Characteristics of Trapping Copper Ions with Scrolled Ferritin Reactor

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Environ. Sci. Technol. 2004, 38, 2476-2481

Characteristics of Trapping Copper Ions with Scrolled Ferritin Reactor in the Flowing Seawater H E - Q I N G H U A N G , * ,†,‡,§ T I N G - M I N G C A O , †,§ A N D Q I N G - M E I L I N ‡ The Center for Proteomics Research and Department of Biology, School of Life Sciences, The Key Laboratory of Chemical Biology of Fujian Province, The Key Laboratory of Marine Environmental Science (Ministry of Education), and The Key Laboratory of MOE for Cell Biology and Tumor Cell Engineering, Xiamen University, 361005, China

Native liver ferritin of Dasyatis akajei (DALF), apoDALF, and reconstituted DALF were employed to construct a ferritin reactor, respectively. An apparatus consisting of a mixer, a ferritin reactor, and a magnetic stirrer was constructed to study capacity and feasibility of trapping Cu2+ in the flowing seawater. The experimental results showed that the numbers of trapping Cu2+ with DALF reactor were higher than these with the reactors of apoDALF and reconstituted DALF, respectively, giving the maximal numbers of 98 ( 5 Cu2+ per molecular DALF in 120 h. We found that the iron layer with a high ratio of phosphate to ion on the surface of the ferritin core played an important role in increasing numbers of trapping Cu2+. In addition, we found two positive relations of dependence of trapping Cu2+ numbers with the reactor on the incubation time and on the Cu2+ concentration in the flowing seawater. Another apparatus consisting of a buoyage, an isolation basket equipped with griddling, and a scrolled ferritin reactor was constructed to study the feasibility of trapping Cu2+ in the sea area. Moreover, the present studies indicated that this apparatus had been used to not only analyze and evaluate the concentration variety of various heavy metal ions such as Cu2+ and Pb2+ diluting by the seawater but also monitor the formation of pollution degree by various small organic molecules during the climax and the neap.

Introduction Iron is an essential element for most living organisms and is abundant in the environment. Iron plays an essential role in the active sites of enzymes responsible for such processes as oxygen transfer, electron transport, DNA synthesis, photosynthesis, and nitrogen fixation (1). Iron is the second most abundant metal in the Earth’s crust (after aluminum). Under a physiological pH range and oxidizing conditions, iron exists as insoluble ferritin hydroxide. Ferritin is a large biological molecule of the ubiquitous iron storage protein, which plays two main physiological roles in detoxification and storage of intracellular iron in living cell (2-5). Ferritin is composed of 24 subunits arranged to form a hollow shell * Corresponding author phone: +86-0592-2186392; fax: +860592-2186630; e-mail: [email protected]. † The Key Laboratory of Chemical Biology of Fujian Province. ‡ The Key Laboratory of Marine Environmental Science (Ministry of Education). § The Key Laboratory of MOE for Cell Biology and Tumor Cell Engineering. 2476

