Journal of Inorganic Biochemistry 104 (2010) 718–725

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Journal of Inorganic Biochemistry

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Complexation of U(VI) with highly phosphorylated protein, phosvitinA vibrational spectroscopic approach

Bo Li, Johannes Raff, Astrid Barkleit, Gert Bernhard, Harald Foerstendorf ⁎Institute of Radiochemistry, Forschungszentrum Dresden-Rossendorf, P.O. Box 510119, 01314 Dresden, Germany

⁎ Corresponding author. Tel.: +49 351 260 3664; faxE-mail address: foersten@fzd.de (H. Foerstendorf).

0162-0134/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.jinorgbio.2010.03.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 November 2009Received in revised form 11 February 2010Accepted 9 March 2010Available online 20 March 2010

Keywords:PhosvitinUranium(VI)ComplexationATR FT-IRInfrared spectroscopy

The complexation of uranium(VI) to variant functional groups of the highly phosphorylated proteinphosvitin in aqueous solution was investigated by attenuated total reflection Fourier transform infrared (ATRFT-IR) spectroscopy. For the verification of the affinity of the actinyl ions to carboxyl and phosphate groups ofthe amino acid side chains, samples with different phosphate to uranium(VI) (P/U) ratios were investigatedunder denaturing conditions as well as in aqueous medium. From a comparative study with other heavymetal ions, i.e. Ba2+ and Pb2+, a strong coordination of U(VI) to carboxyl and phosphoryl groups can bederived. Furthermore, with increasing P/U ratios, a preferential binding of U(VI) to phosphoryl groups isindicated by the spectra of the batch samples. These findings are confirmed by spectra of aqueous U(VI)–phosvitin complexes reflecting an explicit coordination of the uranyl ions to phosphate groups at a high P/Uratio. Our study provides a deeper insight into the molecular interactions between actinyl ions and protein,and can be conferred to other basic biomolecules such as polysaccharides and nucleic acids.

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1. Introduction

Uranium (U) is the heaviest naturally occurring earth element. Thetoxicity of this heavymetal is of both chemical and radiological effects.Under environmental relevant conditions, its chemotoxicity is moresignificant because of its long half life [1]. Because uranium has beenwidely distributed in the environment due to human activities,ranging from uranium mining to storage of nuclear waste ininadequate depositories, it has become necessary to evaluate theimpact of the radionuclide on human health issues. This comprises, forinstance, the assessment of contamination of drinking water, anddefining the adequate limit values as it is currently discussedworldwide [1,2]. To appropriately define such values, it is importantto understand the transportation mechanisms of uranium in livingorganisms at a molecular level. In particular, because of the highsolubility of hexavalent U(VI), that is the uranyl ion (UO2

2+), inaqueous medium, a detailed knowledge of its complexes withbioligand is important.

Extensive research on uranium toxicity has been carried out inthe fields of acute and chronically animal and clinical studies [3,4]. Inaddition, the toxicity effects of uranium on blood transportation [5],kidney [6,7], bones [8], lung [9], skin, reproduction system [10], or inthe embryo development [11] have been recently studied at acellular level. Investigations were carried out to study the impact of

uranium on the local biodiversity due to contamination of theenvironment [12,13]. Also, the microbial biomass/community caninfluence the uranyl species in nature [14].

Proteins hold a key position in any metabolism. Therefore, it isimportant to analyze complexation of U(VI)with protein at amolecularlevel. Frombacterial isolates of U(VI) contaminated sites it is known thattheseorganisms are capable to accumulate high amountsof heavymetalions by S-layer proteins at the cell surfaces. Several U(VI) complexationstudies were carried out in vitro demonstrating the binding of theactinide ionsmainly to carboxylic functional groups [15,16]. In addition,the exposure of a number of metalloproteins to U(VI), such astransferrin, ferritin, and mutated NikR from E. coli were investigated[17–19]. In the case of transferrin and site-directedmodifiedNikR, U(VI)binds specifically in the metal binding pocket, where oxygen fromtyrosine or carboxylic side chains mainly coordinate with U(VI),respectively [17–19]. In the case of ferritin, the protein provides aglobular space for the U(VI) crystals [20].

