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Screening of Human Serum Proteins for Uranium Binding Claude Vidaud,* Alain Dedieu, Christian Basset, Sophie Plantevin, Isabelle Dany, Olivier Pible, and Eric Que´me´neur CEA/VALRHO-Marcoule, Service de Biochimie Post-ge´ nomique et de Toxicologie Nucle´ aire (DSV-DIEP), Bagnols sur Ce` ze 30207, France Received February 11, 2005
About 20% of uranyl ions in serum are associated with the protein pool. A few of them such as transferrin have been characterized, but most still have to be identified to obtain a better explanation of the biochemical toxicology and kinetics of uranium. We designed an in vitro sensitive procedure involving a combination of bidimensional chromatography with timeresolved fluorescence, coupled with proteomic analysis, to identify uranium-binding proteins in human serum fractions. Ten novel targets were identified and validated using purified proteins and inductively coupled plasma mass spectrometry. Of these, ceruloplasmin, hemopexin, and two complement proteins displayed the capacity to bind uranium with stoichiometry greater than 1 mole of uranium per mole of protein. Not all of these targets are metalloproteins, suggesting that uranyl ions can use a wide variety of binding sites and coordination strategies. These data provide additional insights into a better understanding of uranium chemical toxicity.
Introduction Uranium is a naturally abundant element on Earth and the heaviest nucleus found in the environment. It has been heavily used in many chemical forms in numerous civilian and military industries (1). Both radiological and chemical components may contribute to the global toxicity of uranium (1-4). The average body load is ∼100 µg for an adult. On the basis of adverse effects observed in the kidneys of animals with uptakes of 60 µg uranium per kg per day, the World Health Organization has established a tolerable daily intake of uranium of 0.6 µg/ kg body weight per day (5). Natural uranium is mainly composed of three long-lived isotopes and exhibits a specific activity of 1.24 × 104 Bq/g. The ingestion pathway (food and drinking water) is worth considering, but doses are extremely small, i.e., 0.25 µSv per year as a world average. This is a very small contribution as compared to the total ingestion dose of other natural nuclides. Different target organs have been identified (6-14), but the passage of uranium in the human body is poorly described at the molecular level. About 75% of uranium is cleared within 5 min of injection, and 95% within 5 h (1). Whatever the route of entry into the body, i.e., either ingestion, inhalation, or open wound, a major part of internal soluble uranium reaches the blood as uranyl species, where its concentration is about 10 ng L-1 (15). The speciation of uranium at pH 7.4 is complicated, particularly in biological media since its interactions are the results of several equilibria with small ligands in addition to proteins (1, 16, 17). Only a few proteins have been shown to interact with the uranyl ion (18-23). In an aqueous medium, uranium(VI) hydrolyzes and is mostly found in the form of hexavalent uranyl (UO22+) * To whom correspondence should be addressed. Tel: (33)4-66-7967-62. Fax: (33)4-66-79-19-05. E-mail:
[email protected].
ions. As a hard Lewis metal ion, it mainly reacts with oxygen ligands in the equatorial plane. The coordination number is 5-6 in this plane, displaying a bipyramidal geometry with the two oxygen atoms at the top (24). Uranium has no direct chemical equivalent. Does uranium interfere with essential cation pathways or does it take any specific route in the blood? U has been reported to substitute for Fe(III) in transferrin (18, 20-22), and uranium phosphate deposits are also found in remodeling areas of bone surface, which suggests that uranium might sometimes substitute for calcium (11, 25). Both are hard Lewis metals such as UO22+, but their geometries, charges, and ionic strengths are different. One objective of our work was therefore to determine whether broader screening of uranyl targets in serum proteins would validate this chemical assumption. There are two difficulties in isolating metal ion targets. The first is to have good resolution in a separation process preserving the metal-protein complex during this process, and the second is to use a very sensitive detection system. In vitro profiling by capillary electrophoresis has been reported for uranium (20). Peak broadening was observed in elution profiles, but these studies did not lead to identification of new targets. Nondenaturing onedimensional electrophoresis (1-DE) in combination with different detection systems was also a way of isolating metal-containing proteins (26, 27). However, 1-DE has insufficient resolution to isolate serum proteins, because of their high number and posttranslational modifications. Affinity chromatography might also be a powerful tool for isolating toxic metal-binding proteins (28-31). However, although some commercial products are available, no such specific supports are currently available for uranium, and new solid phases with metal specific interactions are difficult to optimize (32). To cope with the complexity of biological matrices such as human serum, sequential techniques based on multidimensional
10.1021/tx050038v CCC: $30.25 © 2005 American Chemical Society Published on Web 05/04/2005
Serum Targets of Uranium
chromatographic methods with specific detection of trace elements have been described. However, most of them have focused on metal speciation issues or distribution profiling (33-37). Inductively coupled plasma mass spectrometry (ICP-MS)1 is often reported to detect element traces. Time-resolved fluorescence (TRF) is also a sensitive method reported to detect uranium traces with a detection limit (DL) ranging from 10-9 to 10-12 M (20). Here, we report on an in vitro search for human serum targets based on two-dimensional (2D) chromatography and off-line TRF for detecting uranyl complexes. Proteomic analysis of interesting fractions enabled us to identify unexpected protein targets, in which uranyl binding capacities were confirmed by TRF and ICP-MS analysis.
