Surface Plasmon Resonance for Rapid Screening of Uranyl Affine

Anal. Chem. 2010, 82, 9797–9802

Surface Plasmon Resonance for Rapid Screening of Uranyl Affine Proteins Olivier Averseng,† Agne`s Hage`ge,†,‡ Fre´de´ric Taran,§ and Claude Vidaud*,† Service de Biochimie et de Toxicologie Nucle´aire/LEPC, DSV/iBEB, CEA Marcoule, BP 17 171, F-30207 Bagnols sur Ce`ze, France, CNRS-UMR 6191, DSV/iBEB, CEA Cadarache, 13108 Saint Paul-les-Durance Cedex, France, and Service de Chimie Bioinorganique et de Marquage, DSV/iBiTecS, CEA Saclay, 91191 Gif sur Yvette, France A sensitive immunoassay based on SPR analysis was developed to measure uranyl cation (UO22+) affinity for any protein in a free state under physiological conditions. The technique involves immobilization of a specific monoclonal antibody (mAb) raised against UO22+ and 1,10-phenanthroline-2,9-dicarboxylic acid (DCP) used as a probe of UO22+ captured by the mAb. Calibration curves were established for accurate determination of UO22+ concentrations with a detection limit of 7 nM. The remaining free UO22+ could be accurately quantified from the different protein-metal equilibrium and a dose-response curve established for KD determination. This generic method was applied not only to proteins such as transferrin and albumin but also to small phosphonated ligands. Its robustness allows the fast UO22+ KD determination of any kind of macromolecules and small ligands using very few amount of compounds, thus opening new prospects in the field of uranium toxicity. Understanding the mechanisms by which a toxic metal exerts its deleterious effects relies on systematic approaches leading to a description of its distribution, localization, and fate in biological systems. The knowledge of how a metal, either exogenous or physiological, is sensed, transported through different biological compartments toward target organs and finally stored, requires the identification of the proteins and other potential ligands interacting with it. Such studies contribute to informing the “metallome”, defined previously as the “entirety of metal and metalloid species present in a cellular compartment, cell or organism”.1,2 Uranium is widespread in the environment, resulting from both natural occurrence and nuclear applications. Uranyl (UO22+) is the predominant form of uranium in aqueous media such as biological ones. It is now recognized that its chemical toxicity is greater than its radiotoxicity. Chemical thermodynamic speciation has been used to model uranium chemistry * To whom correspondence should be addressed: Phone: +33.466.796.762. Fax: +33 0.466.791.905. E-mail: [email protected]. † Service de Biochimie et de Toxicologie Nucle´aire/LEPC. ‡ CNRS-UMR 6191. § Service de Chimie Bioinorganique et de Marquage. (1) Mounicou, S.; Szpunar, J.; Lobinski, R. Chem. Soc. Rev. 2009, 38, 1119– 1138. (2) Thiele, D. J.; Gitlin, J. D. Nat. Chem. Biol. 2008, 4, 145–147. 10.1021/ac102578y  2010 American Chemical Society Published on Web 11/11/2010

