Can Uranium Be Transported by the Iron-Acquisition Pathway? Ur

Mar 17, 2011 - Transferrin (T) is one of the major protein targets of uranyl (Ur) and the Ur-loaded protein (TUr2) interacts with receptor 1. In vitro...
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Can Uranium Be Transported by the Iron-Acquisition Pathway? Ur Uptake by Transferrin Miryana Hemadi,* Nguy^et-Thanh Ha-Duong, and Jean-Michel El Hage Chahine* ITODYS, Interactions, Traitements et Organisation et Dynamique des Systemes, Universite Paris-Diderot, CNRS UMR 7086, 15 rue Jean-Antoine de Baïf, 75205 Paris Cedex 13, France

bS Supporting Information ABSTRACT: Transferrin (T) is one of the major protein targets of uranyl (Ur) and the Ur-loaded protein (TUr2) interacts with receptor 1. In vitro, Ur is transferred from one of the major plasma complexes, tricarbonated Ur (Ur(CO3)34) to T in four kinetically differentiated steps. The first is very fast and accompanied by HCO3 loss. It yields a first intermediate ternary complex between dicarbonated Ur and the phenolate of one of the two tyrosine ligands in the C-lobe; direct rate constant, k1 = (7.0 ( 0.4)  105 M1 s1; reverse rate constant, k1 = (4.7 ( 0.2)  103 M1 s1; dissociation constant, K1 = (6.7 ( 0.6)  103 and an affinity of the T for the dicarbonated Ur (Ur(CO3)22) close to that of the latter to CO32, K0 3 ∼ 1  104. This first kinetic product undertakes a fast ratelimiting conformation change leading to the loss of a second HCO3: direct rate constant, k2 = 33 ( 14 s1. This second ternary complex undergoes two very slow conformation changes (1 and 5 h), at the end of which both C- and N-lobes become loaded with Ur. When unexposed to uranium, the Ur concentrations in the bloodstream are much too low to favor receptor-mediated transport. However, in the case of exposure, these concentrations can grow considerably. This, added to the fast Ur complex formation with the C-lobe and the fast interaction of the Ur-loaded T with the receptor, can allow a possible internalization by the iron-acquisition pathway.

’ INTRODUCTION Human serum transferrin belongs to the transferrin superfamily that includes globular periplasmic transport proteins.1 Transferrins are the most important iron-solubilization system in the biological fluids of vertebrates and invertebrates. All the iron(III) present in the bloodstream is practically completely solubilized by serum transferrin (T).2,3 When this protein is ironloaded, it interacts with transmembrane receptor 1 and the proteinprotein adduct is internalized in the cytosol by receptor-mediated endocytosis.4 This process represents the major iron-acquisition pathway.36 T is a glycoprotein of about 80 kDa. It consists of a single chain of about 700 amino acids organized in two lobes, the N- and C-lobes. Each lobe contains an ironbinding site formed by four protein ligands, two phenolates of two tyrosines, one imidazole of a histidine, and one carboxylate of an aspartate. The binding site also contains a carbonate or bicarbonate adjacent to an arginine that constitutes the synergistic anion, without which the protein loses its affinity for iron. Each lobe consists of two domains containing parts of the ligands. When T is in the apo-form (iron-free), it is in the so-called open structure, in which the protein ligands are in contact with the outside medium. When the protein is in the holo-form (ironsaturated, TFe2), the two domains of each lobe surround the metal like a pair of jaws, burying it 10 Å beneath the surface, in the so-called closed structure.710 Besides Fe(III), T forms stable complexes with no less than 40 different metals, such as cobalt, gallium, bismuth, aluminum, r 2011 American Chemical Society

uranium, titanium, etc.1117 In biological media, uranium is mainly present as uranyl (UO22þ).1821 Uranyl (Ur) forms a rather stable complex with T with an affinity constant estimated to be in the 1071013 range.18,22,23 Moreover, the structure of the UrT complex seems to differ from that of other metals, such as iron or gallium. It is assumed to be in a semiclosed conformation with the metal only partially included in the binding cleft, which is also the case for aluminum.19,24 We recently showed that an interaction between Ur-loaded T and receptor 1 occurs.25 Although weak, this interaction may allow a possible transport of uranium from serum to cytosol by the iron-acquisition pathway .25 Uranium is a heavy metal, traces of which are found in food and water supplies.26 Its use for military and civilian purposes in nuclear weapons and reactors is widely spread.2729 In its less radioactive forms (mainly 238U), uranium is commonly used in military armor and munitions. Thus, exposure of soldiers and civilians is frequent. Uranium is highly toxic in all its oxidation states. It causes damage to bones and to kidney function, is neurotoxic, accumulates in the brain, etc.3033 Therefore, the investigation of the incorporation of uranium and its transport in biological media is a matter of considerable health interest.

Received: December 16, 2010 Revised: February 25, 2011 Published: March 17, 2011 4206

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In the bloodstream, Ur is mainly complexed to carbonate and to T.20,22,3337 In this article, by the use of the methods and techniques of chemical relaxation,3840 we propose a mechanism for Ur uptake by serum transferrin from the carbonateUr complex, in which we take into account the role of bicarbonate and pH. The relation between a possible Ur transport by the iron-acquisition pathway and the proposed mechanism is discussed.

