Characteristics of Trapping Various Organophosphorus Pesticides

Reactor of Shark Liver (Sphyrna zygaena). He-Qing Huang,*,‡,§ ... with the SZLF reactor in the flowing water is described in detail, showing the ma...
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Anal. Chem. 2005, 77, 1920-1927

Characteristics of Trapping Various Organophosphorus Pesticides with a Ferritin Reactor of Shark Liver (Sphyrna zygaena) He-Qing Huang,*,‡,§ Zhi-Qun Xiao,*,† Qing-Mei Lin,†,‡ and Ping Chen*

Department of Biochemistry and Biotechnology, School of Life Sciences, Key Laboratory of Marine Environmental Science, Ministry of Education, The Key Laboratory for Chemical Biology of Fujian Province, and MOE of Key Laboratory for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, China

A reactor is composed of liver ferritin of Sphyrna zygaena (SZLF) and an oscillating bag. A reactive procedure for trapping various organphosphorus pesticides (OPs) with the SZLF reactor in the flowing water is described in detail, showing the maximal trapping numbers of 28 ( 1.0 dichlorovos/SZLF, 42 ( 1.0 dimethoate/SZLF, and 55 ( 1.0 methamido-phos/SZLF determined by a improved spectrophotometric method in 12 h. In addition, it is found that the OP numbers trapped by the reactor increase along with the incubation time and its concentration increment in the flowing water (or seawater), respectively. This trapping capacity is considered to depend on the composition of amino acids on the surface of the ferritin shell interior rather than the available volume within the shell. A novel pathway for trapping various OPs with the ferritin is suggested in reference to unstable characteristics of the protein subunits. We claim that the ferritin reactor will be employed to monitor the contamination level of various OPs in the flowing water continuously. Ferritin is an iron storage protein found in most biological organisms in nature, including bacteria, fungi, plants, and mammalians.1-3 Ferritin roles in vivo are considered to carry out the regulation of iron level within the cell.4,5 Most ferritins coming from the different sources show very similar structures, which are composed of 24 polypeptide subunits to form a hollow spherical structure of 4:3:2 high symmetry.6,7 Moreover, the * To whom correspondence should be addressed. E-mail: hqhuang@ xmu.edu.cn. Tel: 86-0592-2183255. Fax: 86-0592-218663. † Key Laboratory of Marine Environmental Science. ‡ Key Laboratory for Chemical Biology of Fujian Province. § Xiamen University. (1) Stiefel, E. I.; Watt, G. D. Nature 1979, 279, 81-83. (2) Kong, B.; Huang, H. Q.; Lin, Q. M.; Kim, W. S.; Cai, Z. C.; Cao, T. M.; Miao, H.; Luo, D. M. J. Protein Chem. 2003, 22, 61-70. (3) Huang, H. Q.; Xiao, Z. Q.; Chen, X.; Lin, Q. M.; Cai, Z. W.; Chen, P. Biophys. Chem. 2004, 111, 213-222. (4) Lindsay, S. L.; Brosnahan, D., Jr.; Lowery, T. J.; Crawford, K.; Watt, G. D. Biochim. Biophys. Acta 2003, 1621, 57-66. (5) Goto, F.; Yoshihara, T.; Shigemoto, N.; Toki, S. Nat. Biotechnol. 1999, 17, 282-286. (6) Harrison, H. M.; Hoy, T. G.; Macara, I. G.; Hore, R. J. Biochem. J. 1974, 143, 445-451. (7) Harrison, P. M.; Arosio, P. Biochim. Biophys. Acta BioEnerg. 1996, 1275, 161-203.

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ferritins are composed of a protein shell with a diameter of 120130 Å and an iron core with diameter of 70-80 Å located at the center of the protein shell.8 In addition, mammalian ferritins are made up of two subunit types with heavy chain (H, ∼21 kDa) and light (L, ∼19 kDa) chain, such as horse spleen ferritin (HSF). Bacterial ferritins consist of a single subunit type such as ferritin of Escherichia coil. The H and L chains consisting of 182 and 174 amino acids, respectively, are encoded on different chromosomes, sharing ∼55% homology in sequence. It is found that catalytic Fe2+ oxidation is restricted to the H-type subunit and that participating in formation of the iron core is indicated to be an L-type subunit being more efficient than that of an H-subunit.8-11 Recently, Lee et al.9 developed a recombinant ferritin H/L-hybrid by a direct gene fusion between H- and L-chain subunits; these authors found that this hybrid ferritin was more efficient in taking up iron than that of the homopolymers of H- and L-chains, respectively. Previous studies with X-ray technology indicated that various channels with of 0.3-0.5-nm diameter across the ferritin shell were formed by the intersections of two-, three- or four-peptide subunits. These channels are critical to ferritin’s ability to release iron to and store it in the cell. In the kinetic research of iron release, it is indicated that reducing agents such as sodium dithionite and vitamin C and chelating agents of Fe2+ such as R′,R-dipyridyl or 1,10-phenanthroline must pass though the channel consisting of three-peptide subunits across the protein shell for iron reduction and release, respectively.6,12-13 Yang et al.14 used a probe of nitroxide to study the kinetics of molecular diffusion into ferritin, suggesting that the channel diameter of the ferritin be over 0.70.9 nm rather than 0.3-0.4 nm. Interestingly, the Fe2+ compound within the ferritin core can be directly oxidized by big protein oxidants such as cytochrome c and Cu2+ proteins in the absence (8) Theil, E. C.; Takagi, H.; Small, G. W.; He, L.; Tipton, A. R.; Danger, D. Inorg. Chim Acta 2000, 297, 242-251. (9) Lee, J.; Kim, S. W.; Kim, Y. H.; Ahn, J. Y. Biochem. Biophys. Res. Commun. 2002, 298, 225-229. (10) Levi, S.; Yewdall, S. J.; Harrison, P. M.; Santambrogio, P.; Cozzi, A.; Rovida, E.; Albertini, A.; Arosio, P. Biochem. J. 1992, 288, 591-596. (11) Suk, Y.; Kim, I. H. Biotechnol. Lett. 2003, 25, 993-996. (12) Jones, M. M.; Johnston, D. O. Nature 1967, 216, 509-510. (13) Huang, H. Q.; Lin, Q. M.; Kong, B.; Zeng, R. Y.; Qiao, Y. H.; Chen, C. H.; Zhang, F. Z.; Xu, L. S. J. Protein Chem. 1999, 18, 497-454. (14) Yang, X. K.; Burn, N. E.; Thomson, A. J.; Moore, G. R.; Chasteen, N. D. Biochemistry 2000, 16, 4915-4923. 10.1021/ac048753h CCC: $30.25