in simulated human biological fluids,3 thus helping to identify the main bioavailable uranyl species. However, the distribution of uranyl in serum or cell extract proteins is not described, despite the identification of some potential targets.4-6 Indeed, metal distribution relies not only on identification of the proteins that can either bind or transport a metal, but also on accurate determination of their metal binding constants. Despite many research efforts, generic and fast screening techniques allowing the determination of metal affinities under physiological conditions and requiring only very small quantities of proteins remains a challenge. Classical biophysical techniques dedicated to proteinmetal interactions, such as spectroscopic and calorimetric methods, require significant protein quantities and time-consuming procedures not well adapted to systematic studies.7-9 Recently, electrospray ionization mass spectrometry has been used to quantify protein-metal affinities, but was limited to recombinant proteins of low molecular weight.10,11 Surface Plasmon Resonance (SPR) is an efficient and sensitive optical tool for real time biomolecular interaction analysis between immobilized proteins and soluble ligands in minute amounts, with increased applications for low molecular weight entities.12,13 This technique is therefore suitable for sensing metal interaction with various ligands but requires an immobilization step. Direct protein immobilization needs specific optimization, increases its consumption, and may modify its affinity. To approach physiological conditions, proteins must be dispersed in a metallic solution, and once at equilibrium, the remaining free metal must be measured without equilibrium modification. Although conventional SPR provides good detection (3) Sutton, M.; Burastero, S. R. Chem. Res. Toxicol. 2004, 17, 1468–1480. (4) Vidaud, C.; Dedieu, A.; Basset, C.; Plantevin, S.; Dany, I.; Pible, O.; Quemeneur, E. Chem. Res. Toxicol. 2005, 18, 946–953. (5) Vidaud, C.; Gourion-Arsiquaud, S.; Rollin-Genetet, F.; Torne-Celer, C.; Plantevin, S.; Pible, O.; Berthomieu, C.; Quemeneur, E. Biochemistry 2007, 46, 2215–2226. (6) Basset, C.; Dedieu, A.; Guerin, P.; Quemeneur, E.; Meyer, D.; Vidaud, C. J. Chromatogr., A 2008, 1185, 233–240. (7) Myszka, D. G.; Jonsen, M. D.; Graves, B. J. Anal. Biochem. 1998, 265, 326–330. (8) Bornhop, D. J.; Latham, J. C.; Kussrow, A.; Markov, D. A.; Jones, R. D.; Sorensen, H. S. Science 2007, 317, 1732–1736. (9) Myszka, D. Abstr. Pap. Am. Chem. Soc. 2004, 227, U108–U108. (10) Deng, L.; Sun, N.; Kitova, E. N.; Klassen, J. S. Anal. Chem. 2010, 82, 2170– 2174. (11) Sun, J.; Kitova, E. N.; Sun, N.; Klassen, J. S. Anal. Chem. 2007, 79, 8301– 8311. (12) Homola, J. Chem Rev 2008, 108, 462–493. (13) Amarie, D.; Alileche, A.; Dragnea, B.; Glazier, J. A. Anal. Chem. 2010, 82, 343–352.

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Scheme 1. Structure and Binding Constants of Bisphosphonate Ligands

limits for small organic molecules,14 the very small refractive index (RI) variations induced by highly diluted metallic solutions hinders current application. Some examples describe metal detection, but usually this is indirectly demonstrated15,16 and needs specific sensor developments16-21 or major system modification.20,22,23 Since anti-metallic-complex antibodies proved their value in the sensitive and specific detection of very small amounts of metals in various environmental samples,24-27 our goal was to develop a unique and universal system for sensing free uranyl using an immobilized monoclonal antibody (mAb). This paper describes a fast, sensitive, and cost-effective immunoassay based on SPR analysis to measure apparent affinity for UO22+ for any free protein in physiological solutions, by using a mAb directed against 1,10-phenanthroline-2,9-dicarboxylate-uranyl (DCPUO22+) previously obtained in our laboratory.28 MATERIALS AND METHODS Materials. All aqueous solutions were prepared with pure water (18.2 MΩ.cm resistivity; Milli-Q station, Millipore). All the chemicals and proteins were purchased from SIGMA-Aldrich except 1,10-Phenanthroline-2,9-dicarboxylic acid (DCP, MW: (14) Yuan, J.; Oliver, R.; Aguilar, M. I.; Wu, Y. Anal. Chem. 2008, 80, 8329– 8333. (15) Christopeit, T.; Gossas, T.; Danielson, U. H. Anal. Biochem. 2009, 391, 39–44. (16) Wang, J.; Zhou, H. S. Anal. Chem. 2008, 80, 7174–7178. (17) Wu, C. M.; Lin, L. Y. Biosens Bioelectron 2004, 20, 864–871. (18) Wu, C. M.; Lin, L. Y. Sens. Actuators, B 2005, 110, 231–238. (19) Xin, Y.; Gao, Y.; Guo, J.; Chen, Q.; Xiang, J.; Zhou, F. Biosens. Bioelectron. 2008, 24, 369–375. (20) Forzani, E. S.; Zhang, H.; Chen, W.; Tao, N. Environ. Sci. Technol. 2005, 39, 1257–1262. (21) Chen, H.; Lee, Y.; Oh, M. C.; Lee, J.; Ryu, S. C.; Hwang, Y. H.; Koh, K. Sens. Actuators, B 2008, 134, 419–422. (22) Wang, S.; Forzani, E. S.; Tao, N. Anal. Chem. 2007, 79, 4427–4432. (23) Zhang, Y.; Xu, M.; Wang, Y.; Toledo, F.; Zhou, F. Sens. Actuators, B 2007, 123, 784–792. (24) Blake, R. C.; Pavlov, A. R.; Khosraviani, M.; Ensley, H. E.; Kiefer, G. E.; Yu, H.; Li, X.; Blake, D. A. Bioconjugate Chem. 2004, 15, 1125–1136. (25) Sasaki, K.; Oguma, S.; Namiki, Y.; Ohmura, N. Anal. Chem. 2009, 81, 4005– 4009. (26) Reardan, D. T.; Meares, C. F.; Goodwin, D. A.; McTigue, M.; David, G. S.; Stone, M. R.; Leung, J. P.; Bartholomew, R. M.; Frincke, J. M. Nature 1985, 316, 265–268. (27) Blake, D. A.; Jones, R. M.; Blake, R. C.; Pavlov, A. R.; Darwish, I. A.; Yu, H. Biosens. Bioelectron. 2001, 16, 799–809. (28) Reisser-Rubrecht, L.; Torne-Celer, C.; Renier, W.; Averseng, O.; Plantevin, S.; Quemeneur, E.; Bellanger, L.; Vidaud, C. Chem. Res. Toxicol. 2008, 21, 349–357.

