Anal. Chem. 2007, 79, 5760-5762
High-Affinity Binding of Cadmium Ions by Mouse Metallothionein Prompting the Design of a Reversed-Displacement Protein-Based Fluorescence Biosensor for Cadmium Detection Antonio Varriale, Maria Staiano, Mose’ Rossi, and Sabato D’Auria*
Institute of Protein Biochemistry, CNR, Via Pietro Castellino, 111 80131 Naples, Italy
Reported in this study are the experimental design and results of a protein-based biosensor for the detection of the cadmium in water using a reversed-displacement format. This reversed-displacement biosensor methodology has successfully measured cadmium in water by direct injection, eliminating the need for preconcentration or pretreatment of samples. A column containing Chelex resin saturated with Zn2+ and a rodhamine-labeled metallothionein (MT) comprised the assay reactive chamber. In fact, MT are small cysteine-rich proteins that bind heavy metals such as zinc, cadmium, copper, and mercury· Since the affinity for cadmium is higher than zinc and mercury, we used this intrinsic feature of MT to develop a fluorescence biosensor. The rodhamine-labeled MT was incubated with the Zn2+-saturated Chelex resin until binding equilibrium was reached. Under a constant flow, samples containing cadmium were introduced into the flow stream displacing the rodhamine-labeled MT. Limits of detection were lower than 0.5 µM for cadmium in water. Importantly, the addition of 1.0 µM Cu2+, 1.0 µM Zn2+, 1.0 µM Mg2+, or 1.0 µM Ca2+ did not cause the displacement of the rodhamine-labeled MT, indicating that the presence of these ions do not affect the specificity of the biosensor. Furthermore, we also demonstrated that the reversed-displacement format can be used to screen water samples containing cadmium, remains effective after dozens of cycles, and provides significant fluorescence response before regeneration is required. Cadmium is a lustrous, silver-white, ductile, very malleable metal. Its surface has a bluish tinge, and the metal is soft enough to be cut with a knife, but it tarnishes in air. It is soluble in acids but not in alkalis. About three-fourths of the cadmium is used in Ni-Cd batteries; most of the remaining one-fourth is used mainly for pigments, coatings, and plating, and as stabilizers for plastics. Cadmium has been used particularly to electroplate steel where a film of cadmium only 0.05 mm thick will provide complete protection against the sea. Cadmium has the ability to absorb neutrons, so it is used as a barrier to control nuclear fission. * To whom correspondence should be addressed. Tel: +39-0816132250. Fax: +39-0816132277. E-mail:
[email protected].
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Figure 1. Absorption (left) and emission (right) spectra of Rh-MT. The spectra were normalized to 1.0. For fluorescence emission the spectrum was recorded at room temperature with an excitation set at 520 nm.
Naturally a very large amount of cadmium is released into the environment, ∼25 000 tons a year. About half of this cadmium is released into rivers through weathering of rocks, and some cadmium is released into air through forest fires and volcanoes. The rest of the cadmium is released through human activities, such as manufacturing. Human uptake of cadmium takes place mainly through food. Foodstuffs that are rich in cadmium can greatly increase the cadmium concentration in human bodies. Examples are liver, mushrooms, shellfish, mussels, cocoa powder, and dried seaweed. An exposure to significantly higher cadmium levels occurs when people smoke. Tobacco smoke transports cadmium into the lungs. Blood will transport it through the rest of the body, where it can increase effects by potentiating cadmium that is already present from cadmium-rich food. Other high exposures can occur with people who live near hazardous waste sites or factories that release cadmium into the air and people that work in the metal refinery industry. When people breathe in cadmium, it can severely damage the lungs. This may even cause death. Cadmium is first transported to the liver through the blood. There, it is bound to proteins to form complexes that are transported to the kidneys. Cadmium accumulates in kidneys, 10.1021/ac0705667 CCC: $37.00
© 2007 American Chemical Society Published on Web 06/20/2007
Figure 2. Experimental scheme used for the realization of the reversed-displacement protein-based fluorescence biosensor. After labeling, Rh-MT was flowed through a column previously packed with Zn2+-saturated Chelex resin. The column was washed until no fluorescence emission was detected in the flow-through. A water solution containing different amounts of cadmium was applied to the column, and the flow-through was monitored. Experimental conditions: Ex ) 520 nm; Em ) 575 nm; temperature 25 °C.