’ EXPERIMENTAL SECTION Better than 98% pure human serum apotransferrin (Sigma) was further purified by published procedures; its purity was checked spectrophotometrically and by urea polyacrylamide gel electrophoresis.41,42 KCl (Merck Suprapur), NaOH, HCl (Merck Titrisol), EDTA (Merck Titriplex), UO2(OCOCH3)2 3 2H2O (Prolabo), nitrilotriacetic acid (Sigma), acetic acid (96%), sodium acetate (Merck), sodium bicarbonate, glycerol, urea (electrophoresis reagent), SDS, boric acid, ethanolamine, glycine (electrophoresis reagent), Brilliant Blue, and HEPES were used without further purification. Water and glassware were prepared as described previously.43 Stock Solutions. The buffer solution contained 50 mM of HEPES in the presence of 1 mM e [HCO3] e 50 mM. Final pHs were continuously controlled and adjusted to between 7.2 and 8.6 with microquantities of concentrated HCl or NaOH. All final ionic strengths were adjusted to 0.2 M with KCl.6 T concentrations were checked by a Bio-Rad protein assay and spectrophotometrically.44,45 Ur acetate stock solution (5 mM) was prepared in bidistilled deionized water; the pH of the solution was about 5. T (c0) and Ur carbonate (c1) concentrations were obtained by dilution in the final buffers. c0 varied from 1 to 6 μM and c1 from 1 to 500 μM. Metal-Loaded Transferrins. The C-site iron-loaded T was prepared as described elsewhere.46 The C-site Ur-loaded T and Ur-saturated T were prepared by incubating T overnight at 37 °C with 1.3-fold and ∼20-fold its concentration in Ur acetate in the presence of 20 mM HCO3 at pH 7.4. The solution was afterward dialyzed 10 times against HEPES buffer. Protein integrity was checked by SDS-PAGE,42 and the Ur load was checked by inductive plasma coupling by Professor Remi Losno at the LISA laboratory of the Paris Diderot University. pH Measurements. pH values were measured at 25 ( 0.5 °C with a Jenco pH-meters equipped with a Metrohm combined calomel/glass minielectrode. The pH-meter was standardized at working temperature by standard buffers: pH 7.00 and 10.01 (Beckman). At the end of each experiment, final pHs were checked both in the buffer and in the protein solutions. They were reproducible to better than 0.02 pH unit. Spectrophotometric Measurements. Absorption measurements were performed at 25 ( 0.5 °C on a Cary 500 spectrophotometer equipped with a thermostated cell carrier. Twocompartment cuvettes of identical volumes were used for the acquisition of the differential absorption spectra and for kinetics determination. In both cuvettes, the first compartment contained the protein and the other Ur carbonate solutions at the same pH and value of μ. One of these two cuvettes was used as a reference. The protein and Ur carbonate solutions remained unmixed in the reference cell and were mixed only in the sample cuvette. Fluorimetric measurements were performed at 25 ( 0.5 °C on an Aminco-Bowman series 2 luminescence spectrometer equipped with a thermostated cell-carrier. Emission spectra were

Figure 1. Fluorescence emission spectra of apotransferrin T, one uranyl-loaded T (TUr) and two uranyl-loaded T (TUr2) species at 25 ( 0.1 °C and μ = 0.2. Reported at pH = 7.8, c0 = 2 μM for an excitation wavelength λex = 280 nm.

measured in the 300400 nm range. Excitation wavelength was set to 280 nm. The spectra used for the static determination of equilibrium constants were recorded at the final equilibrated state. Kinetic Measurements. Kinetic runs occurring in the 150 s range were performed on a Hi-tech Scientific SF61DX2 stoppedflow spectrophotometer equipped with a thermostated bath at 25 ( 0.5 °C as described previously.12 All signals were accumulated at least 10 times. Data Analysis. The data were analyzed as described previously by linear and nonlinear least-squares regressions, and all uncertainties are twice the standard deviations.12 All the observed kinetics were pure mono- or multiexponentials and were dealt with as relaxation modes.3840 All experimental conditions were set so as to allow the use of the methods and techniques of chemical relaxation.3840

’ RESULTS The Ur donor to T used in our experiment is the carbonate Ur complex.20 Spectrophotometric detection was used in all thermodynamic and kinetic runs. The kinetic processes related to Ur uptake by T were acquired by absorption and fluorescence emission. The excitation wavelength was set to λex = 280 nm. The charge of the protein species is not indicated. Complex formation with Ur is usually accompanied by a fluorescence quenching, which is also observed for the uranyl transferrin complex.4749 Figure 1 presents the emission spectra of T when incubated with Ur in the presence of HCO3 for 48 h. They show two TUr complexes, the presence of which depends on the Ur/T ratio of concentrations (Figure 1). The ICP performed on each of the two complexes indicates 1/1 and 2/1 stoichiometries. When a solution of apotransferrin is mixed with a solution of Ur in the presence of HCO3, four kinetic processes are observed (Figure 2). The first is very fast and occurs in the tens of milliseconds range as an exponential decrease in the fluorescence to yield a first kinetic product (Figure 2A). The latter yields a second kinetic product in a process, which appears as a second exponential decrease of the emission occurring in the second 4207