© 2005 American Chemical Society Published on Web 02/11/2005

of small molecular oxidants.15 It is noted that the molecular diameters of these oxidants are wider than that of the ferritin channels as previously described. Even so, the oxidation pathway and mechanism of Fe2+ conversion by these oxidants are still unclear. In the development of trapping capacity from the ferritin, Webb et al.16 found that HSF can capture a few small organic molecules such as neutral red within the ferritin shell. Huang et al.17 found that AvBF and pig spleen ferritin (PSF) can directly pick up the electrons from the bare platinum electrode at -600 mV versus NHE for iron release in the absence of small molecular reducers. These novel behaviors suggest that there is an electron tunnel across the protein shell that participates in electron transfer between the electrode and the protein core. Unfortunately, the molecular structure of the electron-transferring tunnels has not been elucidated because of its structure complex. In these cases, it is important to study the mechanism for trapping various small organic compounds by the ferritin. Meldrum et al.18reported that Mn3+ oxyhydroxide (MnOOH) cores within the nanoscale cavity of the HSF could be reconstituted in the absence of Fe2+ and phosphate. Pead et al.19 found that various heavy metal ions such as Cd2+, Zn2+, Cu2+, Ni2+, Co2+, and Mn2+ can bind to apo-HSF, holo-HSF, reconstituted HSF, and native HSF, respectively. Moreover, Huang et al.20-21 established a reactor of reconstituted PSF including other fish ferritins and used it for trapping Cd2+, Zn2+, Co2+, Cu2+, and Mn2+ in the flowing water, which have been employed to monitor the contamination level of various heavy metal ions such as Cu2+ in the flowing seawater. Pesticides are the most abundant environmental pollutants found in soil, water, the atmosphere, and agricultural products. OP compounds comprise a diverse group of chemicals that are extensively used as insecticides in modern agriculture.22 These neurotoxic compounds, which are structurally similar to the nerve gases soman and sarin, irreversibly inhibit the enzyme acetylcholinesterase, essential for the functioning of the central nervous system in humans and insects.23 It is for this reason that monitoring the trace levels of OPs is important for human health protection and environmental control. The need for reliable determination of OP residues has led to the extensive applications of sophisticated analytical methods, such as high-pressure liquid chromatography (HPLC), mass spectrometry, gas chromatography (GC), spectrophotometry, thermospray-mass spectrometry, and electrochemical biosensors.23,24 Despite the importance of these problems, very few published studies have been concerned with a biological monitor of pesticide exposure. The reasons for this are, briefly, as follows:25 (15) Watt, G. D.; Jacobs, D.; Frankel, R. B. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7457-7461. (16) Webb, B.; Frame, J.; Zhao, Z.; Lee, M. L.; Watt, G. D. Arch. Biochem. Biophys. 1994, 309, 178-183. (17) Huang, H. Q.; Lin, Q. M.; Wang, T. L. Biophys. Chem. 2002, 97, 17-27. (18) Meldrum, F. C.; Douglas, T.; Levis, S.; Arosio, P.; Mann, S. J. Inorg. Biochem. 1995, 58, 59-68. (19) Pead, S.; Durrant, E.; Webb, B.; Larsen, C.; Heaton, D.; Johnson, J.; Watt, G. D. J. Inorg. Biochem. 1995, 59, 15-27. (20) Huang, H. Q.; Lin, Q. M.; Luo, Z. B. J. Protein Chem. 2000, 19, 441-447. (21) Huang, H. Q.; Cao, T. M.; Lin, Q. M. Environ. Sci. Technol. 2004, 38, 2476-2481. (22) Neufld, T.; Eshkenazi, I.; Cohen, E.; Rishpon, J. Biosens. Bioelectron. 2000, 15, 323-329. (23) Rogers, K. R. Biosens. Bioelectron. 2001, 16, 225-230. (24) Okumura, T.; Nishikawa, Y. J. Chromatogr. 1995, 709, 319-331.