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of highly symmetrical structure. The shell from mammalian ferritin consists of two subunit types, H (heavy chain) and L (light chain), and shows a diameter ranging from 80 to 120 Å (8-12 nm). Two subunit types make up horse spleen ferritin (HSF) and other mammalian ferritins such as pig spleen ferritin (PSF). The H subunit has an apparent molecular weight of 21 kDa and contains a ferroxidase site for iron oxidization. The L subunit has an apparent molecular weight of 19 kDa and plays a role in iron deposition. A difference of 2 kDa for apparent molecular weights for both subunits was distinguished unclearly by a normal SDS-PAGE because of low resolution (6-8). In addition, an iron core of 60-80 Å (6-8 nm) diameter consisted of a few thousand molecules of a hydrated iron phosphate (atomic ratio Fe3+/Pi ) 8∼9:1) (9-10) compound located at the center of the ferritin shell (11), which is observed by electron microscopy because it has typical characteristics of high electron density that can be used as a tracker for identification and orientation of DNA and protein in the cell (12). Ferritin is a robust iron-storage protein that can withstand high temperatures (75-85 °C) (5), a pH ranging from 2.0 to 12.0 (13), and an NaCl concentration ranging from 1 to 6% (14) for the limited periods without significant disruption of ferritin structure consisting of 24 polypeptide subunits. In vitro, reconstitution of iron oxide core can be readily achieved by room-temperature incubation of apoferritin and Fe2+ solutions at moderate pH in the absence of inorganic phosphate (Pi) (15-16). In addition, Apo HSF was reconstituted to various core sizes by depositing Fe(OH)3 within the hollow HSF interior by air oxidation of Fe2+. Fe2+ and Pi were added anaerobically at a 1:4 high ratio of phosphate to iron on the surface of the core for formation of a core (17). Initial investigations of the physiological role of phosphate in ferritin function suggest that phosphate is involved in facilitating ferritin core formation and assisting an accelerated rate of electron transfer from the physical electrode to the ferritin core (14). Chen and Chasteen (18) figured that phosphate accelerated the rate of iron deposition from the apoHSF. Huang et al. (14, 19, 20) reported that phosphate was not only responsible for Fe2+ binding to HSF core but also readily released upon reduction from the core. These results indicate that the phosphate coming from the surface of the ferritin core plays an important role in carrying out iron release and storage as well as binding to various heavy metal ions such as Cu2+. Meldrum et al. (21) reported that ferritin generated nanometer-sized iron sulfide particles by in situ reaction of the iron oxide core of the native ferritin, which indicated that the ferritin was able to carry out synthesis of inorganic nanophase materials within the protein shell. In addition, those authors figured that discrete amorphous MnOOH cores were constituted within the apo HSF shell at pH 8.9 (22), which meant that the ferritin had capacity to store other heavy metal ions for formatting a metal core within the protein shell. Another important role of apo HSF was used to load a uranium core with an average of 800 238U atoms within the protein shell. These biouranium constructions should provide significant advantages over boronated antibodies to meet the requirements for clinical neutron-capture therapy (23). Following research showed that Cd2+, Zn2+, Cu2+, Ni2+, Co2+, Mn2+, and Mg2+ are directly able to bind to apo, holo, reconstituted HSF, and native HSF (168Cd2+/HSF; 244Zn2+/HSF; 99Cu2+/HSF, 37Ni2+/HSF; 56Co2+/HSF; 10 Mn2+/HSF; and 0.8 Mg2+/HSF), respectively (24). Huang et al. (14) reported that an apparatus consisting of two pumps, a mixer, a ferritin reactor, and a spectrometer was constructed 10.1021/es034953j CCC: $27.50

 2004 American Chemical Society Published on Web 03/13/2004

to study the trap of various heavy metal ions and the dynamics of a reconstituted ferrition reactor in flowing seawater. Those authors found that the reactor can trap Cd2+, Zn2+, Co2+, and Mn2+ directly and obtained a 1:2 stoichiometry of the trapped metal ion to the released iron, suggesting that this study provide a convenient means for monitoring the pollution degree of various heavy metal ions in the seawater. Webb et al. (25) developed another technology that HSF traps small organic molecules such as neutral red. Inside the interior of HSF indicated that HSF was first dissociated into subunits by adjustment to pH 2.0 in the presence of the small molecules to be trapped, and then reformed HSF being composed of the dissociated subunits trapped the neutral red while the pH in the medium increased from 2.0 to 7.0. These results show that the ferritin is able to trap the various metal ions and small organic compounds and develop other new physiological functions except for iron storage and release. Here, we emphasize describing a procedure for trapping Cu2+ inside the interior of DALF, methods characterizing a scroll ferritin reactor in flowing seawater, and capacity of trapping Cu2+ under various reaction conditions. Our results show that the ferritin reactor can trap Cu2+ in flowing seawater directly. Moreover, this reactor could be used to provide a convenient means to analyze and estimate the pollution degree of Cu2+ or other heavy metal ions in the sea area.