Experimental Procedures Materials. Human serum of healthy volunteer donors was obtained as a pooled sample without any anticoagulant or citrate treatment (PAA Laboratories GmbH, Pashing, Austria). A 2D chromatography was performed to isolate protein populations. The first step was size exclusion chromatography: 4 × 1 mL of human serum was loaded onto a TSK 3000 column (2.6 cm × 30 cm) equilibrated in 50 mM 4-(hydroxyethyl) piperazine-1ethanesulfonic acid (HEPES) and 0.15 M NaCl, pH 7.4 (flow rate, 4 mL/min). All fractions were collected (fraction volume, 4 mL), and similar fractions from four runs were pooled and then submitted to diethylaminoethyl (DEAE) ion exchange chromatography (IEC) (DEAE FF Hitrap 2 × 1 mL, Amersham Biosciences Europe GmbH, Orsay, France) after dialysis in 20 mM HEPES, pH 8.5 (overnight, 4 °C). A linear 0-0.5 M NaCl gradient in the same buffer was used, and each fraction was dialyzed against 50 mM HEPES and 0.15 M NaCl, pH 7.4. Proteins were obtained from different suppliers: ceruloplasmin, IgG, haptoglobin, R-1-antitrypsin, transthyretin, retinol binding protein, apolipoprotein A1, apo, and holo-transferrins were purchased from Sigma; hemopexin was from BioMac GmbH (Leipzig, Germany); complements C3 and C4 were from BiosPacific; and R-1-acid glycoprotein was from Fluka. Their purities and identities were checked by 2-DE and matrixassisted laser/desorption ionization time-of-flight (MALDI-TOF) analyses. All were more than 95% pure. For ceruloplasmin, OD610/OD280 was 0.055. Tropomyosin was from porcine origin (Sigma), and it displays 60% of homology with the human form. All protein samples were dialyzed against 0.05 M HEPES and 0.15 M NaCl, pH 7.4, prior to use. Uranyl Acetate Solutions. Uranium acetate stock solutions (0.1 M) were obtained by dissolving uranium acetate in pure water. The pH of the solution was within the range of 4-4.5. The 5 mM uranyl solution was obtained just before use by dilutions in 10 mM sodium acetate. Modified Version of the Kryptor Automatic Analyzer. The Kryptor instrument (Cezanne/Brahms, Nıˆmes, France) is an automatic analyzer used in the field of immunology. Briefly, its main specificity is the use of homogeneous phase conditions using energy transfer between antibodies donor and acceptor labels. The original instrument uses a 20 Hz nitrogen laser (337 nm) as the excitation beam; the collection channel involves a beam splitter and two collection channels with 665 and 620 nm band-pass filters. Two photomultipliers are used in photon counting mode, with reading windows for counts summation adapted to the optimal analyte measurement conditions. 1 Abbreviations: TRF, time-resolved fluorescence; IEC, ion exchange chromatography; ICP-MS, inductively coupled plasma mass spectrometry; CPS, counts per second; AU, absorbance units; 2-DE, twodimensional gel electrophoresis; HEPES, 4-(hydroxyethyl) piperazine1-ethanesulfonic acid; CHAPS, 3-[(3-chloroamidopropyl)dimethylammonio]-1-propanesulfonate; DEAE, diethylaminoethyl; MALDI-TOF, matrixassisted laser desorption/ionization time-of-flight; ACN-TFA, acetonitrile-trifluoroacetic acid.
