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Keywords:

  • superoxide radical;
  • Atacama Desert

Abstract

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Materials and Methods
  5. 3. Results and Discussion
  6. Acknowledgments
  7. References

[1] Soils on Mars are thought to accumulate superoxide radicals, as they are formed continually by UV radiation hits on basalt minerals. Yet, beyond electron paramagnetic resonance (EPR) spectroscopy, no other methods exist that detect and quantify superoxide radicals in soils. This study describes two such assays that are based on the reaction of superoxide radical with hydroethidine and oxidized cytochrome c. They are unique in being able to test unambiguously for the presence of superoxide in a soil matrix with a high level of accuracy. Applications with soils from the Atacama Desert showed that the sensitivity of the assays is equal to or greater than that of EPR spectroscopy.

1. Introduction

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Materials and Methods
  5. 3. Results and Discussion
  6. Acknowledgments
  7. References

[2] The Viking missions to Mars in 1976 carried three life detection instruments and a gas chromatograph-mass spectrometer (GCMS) for organic analysis. The results of these instruments were puzzling in three respects. First, was the absence of organics as measured by the GCMS [Biemann, 1979; Biemann et al., 1977]. While the detection sensitivity of the instrument was ppb for organics, Navarro-Gonzalez et al. [2006] have shown that the pyrolysis step limited the detection sensitivity in the soil to ∼ppm levels. The second unexpected result was the rapid release of O2 when soil samples were exposed to water vapor in the Gas Exchange Experiment (GEx) at levels of 70–770 nanomoles per gram [Oyama and Berdahl, 1977]. The third unexpected result was that organic material in the Labeled Release (LR) Experiment was consumed precisely as would have been expected if life was present [Levin and Straat, 1977] -the presence of life being in apparent contradiction with the GCMS results. Although a ppb level of organic material corresponds to more than a million cells per gram soil [Glavin et al., 2001; Klein, 1978] this is true only if there is no extracellular organics present. In most soils there is much more extracellular organic material than living biomass. So the GCMS results do not support a biological explanation. Currently, the most widely held explanation for the reactivity of the Martian soil is the presence of more than one inorganic oxidants [Klein, 1978, 1979; Quinn and Zent, 1999; Zent and McKay, 1994]. A plausible source of the oxidation is H2O2 produced in the atmosphere. However the GEx results cannot be explained by H2O2 alone. Presumably atmospheric H2O2 reacts with soil components creating a series of peroxides and superoxides [Quinn and Zent, 1999].

[3] Superoxide radical was recently proposed as a possible Mars oxidant [Yen et al., 2000]. It is a very reactive molecule (half life 1 μs) and can degrade organics such as polychlorobiphenyls at low temperatures (60–75°C) [Matsunaga et al., 1991]. Moreover, superoxide radical dismutates to molecular oxygen when exposed to moisture, so it can explain one of the Viking results. The possible formation of superoxide radicals on Mars was demonstrated by exposing labradorite to UV under a simulated Mars atmosphere [Yen et al., 2000]. The energetic UV photons eject electrons from the mineral surface, which are subsequently captured by oxygen molecules. The resultant superoxide radical ions, adsorbed on the mineral surfaces, were detected by electron paramagnetic resonance (EPR) spectroscopy. Superoxide radicals can be also formed by UV-irradiation of TiO2 [Konaka et al., 1999], which was identified (together with other metal oxides) in Mars soil during the Pathfinder mission [Foley et al., 2000; Rieder et al., 1997].

[4] Given the high UV influx on Mars, the formation of superoxide radicals there is likely an ongoing process. Under the dry Mars environment, these radicals may diffuse into deeper soil horizons [Yen et al., 2000], where they may convert into a relatively stable form of inorganic superoxides such is potassium superoxide (KO2). Therefore, detecting superoxide radical is key to understanding the soil organic chemistry and biology on Mars. Currently, the only technology being considered for use on Mars is EPR spectroscopy. There is a need for corroborative methods that can detect superoxide with higher sensitivity and specificity.