Materials and Methods Chemicals and Materials. Most chemicals were from Sigma and of analytical and spectral purity. Fresh fish liver of Dasyatis akajei was purchased from Xiamen Fish Company, Xiamen, China. The DALF samples (15-20 mg/mL) were prepared as recently described (5, 26), giving an average ratio of Fe3+/Pi of 1:9 ( 0.5 within the ferritin core before the proteins were purified with PAGE. Analysis of Proteins and Metal Ions. Protein concentration measurement was by the normal Lowry method. Bovine serum albumin of 99% purity was used as a protein standard. Total iron content within the DALF core was determined by a normal spectrophotometry at 520 nm as previously described (9). The Cu2+ numbers trapped by the ferritin reactor were determined by an atomic absorption spectrophotometer equipped with a carbon furnace after the protein was nitrified. Pi content within the ferritin core was determined by normal Cooper technology. Ferritin Reactor and Apparatus of Flowing Seawater. Figure 1 shows a ferritin reactor consisting of DALF and an oscillating bag having up to molecular weight of 3 kDa, which means that the organic compounds, inorganic complexes, and other peptides having molecular weights lower than 3 kDa can pass the bag membrane through freely in the flowing seawater. Accessional experiment showed that a complex of DALF-Cu2+ in the bag can be determined by atomic absorbance spectrophotometer, while the reactor in the absence of Cu2+ was deposited in the seawater containing Cu2+ for 100 s, which meant that DALF in the bag is able to trap Cu2+ coming from the flowing seawater easily because of Cu2+ diffusion. An apparatus consisting of a mixer, a vessel, a pump, magnetic stirrer, and a ferritin reactor was used to study the capacity of trapping Cu2+ in the flowing seawater. The mixer is responsible for establishing various Cu2+ concentrations in the seawater according to experimental desire. Another role of the mixer is that it pumps the seawater into the vessel to drive the reactor move in a scrolled manner. Synthetic Apparatus for Fieldwork. Figure 2 shows a synthetic apparatus for monitoring the pollution degree of various heavy metal ions in the seawater, which is composed of a buoyage (Figure 2A), an isolation basket equipped with griddling (1 × 1 cm) (Figure 2C), two annectent lines (Figure 2B), a scrolled ferritin reactor (Figure 2D), and a iron hammer

FIGURE 1. Apparatus of trapping Cu2+ in the flowing seawater. (A) A mixer that is responsible for supplying flowing seawater containing different Cu2+ concentrations to the reactor. (B) Connective tube for inputting the flowing seawater. (C) Connective tube for outputting the flowing seawater. (D) Flowing seawater. (E) Scrolled ferritin reactor. (F) Magnetic bar. (G) Magnetic stirrer.

FIGURE 2. Apparatus for trapping Cu2+ in the sea area. (A) Buoyage of fixing the reactor. (B) Connective line. (C) An isolation basket equipped with griddling (1 × 1 cm). The basket role prevents the animals from attacking and fixes the reactor in the basket for entrapping Cu2+ in sea. (D) Scrolled ferritin reactor. (E) Equalizer. It allows the basket to move vertically in the sea area. (Figure 2E). The buoyage and the hammer are responsible for fixing the reactor within the specific sea area and for maintaining an identical undersea depth of 10 m during the monitoring period. The hammer role allows the reactor to move vertically during the monitoring period. The important role of the isolation basket rids various halobis that attack the reactor and limits the moving range of the scrolled reactor during trapping Cu2+. Moreover, this apparatus had been used to trap various heavy metal ions such as Pb2+, Ni2+, and Zn2+ and other small organic molecules such as methyl viologen in the flowing water system. In addition, all VOL. 38, NO. 8, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Trapping Cu2+ numbers per molecular ferritin in the reactor against the ferritin samples containing various iron contents within the protein shell. No. 1: Native DALF; No. 2: DALFri; No. 3: ApoDALF; No. 4: DALFrc in the absence of phosphate; and No. 5 DALFhirc in the absence of phosphate. experimental results shown in Figures 3∼8 are established to be average datum for three measurements at the same time. DALF Samples Preparation. DALF samples were prepared as recently described by Huang et al. (26) and Kong et al. (5). Most iron within the DALF shell was moved by a method previously described (9, 27). Then, the apo DALF samples were separated on a Sephadex G-25 column (2 × 10 cm) previously equilibrated with 0.025 M Tris-HCl/0.1M NaCl. The reconstituted DALF was prepared and purified by a combination of two methods as described recently (5, 15). The experimental DALF samples containing different iron contents within the protein shell were prepared according to the methods of Harrison et al. (9) and Huang et al. (26).