Chem. Res. Toxicol., Vol. 18, No. 6, 2005 947 The Kryptor instrument adaptation to uranyl detection was mostly limited to its collection channels, since the nitrogen excitation laser at 337 nm is suitable for uranyl excitation. The beam splitter was removed since only one channel is used, and the corresponding 665 nm band-pass filter was replaced with an Omega Optical 500 ( 25 nm band-pass filter. The photon counting photomultiplier tube was replaced with an Hamamatsu R3896, which had better yield in the uranyl emission range. Optimal individual laser flash counts were found reading window settings of 30 µs gate delay and 970 µs gate length. The total counts per second (CPS) was the summation of a 1 s reading including 20 flashes (20 Hz laser). Protein-Metal Binding Assay. Aliquots of 450 µL of either chromatographic fractions or purified protein solutions were dialyzed in HEPES-NaCl, pH 7.4, buffer and supplemented with 4.5 µL of 0.1 M sodium carbonate and an excess of uranyl ions (30 µL of the 5 mM solution). The molar ratios ranged from 10 to 60 for purified proteins, according to their molecular mass. After overnight incubation at room temperature and under continuous stirring, the unbound uranyl ions and other salts were removed by injection of 200 µL of the solution onto a G25 (10 × 30) column (Amersham Biosciences Europe GmbH) equilibrated in 20 mM HEPES, pH 7.0 (flow rate, 1 mL/min). The proteins were monitored at 280 nm absorbance and collected for further analyses. A rapid sodium carbonate gradient up to 200 mM was applied to elute uranyl ions and to clean the column. Samples were serially diluted (1/10, 1/100, and 1/1000) and put on the U-version of Kryptor board to determine their uranium content by TRF. Further dilutions were automatically carried out by the analyzer (14 µL of sample in 150 µL of 5% phosphoric acid) to generate uranyl dihydrogenophosphate species, which have well-defined and reproducible emission spectra. A 10-7 M uranyl acetate reference and a HEPES blank were placed in each run. Water and HEPES blanks were also placed between two series of measurements to avoid crosscontaminations. The reproducibility was 10% within the same run and 20% after different processes, including metal contact, desalting, and counting. Two-Dimensional Gel Electrophoresis (2-DE). Serum samples were incubated for 1 h in 7 M urea, 4% (w/v) 3-[(3-chloroamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.4% Triton × 100, 2 M thiourea, 1% (w/v) DTT, and 1% IPG buffer and then centrifuged for 30 min at 16000g. First dimension IEFs were conducted using precast 18 cm IPG strips, pH 3-10 (Amersham Biosciences Europe GmbH), and a load of 0.2-0.4 mg. In the gel sample, rehydratations were performed at 50 V for 10 h. Focusing was carried out by steps up to 60000 V h in a Bio-Rad protean IEF Cell. For the second dimension, strips were loaded on a 12% (w/v) acrylamide gel (Bio-Rad Protean 2XI Cell) and run at 25 V constant for 1 h and 12.5 W/gel. Gels were fixed overnight and stained in a Coomassie Brilliant Blue G250 (Bio-Rad) staining solution for 5 days. The gels were digitized at 300 dpi using a UMAX scanner (Amersham Biosciences Europe GmbH) and finally analyzed with the Melanie 4.0 software (GeneBio). In Gel Digestion. Blue Coomassie-stained protein spots of interest were then manually excised from 2D gels and processed through the Montage In-Gel DigestZP 96 Kit (Millipore) according to the manufacturer conditions. Mass Spectrometry Analysis. MALDI-TOF spectra were obtained using a Biflex IV instrument (Bruker Daltonics, Bremen, Germany). A saturated solution of R-cyano-4-hydrocinnamic acid prepared in 0.1% acetonitrile-trifluoroacetic acid (ACN-TFA) (1:1) was diluted 1/4 in the same solvent and used as a matrix. A 0.5 µL amount of the peptide sample and 0.5 µL of the matrix were spotted and air-dried on the target plate. Spectra were acquired over the 600-3500 m/z range according to the manufacturer’s instructions.