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Figure 2. Relative variation of the normalized fluorescence intensity (A,B for λex = 280 nm and emission wavelength λem g 310 nm) and of the differential absorption (C,D) with time when a solution of apotransferrin (A,B, c0 = 6 μM; C, c0 = 4 μM; D, c0 = 2 μM) is mixed with a solution of uranyl (A,B, c1= 30 μM; C, c1 = 40 μM; D, c1 = 20 μM) at pH 8.3 for A,B and pH 7.7 for C,D at 25 ( 0.5 °C with [HCO3] = 20 mM and μ = 0.2.

range (Figure 2B). These two processes are followed by two slow kinetic phenomena during which the Ur-saturated T attains its thermodynamic state. The third is as an exponential increase in absorption or decrease in the fluorescence emission (not shown) in the 1000 s range (Figure 2C). It is followed by another increase in absorption or decrease in fluorescence (not shown) lasting about 20 000 s (Figure 2D). The experimental reciprocal relaxation times related to the first process of Figure 2A depend on the apotransferrin concentration (c0), the Ur carbonate concentration (c1), [HCO3], and pH. The second process (Figure 2B) depends on c1, pH, and [HCO3]. These two processes (Figure 2A,B) are not observed with a C-lobe only iron-loaded T. As for the third and fourth processes (Figure 2, C and D), they seem independent of all our experimental parameters. First Kinetic Process. The facts that the first kinetic process of Figure 2A depends on c0, c1, and [HCO3] and is not observed with a C-lobe only iron-loaded T leads us to assume that, as in the case of cobalt uptake from a mixed carbonate complex, metal uptake occurs at the C-site (TC) of the protein with the carbonated metal species.12 Three series of experiments were performed. The first were undertaken at four fixed pH values (7.5 e pH e 8.3), variable c1 with constant [HCO3] and c0. They showed for each fixed pH a linear relationship between the reciprocal relaxation time and c1 (Figure 3). The second series was performed at fixed pH, c1, and c0 with variable [HCO3].

Figure 3. Plot of τ11 against c1 at four fixed pH values for [HCO3] = 20 mM, c0 = 6 μM. pH 7.5 (crossed triangles): intercept 89 ( 2 s1, slope (1.71 ( 0.08)  105 M1 s1, r = 0.99 844. pH 7.8 (square): intercept 97 ( 6 s1, slope (2.56 ( 0.25)  105 M1 s1, r = 0.99 267. pH 8.0 (triangle): intercept 96 ( 7 s1, slope (3.64 ( 0.44)  105 M1 s1, r = 0.99 111. pH 8.3 (circle with dot): intercept 91 ( 7 s1, slope (5.05 ( 0.43)  105 M1 s1, r = 0.99 647. 4208

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Figure 4. Plot of τ11 against [HCO3] for c1 = 90 μM and c0 = 6 μM at pH = 8.0.

The experimental reciprocal relaxation times showed what appears to be a second-order polynomial relationship with [HCO3] (Figure 4). Finally, all concentrations were fixed and the experiments performed at several pH values. At a fixed pH value we can write to a first approximation eq 1:12 k0 obs

TC0 þ UrðCO3 Þ3 4 s F TC0 UrY þ CO3 2 s R k0 obs

ð1Þ

with TC0 , the C-lobe of T in an unknown state of protonation; TC0 UrY, a T ternary complex intermediate in which the C-lobe is loaded with Ur. In this case, the reciprocal relaxation time equation can be expressed as12,38,39 τ0 1

1

¼ k0 obs ð½TC0  þ ½UrðCO3 Þ3 4 Þ þ k0 obs ð½TC0 UrY þ ½CO3 2 Þ

ð2Þ



Under our experimental conditions ([HCO3 ] = 20 mM, [CO32] = [HCO3]K1a/[Hþ] and c0 < c1 < [HCO3]), eq 2 can be expressed as eq 3: 1 τ0 1

0

0



þ

¼ k obs c1 þ k obs ½HCO3 K1a =½H 

ð3Þ

Figure 5. Plot of 1/kobs against [Hþ]: intercept = (1.35 ( 0.36)  106 M s; slope = 146 ( 20 s; r = 0.99 534.

exists as two different species (eq 5).20 All these indications lead us to propose eqs 5 and 6. UrðCO3 Þ3 4 þ Hþ hUrðCO3 Þ2 2 þ HCO3  k1

UrðCO3 Þ3 4 þ TC s F TC UrZ þ HCO3  s R k1

ðrate-limitingÞ

where TCUrZ is a ternary complex between the C-site of transferrin and a bicarbonated uranyl, with K2a = [Ur(CO3)34] [Hþ]/[ Ur(CO3)22][HCO3] and K1 = [Ur(CO3)34] [TC]/[TCUrZ][HCO3]. In this case, where [HCO3] . c1 > c0, the reciprocal relaxation equation associated with eq 6 is expressed as38,39 τ1 1 ¼ k1 ½UrðCO3 Þ3 4  þ k1 ½HCO3  