Results and Discussion Trapping Cu2+ Capacities of the DALF Samples. Yablonski (28) and Theil (4) indicated that multiple steps occurred in iron core formation, which were described as follows: (1) first, Fe2+ entered the protein; (2) several alternate paths might be followed that include oxidation at a site on the protein, oxidation on the core surface, and mineralization. In addition, our previous study with HSF demonstrated that two different ratios of phosphate to iron within the ferritin core were clearly divided. The iron structures with 1:3 and 1:13 ratios of phosphate to iron were found on the surface of HSF core and in the inside of the core, respectively (19). Moreover, the rate of iron release with dithionite reduction on the surface of the core is quicker than that in the inside of the core, indicating that the phosphate plays an important role in accelerating the rate of iron release (19, 20, 26). Further, a strong oxidizer and ferrous ions at slow rates easily reconstituted a new core formation within the apoferritin shell in the absence of extra phosphate (9, 10, 29). Even so, the mechanism of phosphate role in iron deposition and binding to other heavy metal ions such as Cu2+, Co2+, and Pb2+ is still unclear and disputed. To understand the role of high ratios of phosphate to iron within the ferritin core in trapping Cu2+, five samples of native DALF, DALF with half iron core (DALFhi), apoDALF, reconstituted DALF in the absence of phosphate (DALFrc), and reconstituted DALF with half iron core (DALFhirc) were prepared and purified by a combined technology of the kinetics of iron release and the column chromatography, respectively, according to the experimental desire. The relationship between the Cu2+ numbers trapped by the ferritin 2478

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and the iron content within the protein core was next investigated. Figure 3 shows that average trapping numbers of 50 ( 3 Cu2+/DALF, 37 ( 2 Cu2+/DALFhi, and 15 ( 2 Cu2+/ apo-DALF were calculated in 24 h, respectively. It is for this reason that the numbers of trapping Cu2+ are independent of the available volume within the protein shell due to the volume size; among these ferritins is VapoDALF > V DALFhi > VDALF. In addition, the close numbers of 26 ( 3Cu2+/DALFrc and 28 ( 3Cu2+/DALFhirc are observed in 24 h according to Figure 3, in which both numbers are lower than that with native DALF and DALFhi and higher than that with apoDALF. Even so, the available volume of both DALFrc and DALFhirc is quite different; the Figure 3 results show that the capacities of trapping Cu2+ of both proteins are similar, which indicates that the phosphate role within the ferritin core in improving capacities of trapping Cu2+ numbers would not be ignored, whereas the available volume within the ferritin shell plays a minor role in increasing trapping numbers. Initial investigation of the physiological role of phosphate in ferritin function found that there is a high ratio of phosphate to iron on the surface of the ferritin core and indicated that the phosphate is involved in facilitating ferritin core formation and assisting in redox reaction (17, 30). With the results shown in Figure 3, we found that the sites of storing Cu2+ and the capacities of trapped Cu2+ depended strongly on the high ratio of phosphate to iron on the surface of the DALF core. In addition, it is noted that apo DALF in the absence of phosphate and iron still shows the capacities of trapping Cu2+, which suggests that the composition of amino acids on the inside surface of the protein shell participate in the reaction of trapping Cu2+. We conclude that three main factors involved in the iron structure, the phosphate content, and the composition of amino acid on the inside surface of the protein shell play an important role in bound Cu2+. Moreover, the phosphate may play a most important role in trapping Cu2+ because DALF shows the maximal trapping capacities, which are three times higher than that of apoDALF and double that of DALFrc in the absence of phosphate. Recent observations demonstrate that although phosphate is not essential for core formation, its presence influences the core properties and likely shows a lot of important roles in developing its new function such as a ferritin reactor for monitoring environmental pollution in the flowing water and being considered a marker for diagnosing cancer. Dependence of Trapping Cu2+ on the Incubation Time. Our recent study with a reactor of reconstituted pig spleen ferritin (PSFr) demonstrates that the reactor has three basic characteristics, which are described as follows: (1) the ferritin is able to trap various heavy metal ions such as Pb2+, Co2+, and Mn2+ in the flowing seawater directly; (2) the composition of phosphate and iron within the ferritin core shows high stability in the seawater; and (3) the composition within the ferritin shell appearing to have high stability during the protein is placed in the NaCl solution with a concentration ranging from 1 to 6% (14). In addition, we found that the composition within the DALF core appearing to have a high stability during the reactor shown in Figure 1 is placed in the seawater solution with 4% NaCl for 12 h, specifically in the flowing seawater in 120 h. Figure 4 shows a relation of trapping Cu2+ numbers with the DALF reactor as a function of reaction time, which concludes that the longer the DALF reactor is in the flowing seawater, the more Cu2+ numbers the reactor traps. A parabola curve in both relations in Figure 4 was observed. The maximal trapping capacity shows 98 ( 5 Cu2+ per molecular DALF in 120 h, giving a average trapping rate of 0.82 Cu2+/DALF/h and exhibiting a saturated phenomena for trapping Cu2+. Huang et al. (14) reported that the DALF released two molecular Fe3+ while it traps one molecular