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Figure 1. Variation of fluorescence signal (in CPS) vs uranyl concentration in standards. The results were recorded on a modified Kryptor automatic analyzer. The nitrogen laser excitation was at 337 nm; emission, 500 ( 25 nm; 30 µs gate delay; and 970 µs gate length. Blanks were 20 mM HEPES, pH 7.0, and triplicates of each uranium acetate dilution were analyzed. The data presented here are mean values of triplicates ( SD values ( 1. The three proteins tested (retinol binding protein, transthyretin, and tropomyosin) did not bind any uranyl ions. b TRF measurement, mean value ((20%) of two different runs.
can lead to partial saturation of weaker binding sites (19, 22). These species probably compete with nonspecific interactions of proteins. Metal stripping in the subsequent desalting step was strong enough to eliminate weak binding: Despite an excess of uranyl ions during contact, desalting column efficiency could discriminate between two classes of different proteins, according to their ICP-MS results. TRF and ICP measurements proved essential to validate the potential targets after proteomic analyses. A good correlation between TRF and ICP-MS measurements was obtained in all cases (Table 1), strengthening the initial choice of the modified Kryptor automatic
analyzer for rapid off-line analysis of chromatographic fractions. Unfortunately, at the moment, some potential targets have not yet been investigated due to their unavailability as purified proteins. Large scale purification from human serum or production as recombinant proteins would be necessary to confirm that they are genuine binders. As a general rule, there was no correlation between uranium-binding capability of these proteins and their molecular mass, pI, or relative abundance in serum. For example, transthyretin, which is a negatively charged transport protein, did not show any binding; hemopexin and R-1-antitrypsin have similar molecular masses (46
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and 50 kDa), but their reactivities with uranium are very different. Under our experimental conditions, three potential targets did not show any binding of UO22+ and 12 proteins remained associated with uranium. A first group of proteins displayed sufficient affinity for uranium to pass the desalting step but with results below transferrin as a reference protein. The heterogeneity of human serum proteins and stability in used buffers could explain the uranyl/protein mean ratios below 1. HSA and IgG have already been suspected of binding uranyl species, and their abundance in serum could also explain modifications in capillary electrophoresis profiles (20). However, they displayed a low uranium load after desalting with only 0.6 and 0.5 mol of uranium per mol of protein. IgG recovery in the protein peak elution was low (45%), and partial retention of protein by interactions with G25 could not be excluded. Some authors have reported spectroscopic or chromatographic data supporting molecular interactions of transition metals and lanthanides with IgG (40, 41). HSA is the major Zn2+ transport protein in the blood and is known to bind many metals nonspecifically (42). Haptoglobin displayed 0.65 mol of uranium per mol of protein but had a tendency to precipitate in our binding assay. Studies of different stoichiometries and/or other chemical forms of uranium would be necessary. Few metal-protein interactions have been reported for R-1-acid glycoprotein (43) and apolipoproteins (44), and to our knowledge, there is no description for metal binding to R-1-antitrypsin. Strikingly, holotransferrin did not behave as a “negative” protein as expected. A final molar ratio of 0.3 uranium per mol of protein could not be explained by a few percent of residual free iron sites. These results need further investigations. A second family is composed of apotransferrin, ceruloplasmin, complement proteins C3 and C4, and hemopexin, which displayed stoichiometries greater than one uranyl ion/mol of protein. Apotransferrin is known to bind numerous metals including uranyl ions. Binding constants (log K ) 16) have been reported, but the UO22+-protein complex seems much weaker than other tetravalent actinide complexes (21). The metal content of less than two has also been reported (22, 45). Here, we found a final molar ratio below 2, which could be explained by a weak binding to one site allowing partial metal elimination after desalting. Metal ion effects have been reported on ceruloplasmin oxidase activity (46). Here, the stoichiometry was two uranyls per ceruloplasmin, as demonstrated by both TRF and ICP-MS experiments. Complements C3 and C4 were each able to bind two and four uranium ions, respectively, even after desalting. As for IgG, they both showed a low recovery in protein peak elution and partial elution at higher retention times. Various divalent ions have been tested as activators of C3b and C4b generations (47, 48). An unexpectedly high level of association was obtained with hemopexin. In our experiments, eight uranyl ions remained bound to the protein after the desalting step. A strong interaction with Zn2+ has been described to purify the protein in single step affinity chromatography (49), and recently, binding to various divalent cations has been reported (50). This remarkably high level of metal content for a 50 kDa protein could be explained, for example, by water, carbonate, or a donor atom-linked multimetallic complex building up in a lowered dielectric
Vidaud et al.