[5] In this study, we present two such assays. The first assay is based on the specific and stoichiomitric (1:1 molar) reaction of superoxide radical with hydroethidine and the formation of the fluorescent product 2-OH-ethidium, which can be detected with high sensitivity [Georgiou et al., 2005]. The second assay is based on the reaction of superoxide radical with oxidized cytochrome c. The equimolar formation of reduced cytochrome c is measured photometrically [McCord and Fridovich, 1969]. Both assays were tested on Mars-like soils collected from the hyper arid Atacama Desert [Navarro-Gonzalez et al., 2003]. The assays were performed on soil samples spiked with solid potassium superoxide and superoxide solution made in dimethyl sulfoxide (DMSO). Finally, the assays were used to determine whether there are any naturally formed superoxide radicals in the Atacama Desert soil.

2. Materials and Methods

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Materials and Methods
  5. 3. Results and Discussion
  6. Acknowledgments
  7. References

2.1. Reagents

[6] Xanthine (X) was obtained from Serva (Heidelberg, Germany). Buttermilk xanthine oxidase (XO), hydroethidine (HE), horseradish peroxidase (HRP), superoxide dismutase (SOD) from bovine erythrocytes, DNA (type III from salmon testes), horse heart cytochrome c, sodium dithionite, potassium superoxide, diethylenetriaminepentaacetic acid (DTPA), and Dowex 50X-8 (mesh 400) were obtained from Sigma (St. Louis, MO, USA). Anhydrous dimethyl sulfoxide (DMSO), chloroform, acetonitrile (ACN), absolute methanol (MetOH), hydrogen peroxide, Tris-HCl, and trifluoroacetic acid (TFA) were obtained from Merck (Darmstadt, Germany). Hydrophobic Oasis HLB 1 cm3 (30 mg) extraction cartridges were obtained from Waters (Milford, MA, USA). All reagents and solvents used were of the highest purity.

2.2. Superoxide Radical Sources

[7] The following superoxide radical sources are used in mixture with soil samples from Atacama: (I) DMSO-superoxide stock solutions produced (a) by alkalinization of DMSO and (b) by dissolving solid potassium superoxide in DMSO [Hyland and Auclair, 1981], and (II) solid potassium superoxide.

[8] I(a) Superoxide-DMSO stock solution produced by alkalinization of DMSO: Anhydrous DMSO (995 μl) is mixed with 5 μl 1 N NaOH (in a 1.5 ml-eppendorf tube) and is sonicated for 30–60 sec at 350 W cm−2, using sonicator (model UP-50 H, Dr. Hielscher, GmbH, Teltow, Germany) with 2 mm diameter microtip (type MS2) placed in the center of the NaOH//DMSO solution at 1.5 cm depth, followed by centrifugation at 15,000 g for 5 min. An identical superoxide stock solution, the water content of which is adjusted to 1.5% in order to induce superoxide destruction by dismutation, is used as a blank. Superoxide concentration in the supernatant (stock solution) is determined by using its extinction coefficient 2686 M−1 cm−1 at 256 nm [Hyland and Auclair, 1981]. The concentration of the superoxide radical stock solution was determined to be 4 × 10−5 M.

[9] I(b) Superoxide-DMSO stock solution produced by dissolving potassium superoxide in DMSO: Anhydrous DMSO (2 ml) is mixed with 1.27 mg potassium superoxide and sonicated as above for 30–60 sec, followed by centrifugation at 15,000 g for 5 min. A blank is prepared in an identical manner to that described above. The concentration of superoxide radical in supernatant (stock solution) was determined as in case (a) to be 1.4 × 10−4 M.

[10] Superoxide radical stock solutions (a) and (b) are stored in tightly stoppered bottles to prevent superoxide destruction due to humidity absorption by DMSO. Various volumes from these stock solutions are mixed with Atacama soil samples and treated as described in the ‘Soil Treatment’ subsection.

[11] II. Solid potassium superoxide: Various amounts of it are mixed with Atacama soil samples and treated as described in the ‘Soil Treatment’ subsection. For example, 1 g of soil sample is mixed with 1 mg potassium superoxide, which produces 1.1 × 10−4 moles superoxide.