FIGURE 4. Trapping Cu2+ numbers per molecular DALF of the reactor in the flowing seawater against reaction time. The maximal trapping Cu2+ numbers per molecular DALF is 98 ( 5 in 120 h, giving an average trapping rate of 0.82 Cu2+/DALF/h.

FIGURE 5. Trapping Cu2+ numbers per molecular DALF of the reactor against reaction time in the different Cu2+ concentration. (A) 10 mg/L Cu2+ in the flowing seawater. (B) 1 mg/L Cu2+ in the flowing seawater. (C) 100 µg/L Cu2+ in the flowing seawater.

heavy metal ion such as Zn2+ in the flowing seawater. It is for this reason that the curve orbit of trapping Cu2+ with the DALF reactor as a function of time (Figure 4) is similar to that of iron release from the DALF as recently described (5). This novel behavior means that the reactor not only traps Cu2+ in the flowing seawater but also releases the iron out of the protein shell synchronously. It is indicated that the Cu2+ takes up the site of the released iron within the ferritin core during the reactor trapping. In addition, similar Cu2+ numbers can be trapped by both DALFrc (26 ( 3 Cu2+/DALFrc) and DALFhirc (28 ( 3 Cu2+/DALFhirc) (Figure 3), which affirmed further that the numbers of trapping Cu2+ in the flowing seawater were independent of the structure of the ferritin core in the absence of phosphate. In addition, the amino acid composition on the inside surface of the ferritin shell or the regulation capacity of the protein shell plays a role in improving the capacity of trapping Cu2+. Moreover, a high ratio of Pi to Fe3+ within the ferritin core is also considered to be an important factor in improving the trapping capacity in the flowing seawater, but the factor later appears to be more important for trapping Cu2+ than that in front because the trapping numbers with DALF reactor shown are higher than that with DALFrc or DALFhirc reactors. Dependence of Trapping Cu2+ Numbers on the Cu2+ Concentration and on the Incubation Time. One of most important factors of designing a ferritin reactor to monitor the pollution degree of heavy metal ions in the flowing seawater is whether there is positive dependence of the trapping Cu2+ numbers with the reactor on the Cu2+ concentration and on the incubation time, respectively. To characterize the trapping capacity of Cu2+ in the flowing seawater, the DALF reactor was incubated with different Cu2+ concentrations of 100 µg/L, 1 mg/L, and 10 mg/L according to experimental desire at 30 °C, respectively. The experimental data reported in Figure 5 have revealed two important properties of trapping Cu2+ numbers with the reactor, which are described as follows: (1) the maximal Cu2+ numbers that the reactor traps in the seawater system with Cu2+ concentration of 100 µg/L, 1 mg/L, and 10 mg/L in 24 h are observed to be 55 ( 3 Cu2+/DALF (Figure 5A), 47 ×b1( 3 Cu2+/DALF (Figure 5B), and 22 ( 2 Cu2+/DALF (Figure 5C), respectively. These results indicate that there is a positive dependence of trapping Cu2+ numbers with the DALF reactor on the Cu2+ concentration in the flowing seawater. (2) Like the Figure 4 results, the Cu2+ numbers that the reactor traps in the seawater system with Cu2+ concentrations of 100 µg/L, 1 mg/L, and 10 mg/L, respectively, depends on the different