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278.30, CAS No.: 57709-61-2, Alfa Aesar, Woodhill,MA ref: A18548). The solutions of natural uranyl were from CEA and handled according to the safety rules of the laboratory. DCP solutions (100 µM) were prepared by dilution in pure water (Milli-Q station, Millipore), and stored frozen at -80 °C under aliquots. A 100 mM uranyl acetate solution was obtained by diluting uranyl acetate in pure water (1:100). For biological relevance the metal solutions were prepared in a saline buffer at pH 7.4. Handling UO22+ in such buffers is problematic because the ion, sensitive to hydrolysis, can form colloids and tends to adsorb onto surfaces.29-31 UO22+ solution stability and regeneration steps are therefore crucial for reliable measurements. It is essential to work within a narrow concentration range with a chelating agent to limit both hydrolysis, and speciation evolution. Even in the case of acute intoxication UO22+ions are at significantly lower concentration than carbonate ions in serum.3 A 1000:1 carbonate/UO22+ ratio was thus used to prevent metal hydrolysis. JChess modeling confirmed (UO2)2CO3(OH)3 as the major species (Supporting Information (SI)). A 100 µM uranyl solution was prepared in 100 mM NaHCO3/ Na2CO3, pH 9.5 buffer to constitute the stock solution (storage: one month at 4 °C). Working solutions were prepared daily by diluting stock solution in 50 mM TRIS, 150 mM NaCl pH 7.4 buffer. The final pH values were recorded at 7.4. The uranyl external standard (PlasmaCAL, 1000 µg/mL) was obtained from SCP Science, Baie d’Urfe´, Canada. DCP-UO22+ solutions were prepared according to Blake et al.24 and assumed to form 1:1 complexes. The protein dilution buffer and the running buffer for Biacore experiments were 50 mM TRIS, 150 mM NaCl pH 7.4. Bisphosphonate ligands 1-4 (Scheme 1) were synthesized according to the procedure previously described.32 Proteins were dialyzed in 50 mM TRIS, 150 mM NaCl pH 7.4 prior to use, and their concentration controlled by their molar absorption at 280 nm. Monoclonal antibodies (U08S) were obtained from mouse (29) Baes, C. F.; Mesmer, R. E. The Hydrolysis of Cations; John Wiley and Sons, Inc.: New York, 1976, pp 174-182. (30) Szabo, Z.; Toraishi, T.; Vallet, V.; Grenthe, I. Coord. Chem. Rev. 2006, 250, 784–815. (31) Choppin, G. R. J. Radioanal. Nucl. Chem. 2007, 273, 695–703. (32) Sawicki, M.; Lecercle, D.; Grillon, G.; Le Gall, B.; Serandour, A. L.; Poncy, J. L.; Bailly, T.; Burgada, R.; Lecouvey, M.; Challeix, V.; Leydier, A.; PelletRostaing, S.; Ansoborlo, E.; Taran, F. Eur. J. Med. Chem. 2008.