Apart from carboxylic groups and appropriate binding sites, organicphosphate groups are predestined to form actinidemolecule complexesat a physiological relevant pH level. In particular, since it is estimatedthat 1/3 of the proteins in cells are phosphorylated in order to acquiretheir physiological function properly, such as, regulation of genetranscription, signal transduction throughout the cell, regulation ofenzyme activities, or transport targeting [21,22], a complexation ofphosphate groups with heavy metal ion will lead to a direct damage ofprotein function. Therefore, the study on the complexation is of greatsignificance. From recent investigations, high affinity of U(VI) to organicphosphoester groups is assumed [23]. Unfortunately, the low

719B. Li et al. / Journal of Inorganic Biochemistry 104 (2010) 718–725

concentration of these functional groups in proteins hampers theunequivocal identification of U(VI)–phosphate complexation whichmakes it difficult to verify the high affinity of uranyl ions to organicphosphate groups by means of spectroscopic methods.

Phosvitin, from egg yolk, is a slightly glycosylated, highly watersoluble protein with a molecular mass of 34 kDa [24]. As 123 out of its216 amino acids are serine and most are phosphorylated, 8–10% of themolecular weight composes from the phosphorus, which makes it oneof themost phophorylated proteins in nature [24,25]. Besides its kinasecellular function [26], phosvitin is also known to provide differentfunctional groups to different bivalent and trivalent metal ions. It hasbeen shown that histidine residues of the phosvitin coordinates Cu(II)ions [27], butmainly phosphate groups are complexed by Al(III) [28], Fe(III) [29], Co(II), Mn(II), Ca(II), and Mg(II) due to electrostaticinteractions [30]. With respect to these facts, it became an ideal modelcompound to study the complexation of actinyl ions with organicphosphate residues of proteins. Therefore, it was chosen for theinvestigation of U(VI) complexation at low environmentally relevantconcentrations, where no immediate denaturation occurs. It has beenshown previously that the molecular interactions between proteinfunctional groups and U(VI) can be particularly investigated in situ byvibrational spectroscopy using the attenuated total reflection Fouriertransform infrared (ATR FT-IR) technique [31].With thiswork, the basicknowledge of the interactions between radionuclides and fundamentalbiomolecular components, such as proteins, polysaccharides, nucleicacids, are expanded. It is fundamental for a deeper understanding of thetransportation mechanisms of heavy metal ions in organisms.

2. Experimental

2.1. Materials

UO2Cl2 stock solution (200 mM; pH 1) was prepared following theprotocol as it is already described [32]. Phosvitin was purchased fromSigma-Aldrich, Germany, and was used without further treatment.NaOH, 2 N HCl solution, NaClO4·H2O, and BaCl2·H2O used in the batchexperiments were purchased from Merck, Germany, and were all ofp.a. degree. PbCl2 was purchased from Sigma-Aldrich, Germany, withthe purity of 99.999%.

In the titration experiment, 0.1 M HClO4 was purchased fromMerck, suprapure, and the exact concentration was analyzed withstandardized NaOH, which was purchased from Merck, Titrisol. Thestandard buffers (NIST; pH 4.01 and 6.86) used in the electrodecalibration were purchased from Schott.

2.2. Potentiometric titration

The titration experiments were carried out under CO2 exclusion.The samples were prepared in a glove box under inert gas atmosphere(nitrogen) and then transported immediately in a closed titrationvessel to an automatic titrator (736 GP Titrino, Metrohm). Thedynamic titration procedure was conducted at CO2 free conditionunder inert gas atmosphere (nitrogen bubbling) at 25±1 °C and aconstant ionic strength of 0.1 M (NaClO4). The minimum drift was setto 0.5 mV/min, and a delay time of at least 60 s at each titration pointwas maintained before measuring the pH.

Two potentiometric titrations, comprising about 85 experimentalpoints each, were carried out. In each case, 10 mg phosvitin wasdissolved in 27 mL deionized and carbonate-free water and acidifiedwith 3 mL 0.1 M HClO4 to obtain a starting pH of about 2. The titrationwas performed from pH 2 to pH 12 using 0.1 M NaOH. The pH valuewasmeasured by BlueLine16 pH electrode (Schott). The electrodewascalibrated, for each experiment, with NBS buffers. Because theelectrode was calibrated only up to neutral pH, the determined pKa

value in the alkaline range serves only as an estimation. The sampleswere titrated with an automatic titrator (736 GP Titrino, Metrohm)

and monitored by the accompanying software (TiNet 2.50). The pKa

values and site densities were calculated with the software HYPER-QUAD2006 [33].