incubation time (in 24 h), giving average trapping numbers of 2.29, 1.96, and 1.17 Cu2+/DALF/h in the incubation time of 12 h, respectively. It should be noted that the trapping numbers with the DALF reactor is higher than that with other reactors equipped with apoDALF, DALFhi, DALFrc, and DALF hirc, respectively (Figure 3). In the course of this work, we have noticed that these differences might be connected with the rate regulation capacity of the protein shell and the different composition of iron and phosphate among the ferritins. Accordingly, our present work has largely focused on studying the mechanism that the native ferritin reactor traps various heavy metal ions and its application in monitoring environmental pollution in the flowing water system. Effect of the Incubation Mediums on the Capacities of Trapping Cu2+. Our recent study with the PSF reactor indicated that nontransient metal elements such as Na+, K+, and Ca2+ were scarcely trapped by the ferritin reactor, which suggests that the basic metal ions such as Na+, K+, and Ca2+ rather than the transient elements such as Cu2+, Mn2+, and Pb2+ in the seawater show a weakening capability for taking up the iron binding sites within the ferritin core. In the course of this work, we have noticed that only these transient elements rather than nontransient elements in the flowing seawater are trapped by the reactor directly. The reason for trapping transient elements was considered because those elements might utilize its electron orbit of a d structure for binding to the iron site within the ferritin core while the reactor trapped them. The experiments shown in Figure 6 reported a study that the DALF reactor trapped Cu2+ in the different mediums. Upon standing, the trapping Cu2+ capacities with the DALF reactor were studied under the various reaction mediums with the distilled water, the seawater, and the lake water at 30 °C (pH 6.5), respectively. The data in Figure 6 are particularly revealing of three processes involved in the Cu2+ numbers in that the DALF reactor trap depends on the reaction mediums in the different incubation times, which are described as follows: (1) the numbers of trapping Cu2+ in the distilled water (Figure 6A) are somewhat higher than that in the seawater (Figure 6B) in the incubation time ranging from 0 to 30 h, which suggests that the basic metal elements such as Na+, K+, and Mg2+ in the seawater play a minor role in affecting the capacity that the reactor has to trap Cu2+. Similar results and conclusions of the trapping numbers of 0.8 Mg2+/HSF were reported by Pead et al. (24). (2) The trapping Cu2+ numbers in the lake water (Figure 6C) are evidently lower than these in the distilled VOL. 38, NO. 8, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 7. Trapping Cu2+ numbers per molecular DALF of the reactor in situ measurement in the sea area against the reaction time. FIGURE 6. Effect of various reaction mediums on trapping Cu2+ numbers per molecular DALF of the reactor. The systems of reaction medium: (A) Distilled water; (B) seawater; and (C) lake water. water and in the seawater. To obtain information on how the lake water affects the accumulation of Cu2+ within the ferritin core, the concentration and kinds of various transient metal ions in the lake water were determined by an atomic absorbance spectrophotometer. Our experimental results reveled that the ion concentrations of Zn2+, Cd2+, and Pb2+ in the lake water were at least five times higher than that of the seawater. In addition, we found further that the concentration of various heavy metal ions such as Zn2+, Cd2+, and Pb2+ in the lake water, where the DALF reactor is placed, were higher than those in the distilled water or the seawater. It is for this reason that the lesser accumulation of Cu2+ within the ferritin core is probably that the DALF distributes a few sites of iron binding to trap other metal ions except Cu2+, which shows that the numbers of trapping Cu2+ in the lake water are lower than those in the distilled water and the seawater (Figure 6). (3) Three curves showing that the ferritin reactor traps Cu2+ are dependent on the incubation time reflected in that the trapping number increases with the incubation time, suggesting that the inorganic and organic complexes in the flowing water may affect scarcely the behavior of trapping Cu2+. Trapping Cu2+ with DALF Reactor in Situ Measurement. A synthetic apparatus shown in Figure 2 is composed of a buoyage, an isolation basket equipped with griddling (1 × 1 cm), and a scrolled ferritin reactor. Two important roles of the buoyage are that it acts as a marker exhibit and fixes where the reactor works for trapping Cu2+ at specific sea area under the depth of 10 m below seawater, in which the Cu2+ concentration in the area maintains the relative constant in 1 day (24 h). The isolation basket prevents halobios from destroying the reactor (Figure 2). With seawater movement back and forth, the dissociative reactor as a scrolled manner traps Cu2+ in the basket. To investigate the capacity of trapping Cu2+ as an in situ measurement, the synthetic apparatus was placed in the southern sea area, Xiamen City, China for 12 h. The experimental results of the Cu2+ numbers trapped by the reactor in the sea area are given in Figure 7. Figure 7 shows that the reactor has a capacity to trap Cu2+ in the sea area, giving the maximal trapping numbers of 16 ( 2Cu2+/DALF and indicating an average rate of 1.33 Cu2+/ DALF/h in 12 h. This trapping capacity is lower than that of those in Figure 5A,B and is similar to that in Figure 5C, which means that the Cu2+ concentration in the sea area is calculated to be lower than that ranging from 1 mg/L (Figure 5B) to 100 2480