2.3. Soil Treatment

[12] Superoxide radical is recovered from the soil sample in anhydrous 100% DMSO as follows: The sample (having a weight ≤20 g in this study) is initially impregnated with DMSO in proportion 0.3 ml DMSO per g soil, which is optimum for 100% superoxide radical recovery (after trying different DMSO:soil ratios). Then, approximately 1 g batches of the soil/DMSO mixture are placed in 1 ml-spin filter (porous glass filter disc of nominal max. pore size 0.1–0.16 mm, by Heraeus Quarzglas GmbH & Co.) fitted in a 2 ml-eppendorf collection centrifuge tube (spin-filter/collection tube size depends on the instrumentation used to measure superoxide radical recovery). The sample is then sonicated continuously for 30–60 sec at 350 W cm−2, with the sonicator microtip placed in the center of the soil/superoxide/DMSO mixture at 1.5 cm depth. This sonication step is important for the dissociation of superoxide radical from the soil sample and its dissolution into DMSO. The filter/collection centrifuge tube assembly is then centrifuged at 15000 g for 5 min, and the superoxide/DMSO eluents of all batches are combined and used in the following assays.

2.4. HE-Superoxide Radical Assay

[13] Step A. Superoxide radical assay procedure: The superoxide/DMSO eluent (see ‘Soil Treatment’ subsection) is mixed with the following proportions HE and DTPA: 40μl 5 mM HE stock (1.58 mg HE dissolved in 1 ml 100% DMSO, N2-sparged and kept in a sealed 5-ml serum brown vial at −80°C for several weeks) and 3 μl 3 M DTPA aqueous stock per 1 ml DMSO-superoxide eluent. Under these conditions of low water content, superoxide radical rapidly reacts with HE to form 2-HO-ethidium. Specifically, in this mixture the final water content in DMSO is ∼0.4 % v/v (and DMSO 99.6%), and it is below the 0.9% water limit above which superoxide radical is very unstable since it reacts rapidly with water. Specifically, this water limit was determined by measuring superoxide in solutions with higher concentrations of water. The resulting 2-HO-ethidium/DMSO solution is treated as described in Step B below.

[14] To ensure that 2-HO-ethidium contained in the above 2-HO-ethidium/DMSO solution results solely from the superoxide radical of the soil sample, the following blank procedures are used to correct the superoxide radical value obtained from the soil sample in step E by subtraction.

[15] HE reagent-DMSO solvent-soil sample blank: This blank measures (a) 2-HO-ethidium present as contaminant in the solid HE commercial reagent, and (b) the extremely slight possibility that the soil might contain a contaminant that was isolated by this assay together with 2-HO-ethidium and has the same excitation/emission wavelengths as 2-HO-ethidium. This blank consists of the same soil amount used in its corresponding soil sample, impregnated with the corresponding volume of anhydrous DMSO. The soil/DMSO mixture is sonicated (as in the ‘Soil Treatment’ subsection), then water is added to a final DMSO/water ratio 98.5%/1.5% in order to destroy (by dismutation) any superoxide present, and finally it is mixed with HE and DTPA in proportions 40 μl HE stock and 3 μl DTPA stock per 1 ml DMSO. The resulting mixture is treated as in Step B.

[16] Sonication effect blank: This blank measures any artificially formed superoxide radical due to the sonication of the DMSO solvent (possibly containing dissolved molecular oxygen) as described in the ‘Soil Treatment’ subsection. This blank consists of the same volume of anhydrous DMSO used in its corresponding soil sample. DMSO is sonicated (as in the ‘Soil Treatment’ subsection), then water is added to a final DMSO/water ratio 99.1%/0.9% (in order for any superoxide radical formed during sonication to be preserved dissolved in the DMSO/water solvent), and finally it is mixed with HE and DTPA in proportions 40 μl HE stock and 3 μl DTPA stock per 1 ml DMSO. The resulting mixture is treated as in Step B.