ð7Þ

[Ur(CO3)34] can be easily determined from K2a, [HCO3], and c1, and its substitution in eq 7 leads to eq 8: τ1 1 ¼ k1 K2a c1 ½HCO3  =ð½Hþ  þ K2a ½HCO3  Þ þ k1 ½HCO3  

ð4Þ

where K1a = [CO32][Hþ]/[HCO3] = 4.5  1011 M. Therefore, at a constant pH, c0 and [HCO3], [HCO3]K1a/ [Hþ] must also be constant. From the slope and intercept of each best line in Figure 3, a k0 obs and k0 obs[HCO3]K1a/[Hþ] values are determined for each of the four fixed pH values. The intercepts of the lines of Figure 3 are within the limits of uncertainty identical. In this case, they cannot describe k0 obs[HCO3]K1a/[Hþ]. This, hence, excludes the dependence of the reciprocal relaxation time on [CO32] and subsequently discards the involvement of CO32 (eq 1). It also implies the probable involvement of HCO3 in the first kinetic process, as shown in Figure 4. Furthermore, a good linear least-squares regression of 1/k0 obs against [Hþ]n is only obtained for n = 1 (Figure 5). This indicates the involvement of a single proton transfer prior to metal uptake by T.43 Apotransferrin does not undertake any proto-dissociation in the pH range used in this work,41,50 whereas the Ur complex

ð5Þ

ð6Þ

with CO3 2 þ Hþ hHCO3 

ðfastÞ

which at constant

[HCO3]

ð8Þ

can also be expressed as

τ1 1 ¼ k1obs ½HCO3  c1 þ k1 ½HCO3  

ð9Þ

kobs ¼ k1obs ½HCO3  

ð10Þ

with [HCO3]

At four fixed pH and (20 mM), the plot of τ11 against c1 is linear, as already shown in Figure 3. From the slope of each of the best lines and eq 10, k1obs and k1 are determined. All intercepts are identical within the limits of uncertainty, which, for [HCO3] = 20 mM, lead to k1 = (4.7 ( 0.5)  103 M1 s1. From eqs 8 and 9 we can write 1=kobs ¼ ½Hþ =k1 K2a ½HCO3   þ 1=k1

ð11Þ

As already shown in Figure 5, the plot of 1/kobs against [Hþ] is linear from the slope and intercept of the best line 4209

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Figure 6. Plot of τ11/[HCO3] against K2ac1/([Hþ] þ K2a[HCO3]): intercept = (4.7 ( 0.2)  103 M1 s1; slope = (7.0 ( 0.4)  105 M1 s1; r = 0.99 062. 1 1

7

k1 = (7.4 ( 1.5)  10 M s and K2a = (4.5 ( 1.5)  10 are determined. With K2a known, the experimental reciprocal relaxation times associated with Figure 2A, at 7.5 e pH e 8.5, 40 μM e c1 e 0.5 mM, and 5 mM e [HCO3] e 30 mM, were plotted against eq 8. A good linear least-squares regression was obtained (Figure 6). From the slope and intercept, k1 = (7.0 ( 0.4)  105 M1 s1 and k1 = (4.7 ( 0.2)  103 M1 s1 and K1 = (6.7 ( 0.6)  103 are determined. Both k1 and k1 are, within the limits of uncertainties, identical to those measured from Figures 3 and 5. Second Kinetic Process. The second kinetic process (Figure 2B) seems to depend on c1, [HCO3], and pH. It is not observed with a C-lobe iron-loaded T. Therefore, it very probably concerns the C-lobe of T. In this case, several possibilities can be envisaged. The first leads to kinetic intermediate TCUr0 and involves a second HCO3 loss (eqs 12). The second involves a CO32 loss and leads to intermediate TCUr00 (eq 13). 5

k0 2obs

TC Ur0 þ HCO3  s F TC UrZ s R k0 2obs k00 2obs

TC Ur00 þ CO3 2 s F TC UrZ s R k00 2obs

ð12Þ

ð13Þ

Under our experimental conditions, c1 > c0 and a fixed pH value, the reciprocal relaxation equations associated with eqs 12 and 13 can be expressed as eqs 14 and 15, respectively:38,39 τ0 2 1 τ0 2

1

¼ k0 2obs ½HCO3   þ k0 2obs

ð14Þ

¼ k00 2obs ½HCO3  K1a =½Hþ  þ k00 2obs

ð15Þ

At a fixed pH value, the plot of the experimental reciprocal relaxation times, related to Figure 2B, against [HCO3] is not linear (Figure 7). Consequently, eqs 14 and 15 must be discarded. On the other hand, the dependence of the experimental τ21 on c1 is not compatible with eqs 12 and 13. Indeed, under our experimental conditions and at the end of the first kinetic process (Figure 2A), T has at least partly reacted with Ur

Figure 7. Plot of τ21 against [HCO3] for c1 = 90 μM and c0 = 6 μM at pH = 8.0.