immunization with BSA-DCP-UO22+ conjugates, and selected via a counter selection step using casein-DCP-UO22+ conjugates as described elsewhere.28 The control mAb was from the laboratory. F(ab) fragments were obtained by papain digestion according to laboratory protocols. Surface Plasmon Resonance. A T100 biosensor system (Biacore, GE Healthcare Biosciences) for Surface Plasmon Resonance (SPR) experiments was used to quantify the affinity of UO8S and its F(ab) fragment for UO22+, DCP and DCP-UO22+. The standard EDC (N-[3-dimethyl-aminopropyl]-N′-ethylcarbodiimide)/NHS (N-hydroxysuccinimide) and carbohydrate coupling chemistry (provided by Biacore, GE Healthcare Biosciences) was followed by ethanolamine injection to block any remaining free ester groups. It was performed on both channels of a CM5 biosensor chip. The sample channel was prepared by covalent coupling of the mAbs or their F(ab) fragments to a density of 1700-4000 RU. The same chemistry was applied to the reference channel. The running buffer was 50 mM TRIS, 150 mM NaCl, pH 7.4. The different cycles were processed in duplicates at 30 µL.min-1 working flow rate and by injection of the reactants into both channels. To establish calibration curves, uranyl solutions were used as samples; to establish dose-response curves, series of protein or ligand dilutions were incubated with a uranyl solution at a fixed concentration. In any case, all these samples were injected for 100 s and the DCP solutions for 60 or 200 s according to experiments. The kinetic experiments included 100 s of dissociation phase. Regeneration steps included pulses of 15 µL of 50 mM H3PO4, 0.1 M Na2HPO4, 50 mM NaOH, 1 M NaCl before any new sample analysis. ICP/MS. Uranyl concentrations in the calibration standards were controlled by ICP-MS (Agilent Technologies, 7700 ICP-MS) 24 h after the beginning of the run cycle. After appropriate dilutions with 1% ultrapure nitric acid, the samples were injected via a peristaltic pump at a flow rate of 100 µL · min-1. Samples were nebulized by a microconcentric nebulizer (Micromist). A 7700 ICP-MS (Agilent) was used as elemental detector. Uranium was detected by selecting the most abundant isotope, that is, 238U. Data Evaluation. All the experiments were carried out in duplicate and Biaevaluation 4.1 software (GE Healthcare Biosciences) was used to evaluate the sensorgrams and result analysis. The duplicates were neither consecutive, nor injected in an increasing range of concentrations. This was deliberately chosen to verify the stability of the solutions and the signal levels after a lag time. Signals from the reference surface, and blank injections of buffers were subtracted. When necessary, the sensorgrams were normalized with respect to the maximal signal. The KD values of IgG or its F(ab) fragment were determined using Biaevaluation software. Biological replicates were systematically performed. Graphics of the raw data presentation (RU vs log UO22+) and the linear regression of the calibration curve were produced by using Excel software, version 2003. Calibration curves, dose-response curves and EC50 determination of proteins were produced from nonlinear regression analysis using GraphPad Prism software, version 4.0. Sigmoidal dose-response analysis was performed with variable slope, bottom and top