1. Analytical methods currently available are often very complicated and require highly specialized laboratories. 2. Pure standards for measuring metabolites are not commercially available. 3. In field studies on pesticide exposure, it is difficult to collect representative samples and to define a correct sampling time. 4. There are scarcely any validated analytical methods of chemistry and biological indicators suggested by reference organizations for monitoring trace OP residues in the river or sea continuously coming from the agriculture industry and OP manufacturers. Based on the above consideration, developement of a conventional analytical method for evaluating the contamination level of trace OPs in flowing water continuously for few days or one week is very important. The present studies reported the trapping capacity of various OPs such as dimethoate, methamidophos, and dichlorovos with the SZLF reactor in the flowing water. Several influencing factors for trapping OPs with the reactor are discussed in detail. A ferritin mode is pointed out to elucidate a novel pathway for trapping various OPs in the flowing water, indicating that the reactor is a perfect analytical means for continuously monitoring the contamination level of various OPs in the flowing water. EXPERIMENTAL SECTION Materials and Reagents. Fresh shark, Sphyrna zygaena, was purchased from Xiamen Fish Co. DEAE-cellulose 52 was obtained from Waterman Co. (Berman, Germany). Sephadex G-25 and Sephacryl S-300 HR are products of Pharmacia (Uppsala, Sweden). Sinapic acid (SA), 2,5-dihydroxybenzoic acid (DHB), and all electrophoresis reagents were obtained from Sigma Co.. The inorganic chemicals are analytical grade obtained from commercial sources in China. SZLF was separated and purified as recently described by Huang et al.3 A procedure of electrophoresis purification was employed to purify abundant SZLF or other ferritins.26 Analysis of Protein and Iron Concentration. Total Fe3+ numbers of SZLF were determined by atomic absorbance spectrophotometry (AAS; model PE800) after the ferritin was nitrated fully. Protein concentration measurement was determined by a normal Lowry procedure. Bovine serum albumin of 99.9% purity is used as a protein standard. Analysis of Inorganic and Organic Phosphorus. The content of inorganic phosphate within the ferritin shell was analyzed by a normal Cooper method27 or inductively coupled plasma (ICP).28 However, these methods are not fit for determining variation in quantity of the ferritin organic phosphorus because of their low sensitivity and instability. Here, an analytical method with high sensitivity and stability for determining minimal organic phosphorus within the ferritin shell is optimized and developed. A mixed reagent is prepared for phosphorus analysis manually, which shows that the original reagents for phosphorus analysis as previously described by Copper27 mixs bismuth sulfate dis(25) Aprea, C.; Colosio, C.; Mammone, T.; Minoia, C.; Maroni, M. J. Chromatorgr., A 2002, 769, 191-219. (26) Chen, X.; Huang, H. Q.; Kong, B.; Cao, T. M.; Xiao, Z. Q. Mar. Sci. 2004, 28, 15-19. (27) Cooper, T. Tools of Biochemistry; Wiley: New York, 1997; p 53. (28) Huang, H. Q.; Kofford, M.; Simpson, F. B.; Watt, G. D. J. Inorg. Biochem. 1993, 52, 51-71.

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solved in 5.0 N nitric acid directly before use. An equation for calculating various OP numbers that the SZLF reactor traps is established as follows:

Pt/SZLF - Pi/SZLF ) Pop/SZLF

(1)

where Pt/SZLF is total numbers of organic and inorganic phosphorus per molecular SZLF, Pi/SZLF is total numbers of inorganic phosphorus per molecular SZLF, and Pop/SZLF is total numbers of trapping OPs per molecular SZLF. Trapping OPs with SZLF Reactor. An apparatus consisting of two pumps, a mixer, a spectrophotometer, a ferritin reactor, and a reactive container was constructed to study the numbers of trapping OPs with the SZLF reactor as recently described by Huang et al.20,21 The reactor consists of SZLF and an oscillating bag with retention molecular mass of 3.0 kDa. The chemical and the biological molecules having molecular mass lower than 3.0 kDa can enter and exit the bag freely, which results in SZLF in the reactor directly trapping the OPs in the flowing water. A 2.0-mL sample of SZLF (1.0 mg/mL) was purified by polyacrylamide gel electrophoresis (PAGE) and placed in the oscillating bag. The reactive bag loading the SZLF sample was sealed up by heat technology to construct a ferritin reactor. The reactor is placed in the flowing water (pH 7.25) containing various concentrations of OPs ranging from 100 ppb to 10 ppm or up to 1000 ppm, respectively. The dimethoate (rogor), the methamidophos (tramaron), and the dichlorovos (DDW) were selected to investigate the capacity of trapping various OPs in the flowing water with the reactor, respectively. The experimental results affirm that salt concentration in the seawater hardly affects the trapping capacity of OPs, which means that this trapping process with a ferritin reactor is an irreversible pathway. To separate free OP in the oscillating bag from the OP within the ferritin shell, the reactor must be dialyzed in distilled water for 60 min before being used. Then the SZLF for trapping OP (SZLFop) was employed to determine the content of organic and inorganic phosphorus within the ferritin shell. Referring to the molecular structures of methamidophos, dichlorovos, and dimethoate, all OPs show only one phosphorus molecule per molecule of pesticides. Clearly, the numbers of trapping OPs with the SZLF reactor are directly calculated according to the increasing content of organic phosphorus within the ferritin shell. Ratio of Phosphorus to Iron within the Ferritin Core. A 10.0-mL aliquot of SZLF (pH 7.25) was divided into seven parts, and then the ferritin samples were mixed with 200 µL of solution containing both R′,R-dipyridyl and dithionite under anaerobic conditions. With reference to the kinetic curve of iron release as recently described by others,13 the protein samples of iron release were separated on a Sephadex G-25 microcolumn (0.5 × 2.0 cm) previously equilibrated with 0.025 M Tris-HCl (pH 7.25)/0.1 M NaCl to remove the released iron and phosphate and to collect the ferritin samples consisting of different contents of iron and phosphate according to the desired kinetic reactive time. To analyze iron and phosphate content within the ferritin core AAS and the modified Cooper’s method are employed, respectively. 1922 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

Figure 1. Visible spectra of SZLFp ranging from 500 to 660 nm at pH 7.25. (A) SZLF; (B) SZLFP in 10 min; (C) SZLFP in 24 h; (D) SZLFP in 32 h.