[17] Step B. Isolation of 2-HO-ethidium by microcolumn cation exchange chromatography: The 2-HO-ethidium/DMSO solution from step A is brought to 70% DMSO with 0.2 M phosphate buffer, pH 7.0, and it is passed at a free flow rate through the Dowex 50X-8 cation exchange microcolumn prepared as described elsewhere [Georgiou et al., 2005]. Then, the microcolumn is washed in sequence with 1 ml 4 M NaCl (optional step), 1 ml distilled water, 2 ml 100% ACN, and 2 ml distilled water. The bound 2-HO-ethidium is eluted from the microcolumn with 1 ml 10 N HCl, and the eluent is diluted with distilled water to 3 N HCl.

[18] Step C. Purification of 2-HO-ethidium by microcolumn hydrophobic chromatography: The 2-HO-ethidium eluted from step B is subsequently passed through the HLB microcolumn at an approximate flow rate 2 ml min−1, after the column was activated as described elsewhere [Georgiou et al., 2005]. The microcolumn is first washed off with 1 ml 17% ACN-phosphate through the column to remove any fluorescent impurities. Subsequently, the bound 2-HO-ethidium is eluted with 1.5 ml 25% ACN-phosphate. The resulting eluent is mixed with 1.5 ml chloroform by vortexing, and the resulting chloroform/ACN layer (containing 2-HO-ethidium and possibly ethidium impurity from the HE stock solution) is collected. The 2-HO-ethidium in it is then concentrated by vacuum-evaporation of the chloroform/ACN solvent at room temperature.

[19] Step D. Fluorometric quantification of 2-HO-ethidium: The total fluorescence (F.U.total) of the vacuum-dried 2-HO-ethidium is due to both the 2-OH-ethidium collected from the soil sample plus any ethidium contaminant (from HE) being in mixture with 2-HO-ethidium. Therefore, the fluorescence of ethidium (F.U.Eth) in the mixture must be calculated after eliminating the fluorescence of 2-HO-ethidium by enzymically destroying it with HRP in the presence of hydrogen peroxide. The difference (F.U.total−F.U.Eth) between the two fluorescence values is due to the fluorescence of 2-HO-ethidium. For maximum assay sensitivity, the fluorescence of 2-HO-ethidium can be enhanced by complexation with DNA (it forms a fluorescent 2-HO-ethidium-DNA complex).

[20] Specifically, the 2-HO-ethidium/ethidium dry residue (from step C) is dissolved in 0.05 ml 50 mM phosphate buffer, pH 7.8, containing 1 mM DTPA and 6% DMSO. DTPA in this step (and in ‘Soil Treatment’ subsection) was used to chelate any metals from the soil sample that may inactivate the enzyme HRP used subsequently. In order to obtain maximum sensitivity for 2-HO-ethidium fluorescent quantification, its fluorescence (F.U.total) is measured in a final solution volume 0.3 ml using a fluorescence microcuvette with internal dimensions 4 × 4 × 45 mm in a Shimadzu RF-1501 spectrofluorometer, set at 10 nm excitation/emission slit width and high sensitivity. An additional increase in sensitivity is achieved by enhancing 25 fold the fluorescence of 2-HO-ethidium in the 0.3 ml solution by the addition of 0.02 ml 2 mg ml−1 DNA stock solution (to bring the final DNA conc. to approx. 0.15 mg/ml), and measuring the fluorescence (F.U.total) of the 2-HO-ethidium/ethidium-DNA complex at ex/em 515/567 nm. Subsequently, the same 0.3 ml solution is incubated for 1 min at RT after adding to it 0.025 ml 0.7 mM H2O2 stock (made in 50 mM phosphate buffer, pH 7.8, containing 1 mM DTPA) and 1 unit HRP (both required for 2-HO-ethidium fluorescence destruction), and the fluorescence (F.U.Eth) of the possibly present ethidium contaminant is measured. The fluorescence difference F.U.total−F.U.Eth corresponds to the actual fluorescence of 2-HO-ethidium, the concentration of which is determined by its fluorescence extinction coefficient (in the presence of DNA) described in the following step E.