constant area, enhancing electrostatic interaction. The same could apply to C3 and C4 even though their high molecular masses could potentially correspond to a higher number of binding areas. It is noteworthy that the highest fluorescence levels were observed in fractions H/P2 and H/P3, which were the richest in hemopexin content. Our results clearly demonstrate the in vitro uraniumbinding capability of an unexpected number of serum proteins. Our aim was to identify new targets of uranium to determine whether specific pathways are involved in its distribution after toxic exposure. Some of them are related to iron metabolism, but others are neither metalloproteins nor related to any metal homeostasis. As compared with the very little data concerning uranium targets, these new identifications open new fields of investigation. Because these targets have very different concentrations in serum and considering the complexity of uranium speciation in this medium, the kinetic distribution of uranium under physiological concentrations must be evaluated.
Acknowledgment. We gratefully acknowledge F. Hely-Joly for skillful assistance in proteomic analyses and P. Correze (Service d’Hygie`ne Industrielle, IRSN Pierrelatte) for ICP-MS measurements.
References (1) Taylor, D. M., and Taylor, S. K. (1997) Environmental uranium and human health. Rev. Environ. Health 12, 147-157. (2) ATDSR (1999) Agency for Toxic Substances and Disease Registry. Toxicological profile for uranium. (3) Miller, A. C., Stewart, M., Brooks, K., Shi, L., and Page, N. (2002) Depleted uranium-catalyzed oxidative DNA damage: Absence of significant alpha particle decay. J. Inorg. Biochem. 91, 246-252. (4) Yazzie, M., Gamble, S. L., Civitello, E. R., and Stearns, D. M. (2003) Uranyl acetate causes DNA single strand breaks in vitro in the presence of ascorbate (vitamin C). Chem. Res. Toxicol. 16, 524-530. (5) WHO (1998) World Health Organization: Guidelines for Drinking Water Quality, addendum to Vol. 2, p 283, WHO, Geneva, Switzerland. (6) Wrenn, M. E., Bertelli, P. W., Durbin, N. P., Lipsztein, J. L., and Eckerman, K. F. (1994) A comprehensive metabolic model for uranium metabolism and dosimetry based on human and animal data. Radiat. Prot. Dosim. 53, 255-258. (7) Leggett, R. W. (1989) The behavior and chemical toxicity of U in the kidney: A reassessment. Health Phys. 57, 365-383. (8) McDonald-Taylor, C. K., Singh, A., and Gilman, A. (1997) Uranyl nitrate-induced proximal tubule alterations in rabbits: A quantitative analysis. Toxicol Pathol. 25, 381-389. (9) Zamora, M. L., Tracy, B. L., Zielinski, J. M., Meyerhof, D. P., and Moss, M. A. (1998) Chronic ingestion of uranium in drinking water: A study of kidney bioeffects in humans. Toxicol. Sci. 43, 68-77. (10) Arfsten, D. P., Still, K. R., and Ritchie, G. D. (2001) A review of the effects of uranium and depleted uranium exposure on reproduction and fetal development. Toxicol. Ind. Health 17, 180191. (11) Leggett, R. W., and Pellmar, T. C. (2003) The biokinetics of uranium migrating from embedded DU fragments. J. Environ. Radioact. 64, 205-225. (12) Lemercier, V., Millot, X., Ansoborlo, E., Menetrier, F., FluryHerard, A., Rousselle, C., and Scherrmann, J. M. (2003) Study of uranium transfer across the blood-brain barrier. Radiat. Prot. Dosim. 105, 243-245. (13) Kurttio, P., Auvinen, A., Salonen, L., Saha, H., Pekkanen, J., Makelainen, I., Vaisanen, S. B., Penttila, I. M., and Komulainen, H. (2002) Renal effects of uranium in drinking water. Environ. Health Perspect. 110, 337-342. (14) Taulan, M., Paquet, F., Maubert, C., Delissen, O., Demaille, J., and Romey, M. C. (2004) Renal toxicogenomic response to chronic uranyl nitrate insult in mice. Environ. Health Perspect. 112, 1628-1635.