(eqs 5 and 6). This implies the involvement of one or several other reactions in the process. These can be changes in the conformation of the first protein complex, allowing it to lose a second HCO3 and to yield a second kinetic product. The first possibility (eqs 16 and 17) implies a semifinal conformation change rate-limiting HCO3 loss from TCUrZ TC UrZhTC Ur00 þ HCO3  k

000

TC Ur00 s F TC Ur s R k

ð16Þ

000

2

ðrate-limitingÞ

ð17Þ

2

The reciprocal relaxation time associated to rate-limiting eq 17 is expressed as eq 18 (see Supporting Information): τ

1

000

2

000

¼k

2

000

þ k 2 K 00 2 c1 R=ðK 00 2 c1 R

þ K 00 2 ½HCO3   þ K1 ½HCO3  2 Þ with K00 2 = [TCUr00 ][HCO3]/[TCUrZ] [HCO 3]/(K2a[HCO3] þ [Hþ]).

ð18Þ

and R = K2a

Equation 18 is not respected by the experimental data, as shown in Figure 7, where at a fixed pH, the experimental reciprocal relaxation times grow with [HCO3]. In the second possibility, a conformation change of the first kinetic adduct is required for HCO3 loss (eqs 19 and 20): k2

TC UrΖ s F TC Ur0 s R k2

ðrate-limitingÞ

TC Ur0 hTC Ur þ HCO3 

ð19Þ ð20Þ

where TCUr0 is a kinetic intermediate and TCUr is a kinetic product, in which the C-site is coordinated to one carbonated uranyl. The reciprocal relaxation time equation associated with ratelimiting eq 19 can be expressed as (see Supporting Information) τ2 1 ¼ k2 ½HCO3  =K3 þ k2 c1 RðK3 þ ½HCO3  Þ= ½ðc1 R þ K1 ½HCO3  K3  4210

ð21Þ

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τ4  k0 4 þ k0 4

ð26Þ

These equations are independent of the concentrations of the species present in the medium.

Figure 8. Plot of τ21 (c1R þ K1[HCO3])/(c1R) against [HCO3] (c1R þ K1[HCO3])/(c1R): intercept = 33 ( 14 s1; slope = 766 ( 56 M1 s1; r = 0.98 772.

with K3 = [TCUr][HCO3]/[ TCUr0 ] and K2 = [TCUr0 ]/ [ TCUrZ]. At the end of the second kinetic process (Figure 2B) and before the beginning of the third, TCUr is assumed to be the major product. Under our experimental conditions, the fact that [HCO3] . c1 implies that K3 > [HCO3], which simplifies eq 21 into eq 22 τ2 1 ¼ ðc1 R þ K1 ½HCO3  =c1 R ¼ k2 ½HCO3  ðc1 R þ K1 ½HCO3  Þ=c1 RK3 þ k2 ð22Þ A good linear regression of the data against eq 22 is obtained (Figure 8). From the slope and intercept, k2/K3 = 766 ( 56 M1 s1 and k2 = 33 ( 14 s1 are determined. It should be noted here that the amplitudes of this second process are small, which can explain the rather high uncertainty on the intercept. Nevertheless, this can give an estimate for K3K2 = [TCUr][HCO3]/ [TCUrZ] ≈ 0.04 ( 0.01 M. Third and Fourth Kinetic Processes. The third and fourth kinetic processes seem to be independent of all our experimental parameters. Furthermore, at the end of these final processes, the protein becomes Ur-saturated in both lobes. As in the case of other metals, these two phenomena can describe conformational changes of the protein metal-loaded in the C-lobe which ratecontrols Ur uptake by the N-lobe.6,13,15,16,41 The involvement of other reactions in these kinetic processes, such as proton transfers, cannot be detected under our experimental conditions by the techniques used. All proceeds as if two first-order reactions were occurring (eqs 23 and 24). k0 3 ??

TC Urh unknown and rate-limiting hðTC UrÞ k0 3 ??

ð23Þ

’ DISCUSSION In Table 1, we summarize the mechanism of Ur uptake by transferrin (eqs 5, 6, 19, 20, 23, 24) along with that of the interaction of TUr2 with transferrin receptor 1 (RM; eqs 3840) and compare them to those of iron uptake (eqs 2737) and interaction of iron-saturated or holotransferrin (TFe2) with RM (eqs 4143).6,25,45 Uranium is present in biological media mainly as uranyl.1821 It is, moreover, transported in the bloodstream through interaction with carbonate and proteins, mainly transferrin.21,51,52 The distribution of the uranyl species in the biological fluids between the carbonate complexes and transferrin plays an important role in the solubilization, transport, and incorporation of uranium. This speciation of uranyl carbonate complex species is, therefore, important for a better understanding of the mechanism of Ur uptake by T. Speciation of the Uranyl Carbonate Complexes in Relation with Uranyl Uptake by T. In neutral and slightly basic

media, at least two Ur carbonate complexes coexist: Ur(CO3)22 and Ur(CO3)34.20 The speciation of the Ur carbonate depends on pH,20 which led us to propose eq 5 (Table 1). In our case (μ = 0.2, 7.4 e pH e 8.5, and [HCO3] . c1), CO32 is barely present in the medium. Furthermore, we clearly showed that the first step in uranyl uptake by T depends on [HCO3] (eq 8; Figure 6; Table 1, eq 6). Hence, we assumed that the protonation of the tricarbonate complex leads to a bicarbonate loss (Table 1, eq 5). Knowing K2a and pH, we can easily show that ½UrðCO3 Þ3 4  ¼ K2a ½HCO3  c1 =ðK2a ½HCO3   þ ½Hþ Þ ð44Þ ½UrðCO3 Þ2 2  ¼ c1 ½Hþ =ðK2a ½HCO3   þ ½Hþ Þ

