 (and thus considering superoxide to have quantitatively reacted with MCLA when its concentration falls to 10 pM in a slug of reaction mixture in the flow cell), we calculate minimum required residence times of 2.0 s for quantitative scavenging of 1 nM superoxide and 4.0 s for 100 nM superoxide. Therefore, provided that the total flow rate of reagent and sample is 7, addition of 1.12 mg L-1 SOD causes no appreciable change in CL due to autoxidation of CLA, while between pH 5.5 and pH 7 there is a very small decrease in CL. To investigate the effect of SOD on background (27) Kimura, H.; Nakano, M. FEBS Lett. 1988, 239, 347-350.


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Figure 1. Effect of superoxide dismutase on chemiluminescence due to MCLA autoxidation. Error bars represent the range of duplicate experiments.

CL of MCLA in our system, we added SOD at concentrations ranging from 10 to 50 kUnit L-1 to seawater samples (resulting in SOD concentrations of 5-25 kUnit L-1 in the mixed sample and reagent in the flow cell) and recorded the FeLume signal. Seawater samples already contained 500 Unit L-1 SOD, which had been added approximately 24 h earlier to ensure there was no superoxide present in the seawater during the experiment. As shown in Figure 1, there was no systematic effect of increasing SOD concentration on background CL. The standard deviation of the variation in signal with the different SOD concentrations was 180 counts s-1, equivalent to approximately 50 pM superoxide (based on a single standard addition of 50 nM photochemically generated superoxide and assuming a linear calibration curve). As the specific activity of SOD used by Fujimori et al.20 was not reported, we were unable to directly compare these results with those for CLA. However addition of 50 kUnit L-1 SOD to seawater samples has no significant effect on the background CL due to autoxidation of MCLA at the pH (6.0) of the reaction mixture. Addition of up to 50 kUnit L-1 SOD to samples therefore allows determination of background CL, which can be subtracted from the signal obtained during sample analysis to determine the signal due solely to superoxide in the sample. Potential Interferences. To verify the specificity of MCLA for superoxide in our system, we examined the FeLume response to standard additions of various redox-active elements that may be present in seawater at potentially interfering concentrations, based on similar testing of the luminol method for Fe(II) determination in seawater by Hopkinson and Barbeau.22 Reduced compounds were tested by Hopkinson and Barbeau22 when they could occur at concentrations >100 pM and had redox potentials that could allow their redox cycling to occur in marine waters ranging from fully oxygenated to oxygen depleted (but where sulfide is not present). We did not test oxidized forms of these compounds because laboratory work has shown no noticeable difference between background CL signals obtained in seawater samples (which would contain the oxidized forms of the compounds tested) and those obtained in solutions of 2 mM NaHCO3 with 0.1 M NaCl in Milli-Q water. Since the background CL signal in seawater samples is stable for several hours, which is much longer than the amount of time taken to analyze each sample, and is not affected by the addition of SOD, even if the oxidized 1220


forms of these compounds in seawater do elicit some CL it is routinely subtracted during the determination of background CL. As shown in Table 2, the majority of the compounds that were specifically tested did not elicit responses that were substantially different from background noise even at extremely high concentrations. However high concentrations of Cu(I), V(III), V(IV), Mo(V), and Fe(II) did generate signals that were comparable to that obtained from 10 nM superoxide. Addition of 50 kUnit L-1 SOD quenched the resulting signals by 72%, 93%, 43%, 68%, and 94%, respectively, indicating that the majority of the signal was being produced by superoxide rather than direct interaction of the compounds with MCLA. This conclusion is also supported by the observation that signals resulting from each compound decayed in an approximately first-order manner over the 90-120 s after their addition to seawater, which suggests the likelihood that the compounds were oxidized by dissolved oxygen in the seawater to produce superoxide. Although addition of 50 kUnit L-1 SOD did not fully quench the signal to background levels in the cases of Cu(I), V(III), and V(IV), this is expected when oxidation of the added metal is producing superoxide at a rate that is comparable to the rate of superoxide decay by SOD, resulting in a nonzero quasi-steady-state concentration of superoxide over the duration of the tests. Thus, it is highly likely that the entire signal induced by these metals was due to production of superoxide, rather than due to their direct interaction with MCLA. In addition, when these “interfering” metals were added at concentrations closer to their expected maximum concentration in seawater, the magnitude of the signal produced was much smaller and in many cases indistinguishable from the background. Further support for the proposition that the “interfering” species was superoxide is that addition of the metals produced signals that did not exceed the signal expected from addition of an equivalent concentration of superoxide. (Note that although the signal produced by 10 nM Fe(II) is more than twice the signal produced by 10 nM superoxide, this is due to the fact that a significant amount of superoxide added from the photochemically generated stock decayed in the ∼15 s between its standard addition to seawater and reaching the FeLume flow cell). Although this condition alone is not sufficient to demonstrate that superoxide is the species actually detected by the MCLA method, it must be satisfied if this proposition is correct. Therefore, although high concentrations of several metals did result in a substantial signal from the FeLume, this signal was almost certainly due to superoxide that was produced after standard addition of the metal to seawater. In practice, it is necessary to account for the possibility that superoxide is produced in the sample from oxidation of these reduced metals by oxygen when interpreting analytical results from field samples, particularly if the presence of relatively high concentrations of the metals is known or suspected. The fact that significant concentrations of superoxide are generated on a time scale of a few seconds when these metals are added to seawater indicates that they are unlikely to accumulate in high concentrations in oxygenated waters. However this phenomenon may be problematic if samples from sub-oxic zones are reoxygenated to a significant extent during analysis. Calibration Using Photochemically-Generated Superoxide Standards. Data illustrating the FeLume signal resulting from