Serum Targets of Uranium (15) Dang, H. S., and Pullat, V. R. (1993) Normal concentration and excretion ratio of uranium in serum of normal individuals in India. Health Phys. 65, 303-315. (16) Carriere, M., Avoscan, L., Collins, R., Carrot, F., Khodja, H., Ansoborlo, E., and Gouget, B. (2004) Influence of uranium speciation on normal rat kidney (NRK-52E) proximal cell cytotoxicity. Chem. Res. Toxicol. 17, 446-452. (17) Sutton, M., and Burastero, S. R. (2004) Uranium(VI) solubility and speciation in simulated elemental human biological fluids. Chem. Res. Toxicol. 17, 1468-1480. (18) Taylor, D. (1993) Transferrin complexes with nonphysiological and toxic metals. Perspect. Bioinorg. Chem. 2, 139-159. (19) Taylor, D. M. (1998) The bioinorganic chemistry of actinides in blood. J. Alloys Compd. 271, 6-10. (20) Scapolan, E. A., Moulin, C., and Madic, C. (1998) Investigations by time-resolved laser-induced fluorescence and capillary electrophoresis of the uranyl-phosphate species: Application to blood serum. J. Alloys Compd. 271, 106-111. (21) Sun H. L. H., and Sadler, P. J. (1999) Transferrin as a metal ion mediator. Chem. Rev. 99, 2817-2842. (22) Harris, W. R. (1998) Binding and transport of nonferrous metals by serum transferrin. Struct. Bonding 92, 121-162. (23) Hainfeld, J. F. (1992) Uranium-loaded apoferritin with antibodies attached: Molecular design for uranium neutron-capture therapy. Proc. Natl. Acad. Sci. U.S.A. 89, 11064-11068. (24) Gorden, A. E., Xu, J., Raymond, K. N., and Durbin, P. (2003) Rational design of sequestering agents for plutonium and other actinides. Chem. Rev. 103, 4207-4282. (25) Kurttio, P., Komulainen, H., Leino, A., Salonen, L., Auvinen, A., and Saha, H. (2005) Bone as a possible target of chemical toxicity of natural uranium in drinking water. Environ. Health Perspect. 113, 68-72. (26) Binet, M. R., Ma, R., McLeod, C. W., and Poole, R. K. (2003) Detection and characterization of zinc- and cadmium-binding proteins in Escherichia coli by gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry. Anal. Biochem. 318, 30-38. (27) Petrak, J. V., and Vyoral, D. (2001) Detection of iron-containing proteins contributing to the cellular labile iron pool by a native electrophoresis metal blotting technique. J. Inorg. Biochem. 86, 669-675. (28) Koyama, H., Omura, K., Ejima, A., Kasanuma, Y., Watanabe, C., and Satoh, H. (1999) Separation of selenium-containing proteins in human and mouse plasma using tandem high-performance liquid chromatography columns coupled with inductively coupled plasma-mass spectrometry. Anal. Biochem. 267, 84-91. (29) Sidenius, U., Farver, O., Jons, O., and Gammelgaard, B. (1999) Comparison of different transition metal ions for immobilized metal affinity chromatography of selenoprotein P from human plasma. J. Chromatogr., B: Biomed. Sci. Appl. 735, 85-91. (30) She, Y. M., Narindrasorasak, S., Yang, S., Spitale, N., Roberts, E. A., and Sarkar, B. (2003) Identification of metal-binding proteins in human hepatoma lines by immobilized metal affinity chromatography and mass spectrometry. Mol. Cell Proteomics 2, 1306-1318. (31) Smith, S. D., She, Y. M., Roberts, E. A., and Sarkar, B. (2004) Using immobilized metal affinity chromatography, two-dimensional electrophoresis and mass spectrometry to identify hepatocellular proteins with copper-binding ability. J. Proteome Res. 3, 834-840. (32) Zachariou, M., and Hearn, M. T. (2000) Adsorption and selectivity characteristics of several human serum proteins with immobilised hard Lewis metal ion-chelate adsorbents. J. Chromatogr. A 890, 95-116. (33) de la Calle Guntinas, M. B., Bordin, G., and Rodriguez, A. R. (2002) Identification, characterization and determination of metalbinding proteins by liquid chromatography. A review. Anal. Bioanal. Chem. 374, 369-378. (34) Pomazal, K., Prohaska, C., and Steffan, I. (2002) Hydrophobic interaction chromatographic separation of proteins in human