RESULTS AND DISCUSSION Visible Spectra of SZLFP and SZLFM. Most mammal ferritins such as HSF, PSF, and human serum ferritin29 consist of two subunit types, H and L. Moreover, though shark is a vertebrate, its liver ferritin consists of the single-subunit type,3,30 which is similar to bacterial ferritin rather than mammal ferritins.31 In addition, the visible spectrum of SZLF shown in Figure 1A exhibits a typical absorbance peak unlikely and its absorbance intensity decrement gradually ranging from 500 to 660 nm, which are similar to these of HSF and PSF rather than that of bacterial ferritin of Azotobacter vinelandii (AvBF) with heme composing.31 These phenomena indicate further that SZLF takes the characteristics of mammal ferritin with a visible spectrum and of bacterial ferritin with a single-subunit type. It is known that HSF has the ability to trap inorganic phosphate (Pi) within its shell interior.32 To determine whether SZLF has a similar ability, the ferritin was directly incubated with phosphate in the absence of Fe2+. The experimental results show that absorbance intensity of SZLF with Pi treatment, shown in Figure 1B, in 10 min is similar to that of native SZLF shown in Figure 1A in the visible spectra ranging from 500 to 660 nm. In addition, we find that both ferritins contain as much as Pi concentration, meaning that minimal Pi is only trapped by SZLF, called SZLFp, in 10 min. With prolonged reaction time, other absorbance curves of SZLFp shown in Figure 1C∼D show still similar spectral characteristics and have different absorbance intensity. But, the intensity both curves shown in Figure 1C∼D shows higher than that shown in Figure 1A∼B. Using analysis of Pi, it is found that Pi content in front is higher than that in later, indicating that the numbers of trapping Pi with ferritin increase along with prolongation of the reaction time. The Pi content among ferrtins, shown in Figure A∼D, are divided to be CFigure 1D > CFigure 1C > CFigure 1B g CFigure 1A according to the analysis results. Clearly, SZLF, like (29) Yan, L.; Huang, H. Q.; Jin, H. W.; Chen, P.; Yang, T. C.; Wang, Q. L. Chem. J. Chin. Univ. 2004, 25, 1892-1896. (30) Geetha, C.; Deshpande, V. Comp. Biochem. Physiol. 1999, 123 (B), 285294. (31) Huang, H. Q.; Zhang, F. Z.; Xu, L. S.; Lin, Q. M.; Huang, J. W.; Zeng, D. Bioelectrochem. Bioenerg. 1998, 44, 301-307. (32) Huang, H. Q.; Watt, R. K.; Frankel, R. B.; Watt, G. D. Biochemistry 1992, 32, 1681-1687.

Figure 2. Molecular structure of various organphosphorus pesticides. The molecular widths of methamidophos, dichlorovs, and dimethoate are observed to be similar, but the molecular length in the last than in the first.

Figure 3. Visible spectra of the SZLFM ranging from 500 to 660 nm at pH 7.25. (A) SZLF; (B) SZLFM in 10 min; (C) SZLFM in 24 h; (D) SZLFM in 32 h.

HSF as recently described by Johnson et al.,33 has the ability to trap Pi within the protein shell in the flowing water. The molecular structures of various OPs, e.g,, methamidophos, dimethoate, and dichlorovos, are shown in Figure 2. It is seen that the length and the width of these OPs are bigger than that of the daimeter of the ferritin channel, 0.7-0.8 nm, as recently described by Yang et al.,14 which points out that, apparently, ferritin cannot utilize its channels to trap and store these OPs. To validate this viewpoint, the SZLF is directly incubated with methamidophos in a vessel equipped with a stirrer for the desired time. The experimental results in Figure 3 reveal that the absorbance intensity of SZLF in the visible spectrum ranging from 500 to 660 nm increases along with the incubation time increment. Pi analysis further finds that SZLF shows the novel behavior of trapping methamidophos, called SZLFM, which results in the absorbance intensity of spectrum from SZLFM increasing distinctly. According to the organic phosphorus content among ferritins shown in Figure 3A-D, we can conclude that the contents of trapping methamidophos with SZLFM are considered to be CFigure 2D > CFigure 2C > CFigure 2B g CFigure 2A. Even so, it is indicated

