Hg²⁺-Trapping Beads: Hg²⁺-Specific Recognition through Thymine–Hg(II)–Thymine Base Pairing

Abstract

Mercury pollution poses a severe threat to human health. To remove Hg²⁺ from contaminated water, we synthesized Hg²⁺-trapping beads that include oligo-thymidine functionalities that can form thymine–Hg(II)–thymine base pairs on the solid support. The beads can selectively trap Hg²⁺ even in the presence of other metal cations. More interestingly, Hg²⁺-trapping efficiency was higher in the presence of the co-existing cations. Thus, the developed Hg²⁺-trapping beads can capture Hg²⁺ without affecting the mineral balance of water so much. The Hg²⁺-trapping beads presented here show promise for removing Hg²⁺ from environmental water.

Mercury is a reputable highly toxic element.^1–3) Notably, World Health Organization (WHO), Environmental Protection Agency (EPA), and U.S. Food and Drug Administration (FDA) reported that certain kinds of fishes accumulate high level of mercury which is harmful enough to fetuses, babies and children.^3,4) Such an accumulation of mercury in fishes occurred through a food chain.^2,5) Based on these facts, the provisional tolerable weekly intake (PTWI) for methylmercury and the reference dose (RfD) for mercuric chloride have been set at 1.6 µg/kg/week and 0.3 µg/kg/d, respectively.³⁾ In any case, mercury originates from natural sources (such as volcanoes) and human activities (such as industrial wastes). To reduce mercury contamination in the environment, it is necessary to reduce and remove mercury from environments (oceans, rivers, and lakes).

For this purpose, various techniques have been reported. These include bioremediation,^5,6) precipitation,⁷⁾ non-specific adsorption (using activated carbon,^8,9) aerogels,¹⁰⁾ and chitosan^8,11) as sorbents), and metal chelations (using imidazoles,¹²⁾ thiols,¹³⁾ and DNA/nucleobases^14–19)). However, these techniques generally trap several kinds of metals, resulting in the loss of mineral balance in the treated water. It is thus indispensable to develop methods for the selective removal of mercury.

For this purpose, thymine–Hg(II)–thymine (T–Hg(II)–T) base pairing system which was used in a DNA-based Hg²⁺-specific sensor by the Ono and Togashi²⁰⁾ gave us a hint, since thymine is a highly Hg(II)-specific ligand. The key structural element of the T–Hg(II)–T base pair was originally proposed by Katz,²¹⁾ based on pre-existing studies on Hg²⁺–DNA interactions.^22–25) New insights were recently reported by Ono and colleagues.^20,26,27) Although extensive structural and theoretical studies on the structure of this base pair in a DNA oligomer have been performed,^28–41) the experimental structure of the T–Hg(II)–T base pair from NMR and Raman spectroscopy was recently reported^27,42) (see the chemical structure of the T–Hg(II)–T base pair for that shown within Fig. 1b). Thermal denaturation experiments⁴³⁾ showed that the T–Hg(II)–T base pair is as stable as the Watson–Crick A–T base pairs. More importantly, it was demonstrated that a T–T mismatch can specifically bind to Hg²⁺ without binding to other divalent metal cations.²⁶⁾ Thus, such Hg²⁺-specificity of thymine base is suitable to develop an Hg²⁺-specific trapping system. We thus prepared Hg²⁺-trapping beads based on this concept (Fig. 1), and examined their trapping efficiency.

Fig. 1. Hg²⁺-Trapping Concept

a) Structure of the Hg²⁺-trapping beads. b) Procedure of Hg²⁺ removal from Hg²⁺-contaminated water by the Hg²⁺-trapping beads.

The Hg²⁺-trapping beads consist of oligo-thymidine (T20) attached onto the polystyrene (PS) beads (solid support), as shown in Fig. 1a. Following capture of the Hg²⁺ ions by T20, the Hg²⁺-trapped PS beads can be easily separated from the solution by filtration (Fig. 1b). To give mobility to T20, polyethylene glycol 400 (PEG 400) was used as a linker to attach T20 onto the PS beads, since PEG 400 is long enough to give attached molecules their mobility on the polymer surface.⁴⁴⁾ The detailed preparation is given in Chart S1 and Supplemental Methods in Supplemental Information.

Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used to assess the performance of the prepared Hg²⁺-trapping beads. This technique is suitable for the quantification of low levels of Hg²⁺ as well as the simultaneous determination of co-existing metal cations in solution.

Prior to conducting the Hg²⁺-trapping experiments, we confirmed the linearity between the Hg²⁺-concentration and the ICP-AES peak intensities, measured at the 253 nm and 194 nm mercury emissions (Hg²⁺ concentration range: 0.5–10 µM=0.1–2.0 ppm) (Fig. S1).

Based on the obtained linear range and signal to noise ratio, the Hg²⁺-trapping experiments were conducted at 50 µM (10 ppm) Hg²⁺, and the supernatant solutions after Hg²⁺ removal were diluted 10 times (maximum 5.0 µM Hg²⁺) for Hg²⁺ quantitation. The residual Hg²⁺ ratios (%), monitored at the 253 and 194 nm emissions, as calculated by Eq. 1 in Supplemental Methods in Supplemental Information, are presented in Fig. 2. A ca. 50% reduction in the residual Hg²⁺-concentration was observed for the Hg²⁺-trapping beads (white bars in Fig. 2). It should be also mentioned that capture of Hg²⁺ was negligible in the absence of the oligo-thymidine moieties (PS beads and PS beads+linker samples in Fig. 2 and Table S1). Thus, the Hg²⁺-trapping capabilities of the beads are exclusively reliant on the presence of oligo-thymidine moieties.

Fig. 2. Mercury Removal Ratios by the PS Beads, PS Beads+Linker, and Hg²⁺-Trapping Beads

The experiments were performed in 50 µM HgCl₂, and either in the absence of Mg²⁺ (−Mg²⁺) or the presence of 5.0 mM MgCl₂ (+Mg²⁺). Residual ratios determined at a) 253 nm and b) 194 nm mercury emission. No obvious spectroscopic interferences between mercury and magnesium was observed, since emission intensities for “PS beads” and “PS beads + linker” were not change significantly irrespective of whether Mg²⁺ is present or absent.

The Hg²⁺-selective trapping was initially examined in the presence of Mg²⁺ to simulate the natural conditions of seawater that contains minerals such as Na⁺, Mg²⁺, and Ca²⁺. Because the actual concentration of Na⁺ (481 mM) and possibly Mg²⁺ (54 mM) in seawater exceeds the upper limit of the ICP-AES measurements, the present experiments were conducted under a 5.0 mM Mg²⁺ solution. Unexpectedly, in the presence of Mg²⁺, the residual Hg²⁺ concentration decreased from ca. 50 to ca. 20% (Fig. 2, Table S1). The same trend was observed for both 253 nm and 194 nm mercury emissions (Fig. 2, Tables S1 and S2). Therefore, for the subsequent studies, only the results from the 253 nm emission spectra are presented as the 253 nm emission generated stronger signals.

As seawater includes several kinds of divalent metal cations, we investigated the performance of the beads to selectively trap Hg²⁺ over other divalent metal cations (Ca²⁺, Ba²⁺, Ni²⁺, Cu²⁺, Cd²⁺) in the presence of Mg²⁺ (Fig. 3, Fig. S2). The Hg²⁺-trapping efficiency of the beads, examined in the presence of co-existing metal cations (Ca²⁺, Ba²⁺, Ni²⁺, Cu²⁺, Cd²⁺), was similar or greater than that obtained in the presence of Mg²⁺ only (absence of the co-existing cations) (Fig. 3, Table S1). For the Hg²⁺-trapping efficiencies in the absence of Mg²⁺, see Fig. S2.

Fig. 3. Mercury Removal Ratios by Hg²⁺-Trapping Beads in the Presence of Mg²⁺ Only (Mg²⁺*), and Mg²⁺ and Co-existing Metal Cations

The experiments were performed in 50 µM HgCl₂, 5.0 mM MgCl₂, and 0 or 0.5 mM of the respective co-existing metal cations. Residual ratios were determined from the 253 nm mercury emission.