ð45Þ 

This implies that in our conditions for a typical [HCO3 ] = 20 mM, both Ur(CO3)34 and Ur(CO3)22 are present under the experimental medium at comparable concentrations (Figure 9). This speciation izs at variance with that proposed by simulation and modeling in different biological fluids.20 However, in other proposals, it was assumed that Ur(CO3)22 is the major carbonate complex in biological media and that it has a very low reactivity.34 In more basic media, we can schematically write eq 46:51,52 UrðCO3 Þ2 2 þ CO3 2 hUrðCO3 Þ3 4

ð46Þ

In this case K2a (reported for eq 5) can be expressed as eq 47. K2a ¼ ½UrðCO3 Þ3 4 K1a =½CO3 2 ½UrðCO3 Þ2 2 

ð47Þ

This allows us to write that K0 3 = [Ur(CO3)3 ]/[CO32] [Ur(CO3)22] = K2a/K1a ≈ 1  104. On the other hand, we can also write eqs 48 and 49:51,52 4

0

k 4 ??

TC Urh unknown and rate-limiting hTC ðUrÞ2

ð24Þ

k0 4 ??UrðCO3 Þ3 4

In this case, the associated reciprocal relaxation time equations would be expressed as eqs 25 and 26:38,39,41 0

0

τ3  k 3 þ k 3

ð25Þ

Ur2þ þ 3CO3 2 hUrðCO3 Þ3 4

ð48Þ

Ur2þ þ 2CO3 2 hUrðCO3 Þ2 2

ð49Þ

with log β3 = log([Ur(CO3)3 ]/[Ur ][CO3 ] ) ≈ 21.2 and 4

4211



2 3

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Table 1. Mechanisms of Uranyl and Iron Uptake by Transferrin and Interactions with Receptor 1a direct rate const

reverse rate const

equilib const

Uranyl Uptake by Transferrin UrðCO3 Þ3 4  þ H þ hUrðCO3 Þ2 2  þ HCO3  ð5Þ UrðCO3 Þ3 4  þ TC hTC UrZ þ HCO3 

ð6Þ

TC UrZhTC Ur0

ð19Þ

TC Ur0 hTC Ur þ HCO3 

ð20Þ

?H þ

hTC ðUrÞ2 TC Urh unknown 4

4.6  107 7.0  105 M1 s1

4.7  103 M1 s1

33 s1



6.3  103

ð23,24Þ

UrðCO3 Þ3

Iron Uptake by Transferrin39,43 0

T þ HCO3



hTC H3

ð27Þ

0

TC H3 þ FeL hTC H3 Fe þ L TC H3 FehTC H2 Fe þ H

þ

1 1

8.0  10 M

7.5  10 M

s

1

ð30Þ þ

ð31Þ ð32Þ

6.2  102 s1

T0 Hð4  mÞ Fe þ H þ hT0 Hð5  lÞ Fe

ð33Þ

6.8 nM

0

T Hð4  mÞ FehT}Hð4  mÞ Fe

ð34Þ

T0 Hð4  mÞ Fe2 hT}Hð4  mÞ Fe2

ð35Þ

T0 Hð4  mÞ Fe þ FeLhL þ T0 Hð4  mÞ Fe2 TC H3 þ Fe

1 1

16 nM

T0 Hð5  mÞ FehTHð5  mÞ Fe



s

4

ð29Þ

H3 TN TC H2 FehTH5 Fe TH5 FehTHð5  mÞ Fe þ mH

ð28Þ

4.35 mM 4

hTC H3 Fe

R M þ TUr2 hR M -TUr2 

ð36Þ ∼1  1016 M

ð37Þ Interaction of TUr2 with Transferrin Receptor 1 Subunit (RM) ð38Þ 5.2  106 M1 s1 95 s1

18 μM

R M þ TUr2 hR M -TUr2

ð39Þ

0.3

R M þ TUr2 h 3 3 3 hR M -TUr2

ð40Þ

6 μM

R M þ TFe2 hR M -TFe2 

Interaction of TFe2 with Transferrin Receptor 1 Subunit (RM) ð41Þ 3.2  1010 M1 s1 1.6  104 s1

0.5 μM

R M -TFe2 hR M -TFe2

ð42Þ

4.6  103

R M þ TFe2 hR M -TFe2

ð43Þ

2.3 nM

a

L is the ligand; RM-TUr2* and RM-TFe2* are the proteinprotein adducts, in which the C-lobe of the metal-saturated protein is in interaction with RM; RM-TUr2 and RM-TFe2 are the proteinprotein adducts, in which both C- and N-lobes are in interaction with RM.6,25,41,45