that spectral characteristics of these curves are similar to these shown in Figure 1A-D. We next addressed the question of whether other OPs could be trapped by the SZLF reactor in the flowing water. The experimental results show that SZLF in the reactor has the capacity to trap various OPs actively, showing that the maximal trapping numbers with the reactor are 28 ( 1 dichlorovos/SZLF, 42 ( 1dimethoate/SZLF, and 55 ( 1 methamidophos/SZLF in 12 h. These results show that SZLF in the reactor is a biological container with nanometer size and it undergoes a process of trapping various OPs in the flowing water. However, this trapping mechanism with the SZLF reactor in the flowing water is still unclear because its channel width, based on the reported data as previously described by ferritin workers, shows narrower than that of the molecular diameters of these OPs (Figure 2). The European Community and the United States National Institute of Safety and Health (NIOSH) have recommended that pesticide concentration should be determined by either GC or HPLC. Unfortunately, although sensitive and accurate, both methods are time-consuming and expensive and, above all, require pretreatment of the sample and highly qualified technicians.22,25 In addition, these methods make OPs difficult to analyze in the flowing water continuously and to evaluate total content of trace OPs in the given range. A serious problem for analytical OPs concentration is that the OP trapped by the ferritin reactor will scarcely release out of the protein shell, which make the OP concentration difficult to be analyzed by HPLC, GC, and ampermoetric biosensor. Thus, the OP concentration within the ferritin cavity has to be measured by the modified Copper’s method according to eq 1. Characteristics of Trapping Dichlorovos with the SZLFD Reactor. To understand whether the numbers of trapping various OPs with a ferritin reactor depend on the available volume within the protein shell, the SZLF molecules that are composed of different contents of Pi and iron are constructed and employed to study the capacity of trapping dichlorovos. Figure 4 results reveal a complete process of trapping dichlorovos with the SZLF (SZLFD) reactor in 24 h, which shows biphasic behaviors of trapping dichlorovos, phase A and phase B. To understand the real mechanism, the results shown in Figure 4 are analyzed from right (X-coordinate) to the left. It is noted that the trapping numbers with 19 ( 2 dichlorovos/SZLFD are only determined before the reaction of iron release carries through (Figure 4). However, in phase A (Figure 4A), it is seen that the trapping numbers increase greatly ranging from 19 ( 2 dichlorovos/SZLFD to 81 ( 2 dichlorovos/SZLFD along with iron numbers within the ferritin core decrement ranging from 1890 ( 10 Fe3+/SZLF to 651 ( 10 Fe3+/SZLF. Apparently, the available volume within the SZLFD shell increases along with the iron release increment. Thus, a preliminary conclusion conisdered is that the numbers of trapping dichlorovos increase along with the available volume within the ferritin shell increment. However, in phase B (Figure 4B), we find that the identical trapping numbers with 81 ( 2 dichlorovos/ SZLFD are observed during the further decrease in iron numbers within the ferritin core ranging from 651 ( 10 Fe3+/SZLF to 18 ( 5 Fe3+/SZLF. Unlike the results and the viewpoint shown in Figure 4A, a contrary conclusion is that the trapping capacity with 81 ( 2 dichlorovos/SZLFD is independent of the available volume Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

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Figure 4. Numbers of trapping dichlorovos with SZLFD reactor against the iron numbers within the ferritin core. All filled symbols in represent the numbers of trapping dichlorovos with the SZLFD reactor in 24 h, which are divided into A and B phases. In phase A, the numbers of trapping dichlorovos with the reactor increase as the linear trend decrease during iron content within the ferritiin core ranges from 1890 ( 10 Fe3+/SZLFD to 651 ( 10 Fe3+/SZLFD. In phase B, identical numbers of trapping dichlorovos are observed during iron numbers within the ferritin core decrease ranging from 651 ( 10Fe3+/SZLFD to 18 ( 5 Fe3+/SZLFD.

within the ferritin shell, which means that the available volume plays an minor role in improving the capacity. Thus, we suggest that the numbers of trapping OPs by the ferritin be tightly connected with the content of binding sites for trapping OPs on the surface of the ferritin shell interior rather than the available volume. We hypothesize that this behavior of trapping OPs would stop once the available sites have been held by the OPs completely (Figure 4B); even so the available volume within the ferritin shell interior is enhanced along with the iron’s release increment. In this study, we pointed out that the available sites of amino acid groups on the inside surface of the protein shell rather than the available volume within the shell play an important role in trapping various OPs or other organic small molecules. Previous research showed that the phosphate role was involved in facilitating ferritin core formation33,34 and in accelerating iron release from the ferritin by the reduction of the platinum electrode.17 To study the role of phosphate in improving the capacity of trapping OPs, the SZLF moelcules containing various Pi content within the protein shell interior were prepared and incubated with dichlorovos (0.01 ppm) directly for 24 h. Like the results shown in Figure 4A, Figure 5A results show that biphasic behavior with varying numbers of trapping dichlorovs with the SZLFD reactor are observed. To understand the real mechanism for trapping OPs further, the experimental results shown in Figure 5 are discussed from the right (X-coordinate) of the figure to the left. In Figure 5A, in the initial stages of Pi release, we find that the numbers of trapping dichloroves with the SZLFD reactor, ranging from 18 ( 2 dichlorves/SZLFD to 81 ( 2 dichlorovos/ SZLFD, increase greatly along with the Pi content ranging from a 158 ( 5Pi/SZLF to 95 ( 5 Pi/SZLF decrement. In this process, the available volume within the ferritin shell interior increases along with the iron (Figure 4A) and Pi (Figure 5A) release (33) Johnosn, J. L.; Cannon, M.; Watt, R. K.; Frankel, R. B.; Watt, G. D. Biochemistry 1999, 38, 6706-6713. (34) Cheng, Y. G.; Chasteen, N. D. Biochemistry 1991, 30, 2974-2593.

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Figure 5. Numbers of trapping dichlorovos with SZLFD reactor against the Pi numbers within the ferritin shell. In phase A, the numbers of trapping dichlorovos with the reactor increase as a linear trend during the decrease of Pi numbers within the ferritin core, ranging from 158 ( 5 Pi/SZLFD to 95 ( 5 Pi/SZLFD. In phase B, a constant number for trapping dichlorovos is observed during the decrease of Pi numbers within the ferritin core, ranging from 95 ( 5 Pi/SZLFD to 18 ( 5 Pi/SZLFD.