constraints set at 0 and 100 respectively. Values are given with ±95% confidence intervals. The detection limit (DL) for UO22+ was determined using the formula: DL ) [(Rb)+3SD] where Rb are average signals (RU) in blank, SD is the blank standard deviation. RESULTS AND DISCUSSION UO8S F(ab) as a Specific Biosensor for Uranyl Species in Physiological Conditions. The critical point for developing our technique was the recognition and quantification of free (non chelated) UO22+ species. For that purpose, we used a monoclonal antibody raised against DCP-UO22+ (named UO8S) obtained in our laboratory.28 Both sensitivity and reproducibility of DCP-UO22+ detection were checked first using a U08S F(ab) fragment (one specific binding site) as biosensor. Kinetic binding of the DCP-UO22+ complex was first checked to determine the binding levels. Highly reproducible curves reached a plateau of 6 RU (Resonance Units) for 1700 RU of immobilized U08S F(ab), whereas no signal was observed in the control (Figure 1A and B). Binding isotherms matched a 1:1 model (χ2 ) 0.0272), with KD ) 4.5 ± 0.5 nM (ka ) 6.94 × 105 M-1 s-1; kd ) 3.13 × 10-3 s-1). In Biacore protein assays, stoichiometry is usually determined by correlating mass ratios to the corresponding signal levels. But RI variations of small organic molecules such as DCP complexes can differ from protein RI. However, from the absolute amount of antibody immobilized on the surface, a maximum signal response (Rmax) for a particular analyte can be calculated using eq 1: Rmax ) (mass of the analyte/mass of the ligand) × I × S (1) Where I is the immobilization level of the ligand (RU) and S the stoichiometry. Thus a theoretical calculation based on 1:1 complexes would have given ∼9 RU of bound DCP for ∼1700 RU of immobilized F(ab). Here, saturation values correlated to 70% of this expected signal, in agreement with current efficiencies after covalent mAb coupling. Our objective was to quantify UO22+ and not the DCPUO22+complexes. We contemplated that our mAb UO8S should display significant binding properties for free UO22+ that can be exploited. Indeed, after UO22+ binding, addition of DCP should form a ternary complex that can be detected by SPR (Scheme 2). Injections of 1 µM of a solution of UO22+ followed by increasing concentrations of DCP, or on the other hand, increasing concentrations of UO22+ followed by 1 µM DCP, produced similar saturation curves also reaching 6 RU (Figure 1C, E). In the first protocol (Figure 1C), DCP binding kinetics matched a 1:1 model (χ2 ) 0.0882) with KD )17.5 ± 0.5 nM (ka ) 2.45 × 105 M-1 s-1; kd ) 4.32 × 10-3 s-1). As expected, the binding of DCP to the UO22+-F(ab) displayed a slightly slower ka value than the one observed for the DCP-UO22+ complex but similar kd. In the second protocol, the analysis of kinetic parameters could not be determined by Biacore software (Figure 1E). No DCP binding signal was observed on the control F(ab) even when 1 µM of the UO22+ solution was injected (Figure 1D), which confirmed the binding specificity. Analytical Chemistry, Vol. 82, No. 23, December 1, 2010

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Figure 1. Sensorgrams expressing association and dissociation kinetics: duplicates of signal variations (RU) vs time (s) according to increasing concentrations of DCP-UO2 complex for U08S F(ab) (A) or a nonrelevant F(ab) (B); 1 µM UO22+ followed by increasing concentrations (nM) of DCP binding to U08S F(ab) (C), or a nonrelevant F(ab) (D); increasing concentrations (nM) of UO22+ followed by 1 µM of DCP binding to U08S F(ab) (E). The arrows indicate the end of reactant injections. Scheme 2. Schematic Depiction of the Screeninga

a Variable protein concentrations are contacted with a fixed UO22+ concentration to reach equilibrium (1). After sample injections, the remaining free UO22+ is captured by the immobilized U08S mAb (2). The cycles continue (3) with 1 µM DCP injections. DCP acts as a probe to reveal free-UO22+ capture and the corresponding binding signals are registered. For each protein, dose-response curves (on the right) are established to determine the EC50.

Therefore, UO22+ species formed 1:1 UO22+-F(ab) complexes where DCP did not modify the equilibrium but provided a metal probe in the F(ab) fragment binding site. 9800

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Lastly, whatever the experiment, perfect baseline returns ensured both efficient regeneration steps and reliable small signal measurements.

Table 1. Repeatability of Measurements for DCP(1µM) Binding Isotherms Following the Injections of Increasing Concentrations (0-1000 nM) of UO22+ onto the Immobilized U08S mAba max min Rmax response (RU) response (RU) (max-min) (RU) EC50 (nM)

average values SD

Figure 2. Calibration curve: variations of DCP binding signal (RU) vs log of UO22+ concentrations. UO22+ bound to immobilized U08S mAb was revealed by 1 µM DCP. Experimental values from the same cycle are presented from duplicates of each UO22+ concentration, leading to a sigmoidal shape of the calibration curve Right insert: isotherm of UO22+ binding to immobilized U08S mAb obtained from treatment by nonlinear regression of the signal vs UO22+ concentrations. Left insert: linear regression of UO22+ binding signal variations vs the lower UO22+ concentrations.