[21] Step E. Conversion of the fluorescence of 2-HO-ethidium to superoxide radical concentration: The calculation of the fluorescence extinction coefficient of 2-HO-ethidium for the spectrofluorometer in use is done once in order to calibrate a known concentration of 2-HO-ethidium with its fluorescence in the presence of DNA. For this, a stock solution of 2-HO-ethidium is made in vitro by the X/XO superoxide generating system as follows: 0.1 ml 3.75 mM stock xanthine (made by dissolving 5.7 mg xanthine in 1 ml phosphate buffer containing 0.1 Í NaOH, and then diluting 10 times with phosphate buffer), 7 μl 5 mM HE stock (final HE conc. 35 μM) and 0.00825 units XO (i.e. 3 μl 33 Units/ml XO stock) are added to 0.9 ml 50 mM phosphate buffer, pH 7.8, and incubated for 30 min at RT. The concentration (35 μM) of the resulting 2-HO-ethidium in this stock solution is equal to the concentration of consumed HE, since it has been established that 2-HO-ethidium is the sole product of the reaction of superoxide with HE [Zhao et al., 2003]. Thus, the extinction coefficient of 2-HO-ethidium can be determined by measuring the fluorescence of various dilutions of the 2-HO-ethidium stock (in ±DNA), and the concentration of the 2-HO-ethidium in the 0.3 ml solution (step D) can be quantified from the fluorescence difference F.U.total−F.U.Eth measured in Step D.

[22] In order to ensure correspondence between the concentration of 2-HO-ethidium and the actual concentration of superoxide radical (in the 0.3 ml solution) it is necessary to construct a standard curve of 2-HO-ethidium versus superoxide radical concentration under the actual HE-superoxide radical assay reaction conditions (stated in Step A of this assay procedure, that is, in the presence of 99.1% DMSO and 0.9% water), using either of the superoxide radical sources I(a) or I(b) (described in the ‘Superoxide Radical Sources’ section above). This standard curve is shown in Figure 1. As long as the concentration of 2-HO-ethidium, determined from its fluorescence difference F.U.total−F.U.Eth converted to concentration by its fluorescence extinction coefficient, falls within the concentration range 0–120 nM (i.e., 0–40 pmoles in 0.3 ml solubilizate) where the molar stoichiometry ratio of formed 2-HO-ethidium/existing superoxide radicals is maintained constant at1/60 (Figure 1), the concentration of 2-HO-ethidium is multiplied by 60 and represents the actual concentration of superoxide radical in the 0.3 ml solution.

image

Figure 1. Standard curve of superoxide radical measured in soil by the HE-superoxide radical assay. It is an angled standard curve consisting of subcurves 1 and 2, which represent different ratios of measured/existing superoxide radicals (1/60 and 1/85, respectively).

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2.5. Cytochrome c-Superoxide Radical Assay

[23] Superoxide radical in soil can be quantified spectrophotometrically by its equimolar reduction of cytochrome c absorbing at 550 nm, and it can be unambiguously identified by the inhibition of cytochrome c reduction utilizing superoxide dismutase (SOD) [Hyland and Auclair, 1981; McCord and Fridovich, 1969]. The assay was developed on Atacama Desert soil samples mixed with known quantities of DMSO-soluble and solid (potassium) superoxide radical from the sources stated in the ‘Superoxide Radical Sources’ subsection.

[24] Step A. Reduced cytochrome c extinction coefficient determination: Since in aqueous conditions 1 mole superoxide radical reduces 1 mole cytochrome c, for quantifying superoxide radical in the presence of DMSO (because, for this assay, DMSO is the solvent where superoxide radical is extracted from soil), it was necessary to determine the extinction coefficient of reduced cytochrome c within the range of the DMSO concentrations tested in this assay. For this, 1 mM oxidized cytochrome c stock (1.23 mg cytochrome c in 0.1 ml 50 mM phosphate buffer, pH 7.8) is prepared. A 250 × working dilution (4 μM) of the stock (made in final 1 ml 50 mM phosphate buffer, pH 7.8) was completely reduced with few grains solid sodium dithionite. The extinction coefficient of cytochrome c at 550 nm is calculated by the absorbance (ΔA550 nm) of its reduced 4μM solution in the presence of DMSO 0–40% v/v final concentration where it was found to be constant. As blank, a 4 μM solution of oxidized cytochrome c (in 0–40% DMSO) was used.