increment. With further analysis (Figure 5B), we still find that the identical capacity for trapping numbers with 81 ( 2 dichlorovos/SZLFD is observed while the Pi content within the ferritin core decreases ranging from 95 ( 5Pi/SZLF to 18 ( 3Pi/SZLF. This novel behavior reveals, in the anaphases of Pi release, that the binding sites within the ferritin shell interior have completely been held by dichlorovos, which results in the identical trapping numbers shown in Figure 5B being observed. Based on similar mechanisms for trapping OPs shown in Figure 4 and 5, we indicate that the Pi content and the available volume within the protein shell play a minor role in improving capacity for trapping OPs. Moreover, the binding sites of trapping OPs being composed of the groups of amino acids on the surface of protein shell interior play an important role in trapping various OPs in the flowing water. Effect of Diffusion Rate on the Capacity of Trapping Dichlorves with the SZLF Reactor. An apparatus consisting of one pump, a mixer, and a ferritin reactor was constructed to study the capacity of trapping various heavy metal ions as recently described.20 The reactor is similarly employed to trap various OPs in the flowing water. The reactor consists of the SZLF and a dialyzer with retention molecular mass of 3.0 kDa. The pump and the mixer are employed to supply the flowing water containing 0.01 ppm dichlorves (pH 7.25) to the reactor for accelerating the diffusion rate of the pesticide at the identical stirring speed of 240 revolutions/min (30 °C). Figure 6A shows the capacity of trapping dichlorves with the SZLF reactor in the flowing water, which gives the maximal numbers of 36 ( 2 dichlorves/SZLFD in 8 h and shows an average trapping rate of 4.5 ( 0.5 dichlorves/ SZLFD per hour. In addition, the SZLF reactor exhibits the maximal trapping numbers with 31 ( 2 dichlorves/SZLF in 8 h under the condition of actionless water (Figure 6B), giving an average trapping rate of 4 ( 0.5 dichlorves/SZLFD per hour. Compared to both curves shown in Figure 6, the trapping rate with 0.5 dichlorves/SZLFD/hr is only improved by the stirring manner. With reference to channel diffusion mechanism for trapping heavy metal ions as previously described, if the channel

Figure 7. Effect of pH on the numbers of trapping methamidophos with the SZLFM reactor under the condition of different incubation time. Incubation time: (A) 12 h; (B) 96 h. Figure 6. Numbers of trapping dichlorovos with the SZLFD reactor against the incubation time. Curve A: A process of trapping dichlorovos with the SZLFD reactor under the condition of flowing water (pH 7.25) with stirring speed of 120 revolutions/min. Curve B: A process of trapping dichlorovos with the SZLFD reactor under the condition of actionless water (pH 7.25).

would only be a pathway for molecule diffusion into the ferritin shell, the rate of trapping dichlorves should be accelerated greatly along with the rate of pesticide diffusion increment. It is for that reason that a factor with increasing diffuson rate of the pesticide plays a minor role in improving the rate for trapping dichlorves (Figure 6). Thus, we indicate that the ferritin may utilize a novel manner rather than the channel pathway for trapping various OPs in the flowing water. Effect of pH on the Capacity of Trapping OPs with the SZLFM Reactor. Funk et al.35 reported that the rate for iron release from HSF at pH 4.0 was 100-fold faster than that at pH 7.0, indicating that its rate change depended strongly on the ratio of Pi/Fe3+ on the surface of the core. Huang et al.31 indicated that the concentration’s diffusion of H+ or OH- by variable pH medium not only altered the total net charges within the ferritin core but also affected the ferritin conformation by self-regulation itself, which caused the rate of iron release to vary dramatically and compelled the ferritin to release the unstable irons. We find that SZLF has the maximal iron numbers with 1900 Fe3+/SZLF at pH 7.0, which means that most irons within the ferritin core appear stable at physiological pH. In addition, we find further that the iron numbers within the SZLF core decrease along with the physiological pH increment (or decrement), showing the iron numbers with 1630 Fe3+/SZLF at pH 6.0 and with 1720 Fe3+/ SZLF at pH 8.0, respectively. These results suggest that H+ and OH- compell SZLF to release the instable irons and form various available volumes within the protein shell for trapping OPs. The available volumes are considered to be VpH6.0 > VpH8.0 > VpH7.0 according to remaining iron (N) within the SZLF core determined by AAS at various pHs. Figure 7A results show that pH depends on the numbers of trapping methamidophos with SZLFM reactor in 12 h, which are (35) Funk, F.; Lenders, J. P.; Crichton, R. R.; Schnider, W. Eur. J. Biochem. 1985, 152, 167-172.

calculated to be 13.34 ( 1 methamidophos/SZLFM at pH 6.0, 16.08 ( 1 methamidophos/SZLFM at pH 7.0, and 13.02 ( 1 methamidophos/SZLFM at pH 8.0, respectively. In addition, another similar trend for trapping methamidophos in Figure 7B can be observed, giving 16.67 ( 1 methamidophos/SZLFM at pH 6.0, 18.38 ( 1 methamidophos/SZLFM at pH 7.0, and 16.10 ( 1 methamidophos/ SZLFM at pH 8.0 in 96 h, respectively. A trend for the numbers of trapping OPs is concluded to be NpH7.0 > NpH6.0 > NpH8.0 according to the results shown in Figure 7, which shows that the improved capacity of trapping OPs are still independent of the available volume within the ferritin shell. Previous studies showed that there was high ratio of Pi to Fe3+ on the surface of the ferritin core,32,33 meaning that the high ratio can be found on the surface of the ferritin core. However, this ratio would be disappearing along with the physiological pH increment (or decrement) because of iron release. Based on these research results, both factors, the Pi composition on the surface of ferritin core and the amino acid groups on the surface of the ferritin shell interior, might play an important role in improving capacities of trapping OPs. Even so, we still point out that the binding sites for trapping OPs should be composed of the group’s numbers rather than the Pi composition according to Figure 4 and 5 results. In addition, these sites might decrease (or increase) along with the pH varying due to H+ or OH- strongly affecting the charge or dissociation of amino acid residues from the groups. These behaviors make the capacity of trapping OPs weaken. It is seen that the numbers of trapping OPs with the SZLF reactor in flowing water depend on the medium pH (Figures 4 and 5). Trapping Methamidophos with the DALFM Reactor in Flowing Water. Figure 8B results show that plot of numbers of trapping methanidophos per molecular SZLFM as a function of methanidophos concentration in 2 h is linear. The slope may be used to calculate the increasing rate of trapping methanidophos in the flowing water, which means that the numbers of trapping methanidophos with the SZLFM reactor increase along with the pesticide concentration increment. Like Figure 8B, a plot of the numbers of trapping methanidophos per molecular SZLFM as a function of methanidophos concentration in 7 h is linear (Figure 8A), which indicates that the numbers of trapping methanidophos increase also along with the pesticide concentration increment in Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

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Figure 9. Ferritin model for trapping various OPs. (D) A, protein shell; B, iron core; C, channels; D, ferritin model (7); (E) ferritin model with movement of its unstable subunit somewhat; (F) ferritin model where the unstable subunit has been assembled.