To gain a better signal dynamic, the U08S mAb was then immobilized. UO22+ injections (0-1000 nM) followed by 1 µM DCP injections produced saturation curves (17.5 ± 0.5 RU for ∼4000 RU of immobilized U08S) corresponding to 2 DCP per mAb (Figure 2). The plots of binding signal B (RU), expressed after subtraction of B0 (buffer solution injection for 0 nM UO22+) versus UO22+ concentrations matched a reproducible standard binding isotherm (Figure 2, right insert). Using nonlinear regression analysis, a UO22+ standard diluted to 50 nM was controlled to 45 ± 3 nM, and 5-100 nM UO22+ was selected as the working calibration curve. The analysis of signal variations, corresponding to the first standard concentrations (0 to 31.25 nM) displayed a linear part (Figure 2, left insert). The UO22+ solutions were stable at room temperature for 24 h (ICP-MS analysis, SI). U08S mAb was confirmed to be stable for about three hundred cycles and a good sensor for extremely dilute UO22+ solutions. The different biological replicates, corresponding to different U08S immobilizations proved the robustness of the calibration curve establishment, and the reliability of the results (Table 1). In five interassays, reproducibility of Rmax and EC50 values was less than 5%, and LD ∼7 nM. Sensitive and reproducible small amounts of UO22+ remaining from the protein-metal equilibrium could therefore be measured. Affinities of Proteins and Small Ligands for UO22+. Few protein UO22+ affinities have been previously described and binding constants have been expressed under various experimental conditions with different thermodynamic parameters.33,34 Humantransferrin (HTf), human albumin (HSA) and U08S mAb were tested using the above-described assay. Increasing concentrations of proteins were contacted with a 50 nM UO22+ solution, a concentration leading to low protein consumption and corresponding to a zone of strong slope inducing high sensitivity. Once protein-UO22+ equilibrium was reached (step 1, (33) Montavon, G.; Apostolidis, C.; Bruchertseifer, F.; Repinc, U.; Morgenstern, A. J. Inorg. Biochem. 2009, 103, 1609–1616. (34) Duff, M. R., Jr.; Kumar, C. V. Angew. Chem., Int. Ed. 2005, 45, 137–139.

18.4 17.4 20.9 21.1 21.4

1.3 0.7 3.2 3.8 4.8

17.0 16.7 17.7 17.4 16.6

35.2 34.7 34.7 36.8 34.5

19.8

2.7

17.1

35.2

0.3

0.8

0.6

1.2

a The values are given for five experimental replicates of mAb immobilizations (∼ 4000 RU). Repeatability of experiments for DCP binding isotherms.

Figure 3. Dose-response curves for EC50 determinations. Increasing concentrations of proteins were contacted with 50 nM UO22+. Duplicates of experimental data are expressed in (B-B0)/(Bmax-B0) vs log of protein concentrations, with the corresponding curve fittings: U08S mAb (dotted line), HTf (continuous line) and HSA (dashed line). The approximations of EC50 can be graphically checked by using the intercepts at 50% of (B-B0)/(Bmax-B0). Table 2. Repeatability of EC50 Determination for the Three Selected Proteinsa

average EC50

U08S mAb (nM)

HTf (µM)

HSA (µM)

2.50 2.75 4.30 2.80 3.0 ± 1

3.2 2.0 2.8 3.3 2.8 ± 1

14.3 20.0 15.2 18.9 17 ± 3

a EC50 is the protein concentration leading to 50% of binding signal, calculated from nonlinear regression analysis of dose-response curves. Repeatability of EC50 determination for the three selected proteins.

Scheme 2), the solutions were injected onto the immobilized U08S (step 2, Scheme 2) to quantify the free remaining UO22+ by 1 µM DCP injections (step 3, Scheme 2). Figure 3 illustrates typical results obtained for the three proteins. The dose-response curves matched a four-parameter logistic equation for EC50 (equivalent concentration leading to 50% of binding signal) calculation. The repeatability of four different experiments, corresponding to four different U08S mAb immobilizations is presented in Table 2. U08S displayed 3 ± 1.5 nM as EC50 values. It should be mentioned that, as expected, this EC50 value is significantly lower than the one determined on immobilized F(ab) fragments (see above)since in the present case the UO22+ affinity was determined with U08S mAb used in solution. HTf and HSA displayed Analytical Chemistry, Vol. 82, No. 23, December 1, 2010