[25] Step B. Superoxide radical/cytochrome c reaction conditions and standard curve: Since superoxide radical is initially recovered from soil samples in 100% DMSO, it was necessary to determine the DMSO concentration range within which the 1:1 molar stoichiometric ratio for superoxide radical and reduced cytochrome c is constant. Therefore, a standard curve of reduced cytochrome c versus superoxide radical concentration was constructed in order to accurately quantify the concentration of superoxide radical in a 1 ml reaction volume. This volume is the minimum reaction volume that can be measured in 1 ml-sample cuvettes (with 1 cm light pathway) used by common laboratory bench-top spectrophotometers (in this study, the UV-VIS 1200 Shimadzu spectrophotometer was used). Moreover, the maximum limit of the DMSO concentration range (in conjunction with the 1 ml reaction volume) will determine the maximum soil sample size that can be used for superoxide radical detection by this assay. Specifically, in order to construct the standard curve, certain volumes (0.05–0.4 ml) of DMSO-soluble superoxide radical stock I(b) (containing 1.1 × 10−4 M in superoxide radical in 100% DMSO, see ‘Superoxide Radical Sources’ subsection) are brought to 1 ml final volume with 50 mM phosphate buffer, pH 7.8, containing 15 μM oxidized cytochrome c. The ΔA550 nm (absorbance of sample minus absorbance of blank) is then converted to reduced cytochrome c moles (Figure 2), using the extinction coefficient of cytochrome c determined in Step A. As a blank for the standard curve, the same volumes (0.05–4.0 ml) of DMSO-soluble superoxide radical stock are first mixed with water to a final DMSO/water ratio 98.5%/1.5%, in order to destroy (dismutate) the superoxide radical present, and mixed with oxidized cytochrome c as above. The standard curve shows that a constant 1:1 molar stoichiometry between reduced cytochrome c and superoxide radical is maintained within superoxide radical concentration range 0–4 μM (i.e., 0–4 nmoles in 1 ml reaction volume) for a DMSO concentration range of 0–40% v/v (Figure 2).

image

Figure 2. Standard curve of superoxide radical versus reduced cytochrome c concentration (solid symbol) as a function of DMSO concentration (open symbol).

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[26] Step C. Assay procedure: Having established from Steps A and B that the constancy of the molar stoichiometric ratio 1:1 of superoxide radical to reduced cytochrome c is maintained up to a final DMSO concentration 40% (in 1 ml reaction volume, Figure 2), this means that the maximum DMSO volume that the superoxide radical can be recovered from any soil sample is 0.4 ml in order for its concentration to be determined accurately by this assay. Given the fact that the proportion of DMSO extraction volume/soil weight was established to be 0.3 ml/1 g (see ‘Soil Treatment’ subsection), the maximum amount of soil that this assay can test for extracting and measuring accurately the concentration of any superoxide radical on it is 1.3 g. Therefore, we used 1.3 g soil samples from Atacama in mixture with known quantities of superoxide radical from sources I and II (see ‘Superoxide Radical Sources’ subsection). The samples are extracted with a total amount 0.4 ml anhydrous DMSO as described in the ‘Soil Treatment’ subsection. The resulting DMSO-superoxide eluent is mixed with the following order: to 0.6 ml 50 mM phosphate buffer, pH 7.8, are added ±3.3 Units/ml SOD, and 15 μl 1 mM oxidized cytochrome c stock solution (15 μM oxidized cytochrome c final concentration), and its absorbance is measured at 550 nm against the following blanks. It should be noted that the use of SOD makes the assay specific for the detection and quantification of superoxide radical because this enzyme catalyzes its dismutation reaction to H2O2 and O2 [McCord and Fridovich, 1969]. Therefore, if superoxide radical is present in the 0.4 ml DMSO extract, SOD will dismutate all of it and it will inhibit the reduction of cytochrome c superoxide radical otherwise would have caused. The ΔA550 nm (absorbance of soil sample minus the sum absorbance of blanks) is then converted to reduced cytochrome c (and superoxide radical) moles from the standard curve of reduced cytochrome c versus superoxide radical concentration (Figure 2).