where it damages filtering mechanisms. This causes the excretion of essential proteins and sugars from the body and further kidney damage. It takes a very long time before cadmium that has accumulated in kidneys is excreted from a human body. Other health effects that can be caused by cadmium are as follows: (1) diarrhea, stomach pains, and severe vomiting; (2) bone fracture; (3) reproductive failure and possibly even infertility; (4) damage to the central nervous system; (5) damage to the immune system; (6) psychological disorders; (7) possibly DNA damage or cancer development. Metallothionein (MT) are small cysteine-rich proteins that bind heavy metals such as zinc, cadmium, copper, and mercury· Vertebrate MT is made of a single polypeptide chain characterized by a low molecular mass (∼6 kDa), high cysteine (30% of total residues in the molecule) and heavy metal contents, and the absence of aromatic residues and histidine.1-3 The cysteine residues of vertebrate MT are arranged according to a substantially conserved pattern and are distributed in motifs consisting of CC, CXC, and CXXC sequences.4 All 20 cysteine residues bind 7 metal atoms such that each metal atom is bound to 4 cysteine ligands forming metal-thiolate clusters.5 These clusters are divided in 2 domains: the N-terminal b-domain constituting 9 cysteine residues and 3 metal ions, and the C-terminal a-domain (1) Kagi, J. H. R.; Vallee, B. L. J. Biol. Chem. 1960, 235, 3460-3465. (2) Kagi, J. H. R.; Nordberg, M. Metallothionein; Birkhauser Verlag: Basel, 1979. (3) Kagi, J. H. R.; Schaffer, A. Biochemistry 1988, 27, 8509-8515. (4) Kagi, J. H. R.; Vasak, M.; Lerch, K.; Gilg, D. E.; Hunziker, P.; Bernhard, W. R.; Good, M. Health Perspect. 1984, 54, 93-103. (5) Stillman, M. J.; Cai, W.; Zelazowski, A. J. J. Biol. Chem. 1987, 262, 45384548.
with 1 cysteine residues and 4 metal ions.6 The possible function of MT is still a matter of debate: the induction by bivalent metal ions implies a detoxification role against heavy metals,7-10 whereas the ability to act as a scavenger of superoxide radicals suggests a participation in the defense against oxidative stress.11,12 On the other hand, the remarkable stability of the metal complex and the high kinetic lability resulting in a facile metal release suggest a zinc donor role for MT.13,14 In this report, we show the experimental design and results of a competitive assay based on a fluorescence biosensor that utilizes the mouse MT as fluorescence probe for the detection of the cadmium in water. MATERIALS AND METHODS Materials. All the chemicals used were commercial samples of the purest quality. Mouse metallotheionein was from Sigma. Preparation of Rodhamine-Labeled MT. One vial of TAMR dye (monofunctional NHS ester) as supplied by the manufacturer (Sigma) was suspended in 200 µL of DMS. Then 50 µL of reactive (6) Kagi, J. H. R. In Metallothionein III. Biological Roles and Medical Implications; Suzuki, K. T., Imura, N.; Kimura, M. Eds.; Birkhauser Verlag: Basel, 1993; pp 29-55. (7) Debec, A.; Mokdad, R.; Wegnez, M. Biochem. Biophys. Res. Commun. 1985, 127, 143-152. (8) Cosson, R. P. Biometals 1994, 7, 9-19. (9) De, S. K.; Dey, S. K.; Andrews, G. K. Toxicology 1990, 64, 89-104. (10) Carginale, V.; Scudiero, R.; Capasso, C.; Capasso, A.; Kille, P., di Prisco, G.; Parisi, E. Biochem. J. 1998, 332, 475-481. (11) Iszard, M. B.; Liu, J.; Klaassen, C. D. Toxicology 1995, 104, 25-33. (12) Miura, T.; Muraoka, S.; Ogiso, T. Life Sci. 1997, 60, 301-309. (13) Vallee, B. L. Neurochem. Int. 1995, 27, 23-33. (14) Jiang, L. J.; Maret, W.; Vallee, B. L. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3483-3488.