Table 2. FeLume Response to Potential Interferences redox couplea

compound tested

seawater concn (nM)a

blank superoxide (control)c 3+ HCrO4 /Cr SeO2/SeO 4 3 NO3 /NO2 2+ + Cu /Cu

Cr(NO3)3‚9H2O Na2SeO3 NaNO2 CuBr

Sb(V)/Sb(III) 3+ VO+ 2 /V

SbCl3 VCl3

2 40

2+ VO+ 2 /VO

VOSO4‚xH2O

40

MoO4 /Mo(V)

MoCl5

100

IO3 /I MnO2/Mn2+ CoOOH (s)/Co2+ Fe(OH)3 (s)/Fe2+

KI MnCl2‚4H2O CoCl2‚6H2O Fe(NH4SO4) ‚6H2O

500 0.8 0.1 0.7

H3AsO4/HAsO2

NaAsO2

4 2 2000d 2

20

concn tested

FeLume response (relative to 10 nM superoxide)b

20 nM 10 nM 5 nM 100 nM 100 nM 10 µM 100 nM 10 nM 1 nM 100 nM + SOD 100 nM 1 µM 100 nM 10 nM 1 µM + SOD 1 µM 100 nM 1 µM + SOD 1 µM 100 nM 1 µM + SOD 10 µM 10 nM 10 nM 10 nM 1 nM 100 pM 10 nM + SOD 1 µM

0.16 5.8 1.0 0.43 0.18 0.18 0.19 13 2.8 1.4 3.5 0.57 28 2.6 0.48 1.9 4.5 0.39 2.6 1.2 0.37 0.39 0.20 0.25 0.23 2.6 0.37 0.74 0.14 0.26

a From Hopkinson and Barbeau.22 b FeLume response was determined as the difference between the maximum PMT output during the 120 s of analysis and the average signal over the first 30 s of analysis (prior to the standard addition). The relative response was then determined by comparison to the response to a standard addition of 10 nM superoxide. The blank signal is therefore nonzero due to baseline noise. c Superoxide was generated by the photochemical method. d Typical concentration in sub-oxic zones where denitrification is occurring.22

standard additions of 5, 10, and 25 nM photochemically generated superoxide to 0.22 µm filtered SORS seawater are shown in Figure 2. In order to obtain the signal at time zero, it is necessary to extrapolate the FeLume signal backward to account for the lag time for the sample to enter the flow cell. In general, this is best achieved by use of a log-linear plot of signal versus time, i.e., by assuming pseudo-first-order decay kinetics for superoxide. An alternative method for extrapolation is to plot the reciprocal of the FeLume signal (after subtracting the background signal) against time if superoxide is known to be decaying primarily via second-order disproportionation, i.e., via the apparent reaction +2H +

O2 + O2 98 O2 + H2O2, k1

(1)

Note that this reaction is apparent because the actual reacting species are superoxide and its conjugate acid, hydroperoxyl radical (HO•2). Since the concentration of HO•2 decreases by 1 order of 11 magnitude per pH unit above the pKa for HO•2/O2 () 4.8), the rate constant for the apparent reaction in eq 1 thus varies with pH in seawater according to the equation10

k1 ) (5 × 1012)[H+] M-1 s-1

(2)

This latter extrapolation method is suggested only for standard additions of superoxide at concentrations >25 nM, since even

small variations at low signal values can result in large errors in the extrapolated value due to the nature of the reciprocal plot. Even if decay is truly second-order, at superoxide concentrations 0.15 s-1) and requires the availability of additional equipment. In contrast, the X/XO method is easier to implement but requires more complicated data analysis, and particular care must be taken to ensure that rates of superoxide generation by the enzyme are accurately known. In either case, calibration is required for each sample during field application due to the possibility of matrix effects on the efficiency of CL generation during the analytical procedure. ACKNOWLEDGMENT This work was funded by ARC Discovery Grant DP0558710 to A.L.R. and T.D.W. and a UNSW Faculty of Engineering Early Career Researcher Grant to A.L.R. We thank Mak Saito for the invitation to participate in the CRD cruise and Tyler Goepfert for assistance with shipboard activities. This work could not have been conducted without the assistance of other scientists and crew on the R/V Knorr. Received for review September 10, 2007. Accepted November 23, 2007. AC7018975


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Determination of Superoxide in Seawater Using 2-Methyl-6-(4

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