The non-specific binding of the co-existing metal cations by the Hg²⁺-trapping beads was then examined (Fig. 4, Figs. S3, S4, Tables S3, S4). The residual ratios (%) of the co-existing cations after Hg²⁺-trapping are shown in Fig. 4 and Table S3. As observed, the remaining percentages of all the co-existing metal cations in the corresponding supernatant solutions, after Hg²⁺-trapping, were very high.

Fig. 4. Residual Ratios of the Co-existing Metal Cations after Hg²⁺-Trapping

The emission wavelengths of the corresponding co-existing metal cations are listed in Table S3. All the experiments were performed in 50 µM HgCl₂, 5.0 mM MgCl₂, and 0.5 mM of the respective co-existing metal cation except for sample Mg²⁺* whereby the experiment was conducted in the absence of the co-existing cations.

The DNA-based Hg²⁺-trapping beads effectively removed mercury in both the presence of Mg²⁺ only, and several co-existing metal cations that included Mg²⁺ (Fig. 3). In contrast to this effective Hg²⁺-trapping, non-specific trapping of co-existing cations did not occur so much (Fig. 4). The results are consistent with our previous study that showed the specific binding of Hg²⁺ by thymine bases.²⁶⁾ Thus the herein developed Hg²⁺-trapping beads are suitable for use in natural conditions that involve a variety of mineral ingredients.

The enhanced performance of the Hg²⁺-trapping beads in the presence of co-existing metal cations is explained as follows. The formation of T–Hg(II)–T base pairs implies the formation of a DNA duplex. For the latter to form, electrostatic repulsion between the negatively-charged phosphates in the respective DNA strands needs to be suppressed. The Mg²⁺ and other co-existing cations thus suppress the electrostatic repulsion between the phosphate groups by neutralizing the negative charges (Fig. S5). Hence, in the absence of a charge compensation effect of the co-existing cations, formation of T–Hg(II)–T base pair and the resulting DNA duplex formation cannot occur efficiently. In addition, the competition between Mg²⁺ and Hg²⁺ during their phosphate-binding steps may assist preferential Hg²⁺-binding to thymine bases. This phenomenon was evident in the presence of Mg²⁺, as indicated by the considerable increase in the Hg²⁺-trapping efficiency (Figs. S2, S5c, d). A slight increase in the trapping efficiency was observed in the presence of the co-existing cations under Mg²⁺-depleted conditions (Figs. S2, S5e). These findings and some foregoing studies^45,46) support our hypothesis that co-existing cations stabilize the formation of a DNA duplex following formation of T–Hg(II)–T base pair. The measured residual ratio of the co-existing metal cations after Hg²⁺-trapping (Fig. S3) further supported our hypothesis that co-existing cations served as counter ions for DNA, as explained: in the absence of Mg²⁺, the other co-existing cations were captured by the Hg²⁺-trapping beads, possibly as counter ions for the phosphate groups (Fig. S5e), hence other co-existing cations rarely remained in solution in the absence of Mg²⁺ (Fig. S3). The present experimental data indicate that the Hg²⁺-trapping beads show potential for their usage under natural environmental conditions, particularly in seawater with a high salt concentration.

The present findings were compared to those obtained by other reported methods (bioremediation,^5,6) precipitation,⁷⁾ non-specific adsorbents,^8–10) and some metal chelators^12,13)). In these studies, the Hg²⁺-specificity was not indicated nor was it expected on the basis of metal-specificities of the ligands.^{5–10,12,13)} Only Hg²⁺-trapping beads, containing thymine moieties,^14–19) showed good Hg²⁺-selectivity and these data are consistent with our previous results.²⁶⁾ As an exception, chitosan-based Hg²⁺-trapping beads showed Hg²⁺ selectivity,¹¹⁾ however, the origin of the Hg²⁺-specificity is unclear on the basis of an intrinsic metal-specificity of chitosan.