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Figure 3. Effect of different concentrations of Cd on the displacement of Rh-MT from the Zn2+-saturated Chelex resin. Excitation was set at 520 nm. Emission was recorded at 575 nm. Temperature was 25 °C.
dye was added to 0.5 mg of MT, giving a final solution that was 1 mg/mL, and buffered with sodium borate, 0.04 M, sodium chloride, 0.04 M (pH 9.0). The reaction mixture was incubated for 1 h at 30 °C, and the labeled molecules were separated from unreacted probe by a dialysis procedure against 50 mM phosphate buffer, 100 mM NaCl, pH 7.0. by using dialysis tubes with a cutoff 1000 Da (Spectrum Labs) overnight, at 4 °C. Figure 1 show the absorption and emission spectra of the rodhamine-labeled MT. Preparation Chelex Resin. An aliquot of 15 mL of Chelex resin (Sigma), rinsed in distilled water, was saturated with a solution of Zn2+ and packed in a column (10 cm). Flow Biosensor Design. The flow biosensor consisted of a model 821-FP Intelligent K2 ISS spectrofluorometer with a flow cell, a Gilson peristaltic pump, and a 10-cm column packed with the Zn2+-saturated Chelex resin. RESULTS AND DISCUSSION Metals are both essential and toxic elements to life processes. On the one hand, they are integral, functional components of many enzymes and transcriptional regulatory proteins, while on the other hand, the binding of non-native metals to biological macromolecules may perturb their function and metal-catalyzed formation of oxygen-derived free radicals has been implicated in a wide variety of pathological conditions such as mutagenicity and carcinogenicity. To cope with potentially hazardous levels of heavy metal ions, organisms appear to have developed an integrated, metal-regulatory network to control the concentration and availability of these elements. One of the components of this network is MT, a small protein with high cysteine content. MTs bind both essential (Cu and Zn) and nonessential (Cd and Hg) metals. Metal coordination in MT has a high thermodynamic but low kinetic stability. Thus, metal binding is very tight, but there is facile metal exchange with other proteins. MTs are thought to function biologically as intracellular distributors and mediators of the metals they bind.3 The rationale of this report is that mouse MT can bind metals such as zinc, mercury, calcium, cadmium, copper, etc., with different affinity constants. In particular, mouse MT binds Cd with 5762 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007
higher affinity than other metals such as Zn, Ca, Mg, or Cu. As a consequence, it is not outrageous to design a competitive assay for the Cd determination based on the utilization of mouse MT. Our strategy consisted of the following steps: (1) label the mouse MT with an extrinsic fluorescence probe such as rodhamine (Rh); (2) bind the rodhamine-labeled-MT (Rh-MT) to a previously Zn2+-saturated Chelex resin; (3) perform a competitive assay by flowing heavy metals containing water through a column packed with “Zn2+-saturated Chelex resin”-Rh-MT. Figure 2 reports the strategy scheme used for the realization of the on-line reversed-displacement fluorescence biosensor for cadmium detection. A solution of Rh-MT was flowed through the column previously packed with Zn2+-satured Chelex resin. The column was washed until no fluorescence emission was detected in the flow-through (the excitation and emission wavelengths were set at 520 and 575 nm, respectively). The flow buffer was continuously pumped through the column at 250 µL/min, and the fluorescence of RhMT was monitored downstream using a fluorometer. Samples were not injected until the unbound and weakly adsorbed Rh-MT washed off the resin and the background fluorescence stabilized. Then 100-µL samples of flow buffer or increasing concentrations of Cd were injected by the peristaltic pump. Cd concentrations ranged from 2.5 ng/mL to 10 µg/mL. The peaks observed as a result of the displacement of the fluorescent Rh-MT were recorded (see spectrum in Figure 2). In Figure 3 are reported the fluorescence intensity variations at different concentrations of Cd. The limits of detection of Cd were lower than 0.5 µM for cadmium in water. Importantly, when we tested the effect of other metals on the specificity of the biosensor, we found that the addition of 1.0 µM Cu2+, 1.0 µM Zn2+, 1.0 µM Mg2+, or 1.0 µM Ca2+ did not cause the displacement of the rodhamine-labeled MT, indicating that the presence of these ions at the tested concentrations do not affect the specificity of the biosensor for Cd detection. We also checked the storage conditions as well as the duration of the biosensor. The biosensor that the reversed-displacement fluorescence biosensor is stable over 2 weeks stored in dark conditions at 4 °C, and it remains effective after dozens of cycles providing significant fluorescence responses before regeneration is required. In conclusion, in this work, we have presented useful data for the design of a competitive fluorescence assay for an easy and rapid on-line detection of cadmium in water. Genetic manipulation experiments for improving the MT affinity and selectivity to different metals could greatly expand the operational possibilities to use this class of protein for sensing heavy metals. ACKNOWLEDGMENT This project was realized in the frame of the CNR Commessa “Diagnostica Avanzata ed Alimentazione” (S.D.). This work was also supported by a grant from the Ministero degli Affari Esteri, Direzione Generale per la Promozione e la Cooperazione Culturale (S.D.).
Received for review March 21, 2007. Accepted May 18, 2007. AC0705667