7 h. Moreover, the trapping numbers later are higher than these in front, meaning that the trapping numbers depend on not only the methanidophos concentration in the flowing water but also the incubation time. It is for this reason that SZLFM reactor can be employed to analyze the contamination level of methanidophos or other OPs in the flowing water covering a specific range continuously according to the desired reactive time. We point out that this technology for monitoring contamination level of various OPs in the flowing water still shows great significamce in environmental science, specifically in continuous contamination monitoring.21 Suggested Model for Trapping Various OPs with Ferritin. Ferritin is a very specific protein in nature. It shows a supermolecule of 24 subunits joined by noncovalent bonds with highly symmetrical structure, sharing stability against pH change ranging from 2.0 to 12.00.36 With reference to research progress in ferritin, the problem of understanding the entry and exit of iron across the ferritin shell is one of extraordinary complexity. No protein in the cell can control the phase transition of metal ions in solution to one in a solid phase.8 Iron entry and exit were employed by localized unfolding of one polypeptide at the junction of three subunits, which is called 3-fold channels as a entry and exit pathway of iron including other small molecules. In addition, the biological functions of 2- and 4-fold channels that consist of two and four subunits, respectively, across the ferritin shell are still unclear except for the 3-fold one. Recently Huang et al.3 reported that the laser coming from MALDI and the matrix SA were able to not only make SZLF to release the instable subunit but also to form it into a molecular ion for mass measurement, which resulted in measrement of the molecular mass of the subunit rather than the whole ferritin by MALDI-TOF MS directly, showing 21 066.52 m/z per subunit molecule. In addition, we found further that the dissociate subunits coming from the liver ferritin of Dasyatis akajei under the condition of pH 1.0 is directly recombined into an whole ferritin consisting of the shell and the iron core when the medium pH

goes up 2.0-3.0. According to previous research, the electron donor, such as ascorbic acid and reduced methyl viologen, and the electron acceptor, such as Cu2+ protein, participated in a reaction of electron transfer with the iron core within the protein shell.15,37 Small organic molecules such as natural red16 and various OPs (Figures 2-8) can be still entrapped by the ferritin. Apparently the molecular size of these compounds shows more width than that of the 3-fold channels of the ferritin; they are still able to pass through the channel across the protein shell for iron reduction and release, which means that there is a novel pathway that had not been known for trapping small organic compounds in the ferritin. Based on the research results mentioned above, a model shown in Figure 9 for trapping various OPs within the ferritin shell is described as follows: 1. The molecular structure of ferritin consist of a protein shell [Figure 9D (A)], iron core [Figure 9D (B)], and the channels formed [Figure 9D (C)] by interactions among subunits. With reference to a ferritin model in Figure 9D,6 the ferritin shell constructed by noncovalent bonds among the subunits shows a highly symmetrical and stabile structure. 2. The ferritins, consisting of various channels with different diameters, are easily attacked and disturbed by various chemical factors such as the reaction temperature, the inorganic and organic compounds, and the pH in the medium. These chemical factors compel the ferritin to lose its balance of symmetrical subunits, which make the protein move one or two instable subunits from its original position somewhat to maintain another new balance with dissymmetrical subunit structure (Figure 9E). This departure behavior forms a new alleyway across the protein shell for trapping various OPs (Figure 9E), indicating that this alleyway width is much bigger than that (0.7-0.9 nm) of the ferritin channel as recently described (Figure 9D). It is for this reason that organic small molecules such as reduced methyl viologen and various OPs having molecular width bigger than that of the protein channels can utilize this alleyway to diffuse into the ferritin core. 3. The half-departure subunit would unthread its original position with self-assembly (Figure 9F) while the ferritin pauses to capture these organic small molecules into its protein shell interior and to release the unstable iron or the protons. According to this trapping mechanism and the results shown in Figures 4-6, we find that the longer the molecular length of diffusion into the ferritin core, the less the numbers that the SZLF reactor captures.

(36) Watt, R. K.; Frankel, R. B.; Watt, G. D. Biochemistry 1992, 31, 9673-9679.

(37) Huang, H. Q.; Lin, Q. M.; Xiao, Z. Q. Acta Biophys. Sin. 2000, 16, 39-47.

Figure 8. Numbers of trapping methamidophos with the SZLFM reactor against the methamidophos concentration in flowing water (pH 7.25). Incubation time: (A) 7.0 h; (B) 2.0 h. The methamidophos numbers that the reactor traps increase along with the incubation time and its concentration increment in the flowing water.