In conclusion, the prepared Hg²⁺-trapping beads can exclusively capture Hg²⁺ even in the presence of co-existing cations. More interestingly, the Hg²⁺-trapping efficiency of the beads improved in the presence of co-existing cations. The Hg²⁺-trapping beads are suitable for the elimination of Hg²⁺ from environmental water, especially seawater. As a practical usage of the Hg²⁺-trapping beads, the Hg²⁺-decontamination from industrial wastes would be possible.

Acknowledgment

The authors would like to thank Dr. Hiroshi Naka at Nagoya University for his discussion. This work was supported by two Grants-in-Aid for Scientific Research (B) and (C) (Grant Nos. 24310163 and 18550146 to Y.T.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Y.T. is also supported by a Human Frontier Science Program for Young Investigators from Human Frontier Science Program Organization (HFSPO) (France) and the Intelligent Cosmos Academic Foundation. M.K. is the recipient of a Japan Society for the Promotion of Science (JSPS) Research Fellowship for Young Scientists. ICP-AES measurements were performed at the Integrated Center for Sciences (INCS_Johoku station) at Ehime University.

References

• 1) Clarkson T. W., Magos L., Crit. Rev. Toxicol., 36, 609–662 (2006), and references cited therein.
• 2) Clarkson T. W., Magos L., Myers G. J., N. Engl. J. Med., 349, 1731–1737 (2003).
• 3) World Health Organization, “Guidance for Identifying Populations at Risk from Mercury Exposure.”: 〈http://www.who.int/foodsafety/publications/chem/mercury/en/index.html〉, cited 11th November, 2013.
• 4) Environmental Protection Agency and US Food and Drug Administration, “What You Need to Know about Mercury in Fish and Shellfish.”: 〈http://water.epa.gov/scitech/swguidance/fishshellfish/outreach/advice_index.cfm〉, cited 11th November, 2013.
• 5) Morel F. M. M., Kraepiel A. M. L., Amyot M., Annu. Rev. Ecol. Syst., 29, 543–566 (1998).
• 6) Rugh C. L., Bizily S. P., Meagher R. B., “Phytoremediation of Toxic Metals,” ed. by Raskin I., Ensley B. D., Wiely-Interscience, New York, 2000, pp. 151–169 and references cited therein.
• 7) Porter S. K., Scheckel K. G., Impellitteri C. A., Ryan J. A., Crit. Rev. Environ. Sci. Technol., 34, 495–604 (2004).
• 8) Babel S., Kurniawan T. A., J. Hazard. Mater., 97, 219–243 (2003).
• 9) Krishnan S. V., Gullett B. K., Jozewicz W., Environ. Sci. Technol., 28, 1506–1512 (1994).
• 10) Kadirvelu K., Goel J., Rajagopal C., J. Hazard. Mater., 153, 502–507 (2008).
• 11) Miretzky P., Cirelli A. F., J. Hazard. Mater., 167, 10–23 (2009), and references cited therein.
• 12) Kara A., Uzun L., Beşirli N., Denizli A., J. Hazard. Mater., 106, 93–99 (2004).
• 13) Liu A. M., Hidajat K., Kawi S., Zhao D. Y., Chem. Commun., 1145–1146 (2000).
• 14) Liu X., Qi C., Bing T., Cheng X., Shangguan D., Talanta, 78, 253–258 (2009).
• 15) Dave N., Chan M. Y., Huang P. J. J., Smith B. D., Liu J., J. Am. Chem. Soc., 132, 12668–12673 (2010).