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2.8 ± 1 µM and 17 ± 3 µM, respectively as EC50 values. In previous studies, from experiments based on desalting processes performed to isolate free UO22+ from UO22+-protein complexes,4 we deduced slightly better stability of the UO22+Tf complex than for UO22+-HSA. In addition, similar stability constants expressed by log K ) 14 and 13 (HTf), log K ) 11 and 17 (HSA) for uranyl-protein and uranyl-protein-carbonate complexes, were determined using time-resolved laser-induced fluorescence spectroscopy and speciation modeling at zero ionic strength.33 In recent calorimetric studies, the data obtained for uranyl nitrate binding to BSA at pH 5.5 matched a two-site model, with recorded binding constants of K ) 1.6 ± 0.7 × 107 M-1 and K ) 2.8 ± 2.5 × 105 M-1, respectively,34 leading to an apparent KD within the micromolar range. By removing NaCl from our buffer, we obtained EC50 ) 5 ± 1 µM for BSA and HSA, with no significant influence on HTf and U08S. Therefore, our results for U08S mAb and the two human proteins are coherent with these previous values. The buffer solutions (NaCl, carbonates, pH 7,4) are close to physiological ones so the rank order of the EC50 reflects the rank order of apparent global affinity constants (KD,app) with 1:1 as default stoichiometry. However, considering the limited number of data related to proteins, we decided to extend the application of our assay to small ligands whose UO22+ binding properties were previously determined.32,35 Mono, di and tripodal bisphosphonates were selected as model ligands (Scheme 1) and tested under our conditions, that is, with a 1000:1 carbonate versus UO22+ ratio, and in 50 mM TRIS, 150 mM NaCl, pH 7.4. Even if the screening method and buffering conditions were also different from the cited results, our antibody-based technique agrees perfectly with the expected hierarchy of UO22+ binding as shown in Table 3 and SI Figure S3. The screening assay was thus validated by two different approaches. CONCLUSION We have demonstrated that this two step immunoassay is an efficient method for screening proteins or small ligands for their UO22+ binding capacities. For the first time, affinities for UO22+ (35) Sawicki, M.; Siaugue, J. M.; Jacopin, C.; Moulin, C.; Bailly, T.; Burgada, R.; Meunier, S.; Baret, P.; Pierre, J. L.; Taran, F. Chemistry 2005, 11, 3689– 3697.

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Table 3. Determination of KD,app for Bisphosphonate Ligandsa ligands

MW (Da)

log Kcond pH 7.429,35

KD,app (M)

1 2 3 4

206 647 739 939

9 17.2 17.8 20

14 ± 2 × 10-6 55 ± 1 × 10-9 52 ± 1 × 10-9 4 ± 1 × 10-9

a

The hierarchy of affinity is in agreement with the results given.

can be easily measured within a few hours and under physiological conditions. The analysis requires only few hundred micrograms of proteins. Taking HSA (66 kDa) as the representative molecule (55% of serum proteins) to calculate average protein molarity, human serum led to a value of UO22+ EC50 ) 300 ± 50 nM. Extensive studies dedicated to human serum proteins are currently in progress to focus on more relevant targets. Since this technique has been shown to be also suitable for small ligands, it could be used to measure the interactions of uranyl with varied metabolites, address the metallome of biological fluids, and evaluate the chelating properties of molecules for therapeutic use. The concept can also be extended to other toxic metals. ACKNOWLEDGMENT This work was partly supported by the CEA Program “Toxicologie Nucle´aire et Environnementale”. We thank O. Pible, F. Rollin-Genetet and T. Vercouter for fruitful discussions. SUPPORTING INFORMATION AVAILABLE Figure S1 presents the uranyl speciation diagram obtained by JChess modeling. Table S1 shows the stability of uranyl solutions on board demonstrated by the good correlation between prepared dilutions and ICP/MS measurements. Figure S2 presents the mass spectrum of the 50 nM uranyl solution used for calibration after a 24 h life cycle on board. Figure S3 presents dose-response curves for EC50 determinations of bisphosphonate ligands. This material is available free of charge via the Internet at http:// pubs.acs.org. Received for review July 28, 2010. Accepted October 26, 2010. AC102578Y