[27] DMSO solvent-soil sample blank: It measures whether any substances present in the soil absorb at 550 nm. It consists of the same soil amount used in its corresponding soil sample (1.3 g), extracted with 0.4 ml anhydrous 100% DMSO. The soil/DMSO mixture is sonicated (as in the ‘Soil Treatment’ subsection), the 0.4 ml DMSO extract is collected and mixed with water to a final DMSO/water ratio 98.5%/1.5% (in order to destroy any superoxide present by dismutation), it is mixed with 0.6 ml 50 mM phosphate buffer, pH 7.8, containing 15 μM oxidized cytochrome c, and finally its absorbance is measured at 550 nm.

[28] Sonication effect blank: It is similar to the corresponding blank of the HE-superoxide radical assay, and measures any artificially formed superoxide radical due to the sonication of the DMSO solvent as described in the ‘Soil Treatment’ subsection. This blank consists of the same volume of anhydrous DMSO used in its corresponding soil sample. DMSO is sonicated (as in the ‘Soil Treatment’ subsection), then water is added to a final DMSO/water ratio 99.1%/0.9% (in order for any superoxide radical formed during sonication to be preserved dissolved in the DMSO/water solvent), it is mixed with 0.6 ml 50 mM phosphate buffer, pH 7.8, containing 15 μM oxidized cytochrome c, and finally its absorbance is measured at 550 nm.

3. Results and Discussion

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Materials and Methods
  5. 3. Results and Discussion
  6. Acknowledgments
  7. References

[29] In the simulation experiment of Yen et al. [2000], EPR could detect superoxide radicals accumulated on minerals because there is no sink for the radicals. On Mars, however, there are processes that continually consume superoxide radicals as they are produced. At equilibrium, the concentration of superoxide radicals is likely to be too low to be detected by standard laboratory EPR spectroscopy. In the present study, we explored alternative methods that can detect lower concentrations of superoxide ions in a soil matrix.

[30] For developing our superoxide radical specific assays and investigating their sensitivity, we spiked soils from the Atacama Desert either with a naturally occurring solid superoxide source (potassium superoxide) or with superoxide radical solution prepared in DMSO (a) by alkalinization with NaOH or (b) by solid potassium superoxide. Superoxide radical is stable in anhydrous DMSO for several days due to its weak solvation in the alkaline aprotic solvent [Hyland and Auclair, 1981]. In contrast, water rapidly catalyzes the dismutation of superoxide radical to hydrogen peroxide and molecular oxygen by the reaction O2 + O2 + 2H+ [RIGHTWARDS ARROW] H2O2 + O2 [Halliwell and Gutteridge, 1999].