2.3. Batch experiments under denaturing conditions

All samples were prepared under ambient conditions. Phosvitinstock solutions were prepared by dissolving the protein in MilliQwater to reach a concentration of 10 mg/mL. The stock solution wasfrozen and kept at −20 °C. All complexation experiments wereconducted at pH 4. At this pH, aqueous solutions of U(VI) aresufficiently stable and the formation of polymeric hydrolysis productsis prevented. Furthermore, it can be shown that the molecularstructure of the protein is also not affected at this pH (see Section 3.1).The pH value of the solutions was adjusted with NaOH and HClsolutions. For the thermal denaturation, 5 mg/mL phosvitin at pH 4was incubated in 80 °C water bath for 1 h. For the phosvitin metalbinding batch experiment, 5 mg of phosvitin was incubated withvarious concentration of UO2Cl2 (10−3 M/10−4 M), 10−4 M BaCl2, or10−4 M PbCl2, in 50 mL aqueous solution with 0.1 M NaCl, at pH 4 fortwo days on a horizontal shaker (Promax 2020 Heidolph Instruments,Germany) at room temperature under normal atmosphere. Threeparallel samples were prepared for each experimental setup. Aftertwo days, the solutions were centrifuged (Optima XL-100K, BeckmanCoulter) at 6000 g for 1 h at room temperature and the pellet ofphosvitin–U(VI) complex was rinsed twice with 0.1 M NaCl at pH 4(blank solution), then followed by ATR FT-IR measurements. Theprotein concentration of the supernatant was determined usingLowry's method [34]. The U(VI) concentrations in the supernatantweremeasured by using inductively-coupled-plasmamass-spectrom-etry (ICP-MS; ELAN 9000, Perkin Elmer SCIEX). These values werefurther used to calculate the protein U(VI) binding capacity.

2.4. Preparation of the soluble phosvitin–U(VI) complexes

U(VI) concentration in the solution was fixed to 10−5 M, and allthe solutionswere prepared in 50 mLwith 0.1 MNaCl. The amounts ofphosvitin protein added to the solution were 0.048 mg, 0.155 mg,0.665 mg, and 4.845 mg resulting in phosphate/U(VI) ratios of 0.3:1,1:1, 4:1, and 30:1, respectively, based on 104 phosphate groupsderived from the titration experiment. The pH value of the solutionswas titrated to pH 4 and samples were shaken at room temperaturefor 1 h to facilitate complexation. Before the IR experiments, the pHvalues were verified and readjusted to pH 4 if necessary. All thesolutions were freshly prepared and the complex stayed soluble overat least one day.

2.5. ATR FT-IR spectroscopy

Single beam ATR FT-IR spectra were recorded in the range between4000 and 600 cm−1 on a Vertex 80/v vacuum spectrometer (Bruker)equipped with a mercury cadmium telluride (MCT) detector. Spectralresolution was 4 cm−1 and each single beam spectrum was averagedfrom 256 scans. The used ATR accessory was a horizontal diamondcrystal with 9 internal reflections (DURA SamplIR II, Smiths). The ATRcellwaspurgedwitha currentof dryair (dewpointb213 K). The spectraof the native phosvitin protein at pH 4 and 8 and of the batch sampleswere subtracted by spectra of thepure solvent. For themeasurements ofthe batch samples, the fresh wet pellets were deposited directly on theATR crystal.

Because of the low concentrations of the aqueous U(VI) solutions,the small spectral changes become only observable when differencespectra were calculated from spectra of the phosvitin–U(VI) complexesand the pure phosvitin solutions. It was found that an accuratesubtraction of the spectra can only be achieved by using a flow cell ontheATRdevicewhich allows a fast exchange of the sample volumeusing

720 B. Li et al. / Journal of Inorganic Biochemistry 104 (2010) 718–725

syringes and a subsequent recording of spectra of the solutions underidentical conditions,which are constantprotein concentration, pHvalueand ionic strength. Otherwise, the subtraction of the spectra mightgenerate strong baseline drifts interfering with the small absorptionchanges in the spectra of the solutions. The software OPUS6.0 (Bruker)was used for spectra acquisition and data evaluation. The differencespectra were calculated from 4 single beam spectra for the aqueoussolutions containing no less than 10−4 Mmetal ions, and from 16 singlespectra for the solutions containing less than 10−4 M metal ions toreduce the background noise.

3. Results and discussion

3.1. Potentiometric titration of phosvitin in aqueous solution

The potentiometric titration provides an accurate determination ofthe pKa values of functional groups, also for macromolecules.

The data of the phosvitin protein titration were analyzed based onthe deprotonation reaction of discrete acid.