log β2 = log([Ur(CO3)22]/[Ur2þ][CO32]2) ≈ 17.51,52 This implies that K0 3 = β3/β2 ∼ 4  104. This value is in the same range as that determined above, which further confirms our proposals. Synergistic Anion and the Mechanism of Uranyl Uptake by Transferrin. A synergistic anion is indispensible for metal uptake by transferrin, as it is directly involved in the coordination sphere of the metal in the complex. In vertebrates, the natural synergistic anion is carbonate or bicarbonate.8,50,53,54 Furthermore, in physiological media, 30% of the C-lobe of apotransferrin and less than 2% of the N-lobe are in interaction with bicarbonate.50 This always led to a first metal-uptake by the C-lobe in interaction with the synergistic anion (TCH3; Table 1, eqs 27, 28), followed by proton-losses triggering a series of conformation changes, which lead to a second metal uptake by

the N-lobe and to thermodynamic equilibrium (Table 1, eqs 3037).11,15,16,41 Four kinetic steps are observed for Ur and Fe(III) uptakes by T.45 Nevertheless, the first two steps of these metal uptakes are different. Indeed, the first step in iron uptake by T consists of a metal transfer from Fe(III) chelators to the C-lobe of T in interaction with carbonate or bicarbonate.41,45 With Ur(CO3)34, a first ternary complex involving the loss of one bicarbonate occurs very rapidly with the C-lobe (Table 1, eq 6). This first kinetic product loses another bicarbonate to yield a second ternary complex (Table 1, eqs 19, 20). This latter undergoes a series of changes in conformation, which ratecontrol a second Ur uptake by the N-lobe and lead to the final thermodynamic product (Table 1, eqs 23, 24), where the log of the affinity of the protein for Ur attains 1213.18 Whether these conformation changes are accompanied by other processes such 4212

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52:11,12,16,41

Figure 9. Normalized uranylcarbonate complexes distribution against pH for [HCO3] = 20 mM.

as proton losses cannot be detected by our techniques. These two final processes are comparable to those detected with other metals, such as, iron (Table 1, eqs 3337) aluminum, cobalt, gallium, and bismuth, where there is a cooperativity between the C- and N-sites upon metal loading, as the N-lobe cannot complex Ur unless the C-lobe is already Ur-loaded.6,13,15,16,41 On the other hand, the mechanism of uranyl uptake is close to that established for cobalt from the cobalt nitrilotriacetate carbonate mixed complex (CoNtaCO32).12 Indeed, with CoNtaCO32, the C-lobe of T directly reacts with the carbonated cobalt complex and does not require the presence of any external synergistic CO32 or HCO3. Furthermore, with Ur(CO3)34 and CoNtaCO32, metal transfers to C-lobe occur much more rapidly than that of iron(III) or other metallic elements from, e.g., acetohydroxamic acid, nitrilotriacetate (Nta), or citrate chelators in the presence of bicarbonate.13,41 Therefore, the presence of carbonate in the metal donor complex accelerates metal uptake by the C-lobe of T. Furthermore, in the Ur tricarbonate complex, the Ur cation occupies a central position with the three bidentate ligands located in the plane equatorial to the axis formed by the Ur ion.55 Does this render the carbonate more accessible to the protein ligands in Ur(CO3)34 than in Ur(CO3)22, thus, favoring ternary complex formation with the C-Lobe of the protein? This question is difficult to answer, because it would be more logical to assume that TCUrZ intermediate complex formation is more favored with Ur(CO3)22 (eq 50). TC þ UrðCO3 Þ2 2 hTC UrZ þ Hþ

ð50Þ

Nevertheless, the dependence of the reciprocal relaxation times on [HCO3] clearly indicates that this is not the case (Table 1, eq 6 and reciprocal relaxation eq 8). However, complex formation with the C-lobe of T in interaction with CO32 usually leads to proton losses from the phenol ligands of the two tyrosines (Table 1, eqs 2831).41,50,53 The Ka of these acid base reactions are in the 10 nM range for iron, gallium, and cobalt.11,12,16,41 If we assume that the proton involved is provided by one of these ligands, the transfer of Ur(CO3)22 from Ur(CO3)34 to the C-lobe, by comparison with other metal uptakes, can schematically be expressed as eqs 51 and