Chem. Res. Toxicol., Vol. 18, No. 6, 2005 953
(35)
(36)
(37)
(38)
(39)
(40)
(41)
(42)
(43)
(44)
(45)
(46)
(47)
(48)
(49)
(50)
blood fractions hyphenated to atomic spectrometry as detector of essential elements. J. Chromatogr. A 960, 143-150. Szpunar, J., and Lobinski, R. (2002) Multidimensional approaches in biochemical speciation analysis. Anal. Bioanal. Chem. 373, 404-411. Ferrarello, C. N., Fernandez de la Campa, M. R., and Sanz-Medel, A. (2002) Multielement trace-element speciation in metal-biomolecules by chromatography coupled with ICP-MS. Anal. Bioanal. Chem. 373, 412-421. De Cremer, K., Van Hulle, M., Chery, C., Cornelis, R., Strijckmans, K., Dams, R., Lameire, N., and Vanholder, R. (2002) Fractionation of vanadium complexes in serum, packed cells and tissues of Wistar rats by means of gel filtration and anionexchange chromatography. J. Biol. Inorg. Chem. 7, 884-890. Scapolan, S. E. A., Moulin, C., and Madic, C. (1998) Uranium(VI)-transferrin system studied by time-resolved laser induced fluorescence. Radiat. Prot. Dosim. 79, 505-508. Pieper, R., Gatlin, C. L., Makusky, A. J., Russo, P. S., Schatz, C. R., Miller, S. S., Su, Q., McGrath, A. M., Estock, M. A., Parmar, P. P., Zhao, M., Huang, S. T., Zhou, J., Wang, F., Esquer-Blasco, R., Anderson, N. L., Taylor, J., and Steiner, S. (2003) The human serum proteome: Display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins. Proteomics 3, 13451364. Fitzgerald, J. J. (1980) Preliminary ESR investigations of metalimmunoglobulin G interactions. Proc. S. D. Acad. Sci. 59, 242257. Ozkara, S., Yavuz, H., and Denizly, A. (2003) Purification of immunoglobulin G from human plasma by metal-chelate affinity chromatography. J. Appl. Polym. Sci. 89, 1567-1572. Stewart, A. J., Blindauer, C. A., Berezenko, S., Sleep, D., and Sadler, P. J. (2003) Interdomain zinc site on human albumin. Proc. Natl. Acad. Sci. U.S.A. 100, 3701-3706. Herve, F., Millot, M. C., Eap, C. B., Duche, J. C., and Tillement, J. P. (1996) Two-step chromatographic purification of human plasma alpha(1)-acid glycoprotein: its application to the purification of rare phenotype samples of the protein and their study by chromatography on immobilized metal chelate affinity adsorbent. J. Chromatogr., B: Biomed. Sci. Appl. 678, 1-14. Hill, B. C., Becker, L., Anand, V., Kusmierczyk, A., Marcovina, S. M., Rahman, M. N., Gabel, B. R., Jia, Z., Boffa, M. B., and Koschinsky, M. L. (2003) A role for apolipoprotein(a) in protection of the low-density lipoprotein component of lipoprotein(a) from copper-mediated oxidation. Arch. Biochem. Biophys. 412, 186195. Congiu-Castellano, A., Boffi, F., Della Longa, S., Giovannelli, A., Girasole, M., Natali, F., Pompa, M., Soldatov, A., and Bianconi, A. (1997) Aluminum site structure in serum transferrin and lactoferrin revealed by synchrotron radiation X-ray spectroscopy. Biometals 10, 363-367. Younis, M., and Birch, N. (1985) The effects of metal ions on the oxidase activity of caeruloplasmin. Appl. Sci. Polytech. Wolverhampton 13, 768. Acevedo, F., and Vesterberg, O. (2003) Nickel and cobalt activate complement factor C3 faster than magnesium. Toxicology 185, 9-16. Blom, A. M., Kask, L., Ramesh, B., and Hillarp, A. (2003) Effects of zinc on factor I cofactor activity of C4b-binding protein and factor H. Arch. Biochem. Biophys. 418, 108-118. Andersson, L. (1984) Fractionation of human serum proteins by immobilized metal affinity chromatography. J. Chromatogr. 315, 167-174. Mauk, M. R., Rosell, F. I., Lelj-Garolla, B., Moore, G. R., and Mauk, A. G. (2005) Metal ion binding to human hemopexin. Biochemistry 44, 1864-1871.
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