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FIGURE 8. Trapping Cu2+ numbers per molecular DALF of the reactor in situ measurement in the sea area against reaction time during the climax and the neap. No. 1 (A): reaction time 0∼1.5 h. No. 2 (B): reaction time 1.5∼3.0 h; No. 3 (C): reaction time 3.0-4.5 h. No. 4 (D): reaction time 4.5∼6.0 h. mg/L (Figure 5A) and close to 1 µg/L (Figure 5C, equal to 1.00 ppb) according to Figures 5 and 7. These data further indicate that the reactor be fit for trapping dissociative Cu2+ in the sea area and that this technology would be considered to be an available monitoring method for analyzing the pollution degree of Cu2+ in an in situ measurement. To further confirm the function and efficiency of the DALF reactor for trapping Cu2+, four ferritin reactors we use are employed to study the varying concentration of Cu2+ in turn in the specific period ranging from the climax and the neap in the sea area of Huang Cu, Xiamen City, China. The DALF reactors are placed 10 m below sea level for studying the capacity of trapping Cu2+. The working time of each reactor is controlled to be 1.5 h, and the total reaction time of four reactors is up to 6 h, covering the period from flood tide and ebb tide. In addition, the authors tried to utilize those reactors to understand primarily the Cu2+ diluting capacity of the seawater in the appointed sea area during the climax and neap. Figure 8 shows the Cu2+ numbers that each DALF reactor takes 1.5 h to trap in turn in the sea area of Huang Cu, Xiamen City, China during the climax and the neap, in which the trapping numbers ranging from 7.0 ( 1 to 10 ( 1 Cu2+/ DALF/h are obtained during the climax and the neap. These findings indicated that the Cu2+ content of the seawater in

the flood tide was lower than that in the ebb tide. Moreover, these data may be used to elucidate the Cu2+ diluting capacity by the seawater in the specific sea area. The significant research here indicates that the scrolled ferritin reactor shown in Figure 2 is a useful tool for monitoring pollution degrees that various heavy metal ions form in the specific sea area in 1 day or longer.

Acknowledgments This project is supported by the State Natural Science Foundation of China (40276033), by the Foundation of Fujian Natural Science (C0310006), and by the Natural Science Foundation of Xiamen State in China (3502Z2001263), respectively.

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Received for review August 31, 2003. Revised manuscript received January 28, 2004. Accepted February 6, 2004. ES034953J

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