[31] The HE-based assay takes advantage of the specific reaction of superoxide radical with hydroethidine (HE) and the formation of the fluorescent product 2-OH-ethidium [Georgiou et al., 2005]. The specific enzymatic dismutation of superoxide radical by superoxide dismutase (SOD) was not used as positive control for its identification with the HE-superoxide radical assay (as it was used for the cytochrome c-superoxide radical assay) because SOD is inactive under the extremely limited water conditions of the assay (data not shown). Nevertheless, the specificity of the reaction of superoxide radical with HE (and the resulting 2-HO-ethidium product) has already been established [Zhao et al., 2005, 2003]. Under DMSO/H2O proportion 99.1%/0.9%, which it was found to be the maximum solvent proportion allowed for superoxide radical stability, only 1 out of 60 moles of superoxide radical (from all superoxide radical sources used in this study) reacts with HE. This partial reaction may be caused by the formation of a byproduct(s) that inhibits further the reaction between HE and the remaining superoxide radicals. Alternatively, the equilibrium of this reaction (i.e. 2-HO-ethidium/superoxide = 1/60) may be affected by the extremely limited aqueous reaction conditions (i.e. ≤0.9% water). The 1/60 ratio, as evident from the superoxide radical standard curve (Figure 1), is constant over soil superoxide radical concentrations from 0 to 2500 pmoles. Above this range, the reaction efficiency changed to 1/85 (Figure 1). In the Atacama soil, the smallest amount of superoxide radical that can be detected by this assay was 180 pmoles. Since in our assay configuration we were able to spike a 20 g minimum amount of soil with superoxide radical, by expressing the assay sensitivity in pmoles per g soil for a 20 g soil sample it would be 50 fold higher than that of the EPR method [assuming an approximate EPR sensitivity 500 pmoles per g labradorite, derived by using the reported by Yen et al. [2000] superoxide concentration on a 0.1 g labradorite sample]. Given the fact that soil amount (and thus DMSO-superoxide extract volume) is not a limiting factor for the HE-superoxide radical assay, in principle its sensitivity increases by increasing the amount of soil sample.

[32] The second superoxide radical assay is based on the specific and stoichiometric reaction of this radical with oxidized cytochrome c. The resultant reduced cytochrome c is then measured photometrically at 550 nm [McCord and Fridovich, 1969]. Compared to the HE-superoxide radical assay assay, this assay can tolerate very high water content in the presence of DMSO, up to DMSO/water 20/80% (see superoxide radical vs reduced cytochrome c standard curve in Figure 2), due to the fact that the reaction of superoxide radical with oxidized cytochrome c can outcompete the dismutation of superoxide radical by water. However, the stoichiometry of the reaction (1:1) is constant only up to 40% DMSO/60% water (Figure 2), which limits the maximum amount of soil (1.3 g) that can be tested by this assay. We used 0.4 ml DMSO and 1.3 g soil to produce a reaction volume of 1 ml. Using this configuration with laboratory generated superoxide radical, it was established that this assay can recover all of the superoxide radical added, and that it could detect superoxide radical as low as 1 nmole with sensitivity approximately 1.5 fold lower than the EPR method. The detection limit can be improved e.g. by doubling the size of the cuvette and the soil sample. Obviously, the 1 ml and 1 cm light path cuvette size used in this study imposes an artificial limitation on the assay's detection limit. In a flight instrument, the dimension of the cuvette could be increased to accommodate large samples to the extent they are permitted by the size and weight restrictions of the payload.

[33] It is worth noting that we were unable to detect superoxide radical in Atacama Desert soils with either of the two methods. This result does not mean that superoxide radical is not formed in that desert. Given the extreme lability of the radical in moisture, radicals may have been actually produced in situ but were destroyed (by dismutation) by traces of humidity either on site or during transport and storage.

[34] In conclusion, both assays can detect superoxide radical in soil matrix with sensitivities equal or at least 55 fold higher (the HE-superoxide radical assay) than the EPR method. Furthermore, both assays are unique in being able to test unambiguously for the presence of superoxide with a high level of accuracy. Both assays can be used for the in situ detection of superoxide radical in future expeditions to Mars and other planets or on soil samples returned to Earth in sample-return missions. In the former case, the assays can be even completed on Earth on the DMSO-superoxide extract, presuming that it does not decay during return (e.g. stored at liquid nitrogen temperature). Nevertheless, the in situ test option is possible because the assays require small and inexpensive instrumentation currently available (e.g. Turner BioSystems Picofluor for the HE-superoxide radical assay), which will allow -especially for the cytochrome c-superoxide radical assay- their automated miniaturization based on a combination of automatically controlled multi reagent delivery pumps.

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Materials and Methods
  5. 3. Results and Discussion
  6. Acknowledgments
  7. References

[35] This research was financially supported by the Greek Ministry of Education.

References

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Materials and Methods
  5. 3. Results and Discussion
  6. Acknowledgments
  7. References
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