TC þ UrðCO3 Þ3 4 hTC UrZ0 þ CO3 2

ð51Þ

TC UrZ þ Hþ hTC UrZ0

ð52Þ

with K3a = [TCUrZ][Hþ]/[TCUrZ0 ] ≈ 510 nM, and K4 = [TCUrZ0 ][CO32]/[TC][Ur(CO3)34]. It can be easily shown that K4 = [TcUrZ0 ][HCO3]K1a/ [TC][Hþ][Ur(CO3)34] = [TcUrZ]K1a[HCO3]/[TC][Ur(CO3)34]K3a = K1K1a/K3a ∼ 6.3  105 to 1.3  104. This value implies that under typical experimental condition, e.g., 20 mM [HCO3], pH 7.4, at the end of the first kinetic process (Figure 2A) 1020% of TC will be loaded with Ur. Furthermore, under these same typical conditions, K3K2 = [TCUr] [HCO3]/[TCUrZ] ≈ 0.04 M reported for the second kinetic process (Table 1, eqs 19, 20) allow to write that at the end of this process and before the beginning of the third one (Figure 2B), [TCUr] = c0/(1 þ [HCO3]/1.5). This implies that 7080% of the C-lobe of T is loaded with Ur. The final conformational changes (Table 1, eqs 23, 24) would lead to an increase in the affinity of T for Ur up to 10121013 where both lobes will be saturated with Ur.18 Uranyl and the Iron Acquisition Pathway. Apotransferrin is in the so-called open structure, in which the ligands are in direct contact with the bulk. When loaded with metals other than aluminum or uranium, the protein becomes in the so-called closed structure, in which the metal is complexed to four protein ligands: two phenolates of two tyrosines, the imidazole of a histidine, the carboxylate of an aspartate, and finally a synergistic carbonate adjacent to an arginine.8,9,13,19,24 The Ur molecule is large as compared to other metallic cations. It is coordinated in T to two tyrosines, one aspartate, and one carbonate. This excludes the histidine and leads to an assumed semiclosed structure of the Ur-loaded lobe, in which Ur is partially in contact with the bulk.19,37This can explain the differences that may occur between the mechanisms of Ur uptake by T and those of other metals.13 This semiclosed structure is also reported for Al(III)-loaded T, despite the rather small size of this metal24 It is also considered responsible for the lack of recognition of the aluminum-loaded protein by receptor 1.15,24 However, the Ur-loaded T is assumed to possess a high chemical reactivity as both an electrophile and a nucleophile, thus facilitating binding to different receptors and entry into a number of target organs and the blood-brain barrier.19 Although there is a discrepancy in the literature between the affinity constants of transferrin for Ur, it seems clear that this still remains several orders of magnitude higher than those reported for other plasma-circulating proteins such as albumin, or metalloproteins, such as metallothionein, apoequine, and ferritin18,22,49 This implies that transferrin is a primary target for Ur. There are several interesting aspects in the mechanism proposed here (Table 1). The first concerns the transfer of Ur from a very stable complex, which is probably one of the forms in which Ur is solubilized in the plasma.20 The second concerns the rapidity of this transfer, at least to the C-lobe of transferrin (Table 1, eqs 6, 19, 20). Finally, the affinities involved seem quite high, as that of second intermediate ternary complex formation is of the same order of magnitude as that of Ur(CO3)22 to CO32 (Table 1, eqs 5, 6, 19, 20). This implies that, in vitro, at uranium concentrations near those found in the plasma (∼15 ng/L),56 the concentration of the available iron-free transferrin (3040 μM), along with that of [CO32] (∼10 μM) and the affinities of both 4213

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The Journal of Physical Chemistry B ligands for Ur, can imply that 2040% of the circulating uranium is in the TUr complex form,36,37 whereas the rest is complexed to carbonate. Is this concentration sufficient to trigger uranium delivery by receptor-mediated endocytosis? We have shown recently that, despite its semiclosed structure, the TUr complex is very rapidly recognized by receptor 1 with a dissociation constant of 6 μM (Table 1, eqs 39, 40, 42, 43).6,25 This implies that uranium can be delivered by the iron-acquisition pathway with, however, a low probability. This was shown in K562 cell lines, where Ur is not easily transported by the iron-acquisition pathway.36,37 On the other hand, it was also shown that despite the high affinity of TFe2 for the receptor, metal delivery to the cytosol by the iron-acquisition pathway is always possible.13 Indeed, only 40% of transferrin is iron-saturated in the bloodstream and iron acquisition occurs mainly with the receptor interacting with the C-lobe of the metal-loaded protein.5 In this case a competition between TUr2 and TFe2 toward the interaction with the receptor becomes possible at high uranium concentrations. Indeed, the affinity of the C-lobe of TFe2 for the receptor is less than 2 orders of magnitude higher than that of the same lobe of TUr2.25 In the case of exposure to uranium, the Ur concentration in blood can grow considerably.57,58 This increases the concentration of Ur-loaded T and, thereby, the probability of an interaction with receptor 1 and delivery by the iron-acquisition pathway. On the other hand, one of the reviewers brought our attention to the possible internalization by receptor-mediated endocytosis of a mixed-metal transferrin, in which the C-Lobe is iron-loaded and the N-lobe uranyl-loaded. The interaction of such a transferrin with transferrin receptor 1 is not proven and would, if it occurs, probably favor uranium transport by the iron-acquisition pathway. This, of course, requires further investigations.

’ CONCLUSION In vitro, Ur is distributed between T, and the tri- and the dicarbonate complex. The first steps in Ur transport from the tricarbonate complex to the C-lobe of T are extremely fast, as they occur in less than 1 s. On the other hand, the interaction of the Ur-loaded C-lobe of T with receptor 1 is also a very fast process occurring in the milliseconds range. This implies that, if this interaction were to occur in vivo, in the case of exposure to uranium, the concentration of the Ur-loaded T will remain in a steady state. This can favor the interaction with the receptor, which in this case can be modulated by the overall concentrations of the Ur tricarbonate and dicarbonate complexes. ’ ASSOCIATED CONTENT

bS

Supporting Information. Information dealing with the derivations of eqs 18 and 21. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (J.M.E.H.C.); Hemadi@ univ-paris-diderot.fr (M.H.). Tel: 33157277238 (J.M.E.H.C.); 33157278839 (M.H.). Fax: 33157277263.

’ ACKNOWLEDGMENT The authors are grateful to Professor Remi Losno for the ICP measurements as well as to Dr. John S. Lomas for helpful discussions.

ARTICLE

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