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4. Yamashita38 reported that a well-ordered array of iron oxide loaded ferritin molecules formed by self-assembly at the Si surface while the protein shell of ferritin was eliminated by heat treatment at 500 °C under nitrogen. Imai et al.39 found that iron core was precipitated in 0.1 M NaOH solutions. These phenomena show that there is weakened interaction of noncovalent bonds between iron composition on the surface of the core and amino acid on one surface of the protein shell interior because whole iron core at the Si surface38 or a bare core39 is easily formed. It is for this reason that the ferritin has the ability to utilize its unstable subunits to build one or more alleyways by self-assembly for trapping organic small molecules and other heavy metal ions in the flowing water. If the suggested model shown in Figure 9 is scientific and reasonable, the fact that the ferritin traps various OPs shown in Figures 2-8 will be explained correctly, which guides the scientists to look for more new biological functions of the ferritin other than iron release and storage. Interpretation of the Ferritin Reactor. Pesticides including various OPs are chemicals manufactured specifically to be toxic to living species and are released deliberately in the environment. In a recently completed U.S. Geological Survey study, the widespread presence of trace amounts of these pesticides was found in surface and groundwaters across the United States.40 Despite analytical techniques with high sensitivity and accuracy such as HPLC, GC, and electrochemical biosensor for trace OP measurement having been applied widely,23-25,40 these technique make evaluating total amount or contamination level of OPs difficult to analyze in the flowing water or seawater during a few hours, days, and weeks. In this cases, biological indicators from living animals, such as oyster and fish skin, currently available for monitoring pesticide exposure could be expected to evaluate the contamination level of OPs in the flowing seawater. However, the OPs that were inhaled by aquatic animals could be metabolized by their tissues, secretions, and shell, which results in the real amount and exposure level of OPs, is not usually obtained. To overcome the above problems, a ferritin reactor has been established to monitor or evaluate the level of OPs in the flowing seawater. A homemade monitoring system consisting of 12 ferritin reactors, two flow meters, and a buoy is established for monitoring the variable level. Based on the above considerations, we indicate that the technology for OPs on a level with a ferritin reactor in the continuously monitored flowing water is better than those of current analytical methods such as HPLC, GC, and electrochemical biosensor including other biological indicators.22-25 With regard to monitoring contamination level in the flowing water or seawater, OPs concentrations, ranging from 100 ppb to 10 ppm, were intentionally designed to study the trapping capacity with a ferritin reactor. This level might reflect actual concentrations of various OPs in surface waters and groundwaters across Xiamen City, China, which mean that this analytical technology can be used to monitor the contamination level of OPs in the flowing seawater in Xiamen. In addition, from this process, our experimental results show that the ferritin reactor still has the

capacity for constantly monitoring OPs levels of about 1000 ppm or more in the flowing water or seawater. In another approach, using a proteomic analysis, we examined different OPs levels in flowing seawater and found them capable of making shark liver express a few proteins as biological indicators for evaluating the contamination level. We indicate that both the ferritin reactor and proteomics are fit to monitor the level of OPs contamination in the flowing seawater for a few days or weeks ceaselessly because similar analytical results and conclusions can be directly obtained. Recently, authors found that there were one or more electrontransfer tunnels (ETT) across the protein shell able to pick up the reduction electron from a platinum electrode for iron reduction in the absence of a chemical redox reagent.31 Similar phenomena of electron transfer between the gold electrode and ferritin have been reported in detail.41,42 Though the molecular structure of the tunnel has hardly been elucidated, we suggest that the ferritin subunit as a switch plays an important role in controlling the rate of electron transfer for iron release because the kinetics of iron release from ferritin are still observed by chemical and electrochemical methods, respectively.17 The pathways of both ETT and the subunit association of ferritin (Figure 9) are fit for explaining the process of iron release and trapping small organic molecules such as OPs. Abbreviations: SZLF, Liver ferritin of Sphyrna Zygaenan; HSF, Horse spleen ferritin; PSF, Pig spleen ferritin; PAGE, Polyacrylamide gel electrophoresis; SA, Sinapic acid; OP, Organphosphorus pesticides; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; MALDI-TOF MS, Matrix-assisted laser desorption/ionization with time- of-flight mass spectrometry; SZLFOP, Trapping various OPs with the SZLF; SZLFP, Trapping inorganic phosphate with the SZLF; SZLFM, Trapping methamidophos with the SZLF; SZLFD, Trapping dichlorovos with the SZLF; TFA, Trifluoroacetic acid; AvBF, Bacterial ferritin of Azotobacter vinelandii; ESI, Electrospray ionization; AAS, Atomic absorbance spectrophotometry; Pt/SZLF, Total numbers of organic and inorganic phosphorus per molecular SZLF; Pi/SZLF, Total numbers of inorganic phosphorus per molecular SZLF; Pop/SZLF, Total numbers of trapping OPs per molecular SZLF; ETT, Electrontransfer tunnel. ACKNOWLEDGMENT This work was funded by grants from the State Natural Science Fund (40276033 and 30470372), by Foundation of Xiamen University (2004xdcx 207) and by the Foundation of Xiamen State Natural Science in China (350 2Z2001262). We thank Drs, Zongwei Cai and Bo Kong for expert technical assistance and discussion. We thank also graduate students Xiaodong Bao and Jinyong Zhu for doing proteomic research for validating the capacity for monitoring levels of OPs contamination with the ferritin reactor in the flowing seawater. Received for review August 20, 2004. Accepted January 6, 2005. AC048753H

(38) Yamashita, I. Thin Solid Films 2001, 393, 12-18. (39) Imai, N.; Umeza, Y.; Arata, Y.; Fujiwara, S. Biochim. Biophys. Acta 1980, 626, 501-506. (40) Mulchandani, A.; Chen, W.; Mulchandani, P.; Wang, J. Biosens. Bioelectron. 2001, 16, 225-230.

(41) Zaoien, D. C.; Johnson, M. A. J. Electroanal. Chem. 2000, 494, 26232645. (42) Masato, T.; Akihiro, O.; Yoshihisa, Y.; Massashi, K. J. Electroanal. Chem. 2004, 556, 323-329.

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