• 16) Yu Y., Zhang B. W., Yu M., Deng B., Li L. F., Fan C. H., Li J. Y., Sci. China Chem., 55, 2202–2208 (2012).
• 17) Wang Y. S., Cheng C. C., Chen J. K., Ko F. H., Chang F. C., J. Mater. Chem. A, 1, 7745–7750 (2013).
• 18) Huang P.-J. J., Liu J., Chem.-Eur. J., 17, 5004–5010 (2011).
• 19) He D., He X., Wang K., Zhao Y., Zou Z., Langmuir, 29, 5896–5904 (2013).
• 20) Ono A., Togashi H., Angew. Chem., Int. Ed., 43, 4300–4302 (2004).
• 21) Katz S., Biochim. Biophys. Acta, 68, 240–253 (1963).
• 22) Katz S., J. Am. Chem. Soc., 74, 2238–2245 (1952).
• 23) Thomas C., J. Am. Chem. Soc., 76, 6032–6034 (1954).
• 24) Yamane T., Davidson N., J. Am. Chem. Soc., 83, 2599–2607 (1961).
• 25) Yamane T., Davidson N., Biochim. Biophys. Acta, 55, 780–782 (1962).
• 26) Miyake Y., Togashi H., Tashiro M., Yamaguchi H., Oda S., Kudo M., Tanaka Y., Kondo Y., Sawa R., Fujimoto T., Machinami T., Ono A., J. Am. Chem. Soc., 128, 2172–2173 (2006).
• 27) Tanaka Y., Oda S., Yamaguchi H., Kondo T., Kojima C., Ono A., J. Am. Chem. Soc., 129, 244–245 (2007).
• 28) Simpson R. B., J. Am. Chem. Soc., 86, 2059–2065 (1964).
• 29) Tanaka Y., Ono A., “Metal Complex-DNA Interactions,” ed. by Hadjiliadis N., Sletten E., John Wiely & Sons Ltd., Chichester, 2008, pp. 439–462 and references cited therein.
• 30) Kosturko L. D., Folzer C., Stewart R. F., Biochemistry, 13, 3949–3952 (1974).
• 31) Mansy S., Wood T. E., Sprowles J. C., Tobias R. S., J. Am. Chem. Soc., 96, 1762–1770 (1974).
• 32) Mansy S., Tobias R. S., Inorg. Chem., 14, 287–291 (1975).
• 33) Gruenwedel D. W., Cruikshank M. K., Biochemistry, 1990, 2110–2116 (1990).
• 34) Morzyk-Ociepa B., Michalska D., J. Mol. Struct., 598, 133–144 (2001).
• 35) Buncel E., Boone C., Joly H., Kumar R., Norris A. R., J. Inorg. Biochem., 25, 61–73 (1985).
• 36) Buncel E., Boone C., Joly H., Inorg. Chim. Acta, 125, 167–172 (1986).
• 37) Kuklenyik Z., Marzilli L. G., Inorg. Chem., 35, 5654–5662 (1996).
• 38) Johannsen S., Paulus S., Düpre N., Müller J., Sigel R. K. O., J. Inorg. Biochem., 102, 1141–1151 (2008).
• 39) Benda L., Straka M., Tanaka Y., Sychrovský V., Phys. Chem. Chem. Phys., 13, 100–103 (2011).
• 40) Benda L., Straka M., Sychrovský V., Bouř P., Tanaka Y., J. Phys. Chem. A, 116, 8313–8320 (2012).
• 41) Šebera J., Burda J., Straka M., Ono A., Kojima C., Tanaka Y., Sychrovský V., Chem.-Eur. J., 19, 9884–9894 (2013).
• 42) Uchiyama T., Miura T., Takeuchi H., Dairaku T., Komuro T., Kawamura T., Kondo Y., Benda L., Sychrovský V., Bour P., Okamoto I., Ono A., Tanaka Y., Nucleic Acids Res., 40, 5766–5774 (2012).
• 43) Torigoe H., Ono A., Kozasa T., Chem.-Eur. J., 16, 13218–13225 (2010).
• 44) Terenzi S., Biala E., Nguyen-Trung N. Q., Strazewski P., Angew. Chem., Int. Ed., 42, 2909–2912 (2003).
• 45) Kiy M. M., Zaki A., Menhaj A. B., Samadi A., Liu J., Analyst, 137, 3535–3540 (2012).
• 46) Kiy M. M., Jacobi Z. E., Liu J., Chem.-Eur. J., 18, 1202–1208 (2012).