R� AiHnX R� An−1

i + H + R = Proteinbackbone;n≥0 ð1Þ

The corresponding proton binding constant Ka can be written as

Ka =R�An−1

i

h iH +h i

R�AiHn½ � ð2Þ

[R-Ain− 1] and [R-AiH

n] represent the concentrations of thedeprotonated and protonated form of the functional group Ai,respectively, and [H+] is the proton concentration in the solution.

The following reactions can be considered to take place in thephosvitin solution:

R� COOH X R� COO� + H + ð3Þ

R� OPO3H2 X R� OPO3H� + H + ð4Þ

R� OPO3H�X R� OPO2�

3 + H + ð5Þ

R� NH +3 X R� NH2 + H +

: ð6Þ

The titration results are summarized in Table 1. The pKa ofreaction (5), that is the deprotonation of hydrogen phosphate ester tophosphate ester, is determined to be 6.01±0.04. This result is inagreement with the estimation provided by results from ATR FT-IRexperiments in aqueous solution [35]. The site density of thephosphate groups is 103.7±1.4 mol/mol protein resulting in aphosphorus content of the protein of 9.5% (w/w) which is withinthe range of 8–10% provided by Sigma-Aldrich.

The pKa value of amino groups is 9.1±0.3, with a site density of18.7±1.7 mol/mol protein. Although this value can only serve as anestimation due to insufficient calibration of the pH electrode in the

Table 1The pKa values of the native phosvitin protein (34 kDa) determined by potentiometrictitration.

Functional group pKaa Site densitya

[mol/mol protein]Content[mol/mol protein]

Phosphoryl (–OPO3H−) 6.01±0.04 103.7±1.4 113 (±∼10)bCarboxyl (–CO2H)Phosphoryl (–OPO3H2)

2.60±0.20 n.d. 28 (±2)c

Amino (–NH2) 9.10±0.30 18.7±1.7 19c

a Determined by HYPERQUAD 2006.b Calculated from phosphorus content (10% w.t.).c From protein sequence.

alkaline pH region, it is in excellent agreement with the number ofamino groups derived from the protein sequence.

Another pKa at around 2.6±0.2 was obtained but only when thetwo previous calculated pKa values at 6.0 and 9.1 were given. The sitedensity of this pKa is very small and could not be determined exactly.This pKa value can be assigned to carboxyl (3) as well as to dihydrogenphosphate groups (4). Although the pKa values of carboxylic acid inproteins are generally found around 4–5 [36], there are exceptions,where low pKa values of carboxyl groups were determined, e.g. sialicacid residues and S-layer protein [37]. It has to be noted that,according to product information (Sigma-Aldrich), phosvitin does notundergo significant conformational changes above pH 2 [38].

In the pH range from 4 to 5, the pKa values of carboxyl groups areobviously covered by the phosphate groups, because of the four foldhigher content in the phosvitin molecule (Table 1). In fact, thepresence of carboxyl groups showing pKa values in the ambient pHrange is demonstrated by the IR measurements presented in thefollowing section.

3.2. IR characterization of phosvitin in aqueous solution

Based on the pKa value of the phosphate groups, IR spectra ofphosvitin solution at pH 4 (Fig. 1a) and pH 8 (Fig. 1b) were recorded.From the spectra, the pattern of band is expected to represent mainlythe two protonation states of the phosphate groups, namely hydrogenphosphate ester (ROPO3H−) andphosphate ester groups (ROPO3

2−) (seeTable 1). In order to demonstrate the spectral change occurring duringthe deprotonation, a difference spectrum was calculated (Fig. 1c).

Vibrational modes from both functional groups of the protein sidechains and the protein backbone have been identified from the spectra(pH 4 and pH 8, Fig. 1a,b). Bands at 1658 cm−1 and 1546 cm−1 reflectthe amide-I and amide-II modes of the peptide bonds from the proteinbackbone. The region from 1240 cm−1 to 900 cm−1 mainly shows thevibrational modes of phosphoryl groups. The bands at 1180 cm−1,1080 cm−1, and 936 cm−1 in the spectrum recorded at pH 4 areassigned to νas(P–O), νs(P–O), and νas(P–OH), respectively [39]. Thischaracteristic band pattern obviously represents organic phosphategroups at lowpH levels showingC2ν symmetry [40]. From the symmetryit can be assumed that the monoanionic species, ROPO3H−, is thedominant species present at pH 4.

At pH 8, the peaks described abovemerge into two bands at position1094 cm−1 and 982 cm−1, which are assigned toνas(P–O) and νs(P–O),respectively (Fig. 1b). The change of this band pattern reflects thechange of the symmetry of the organic phosphate residues upondeprotonation, where ROPO3

2− becomes the dominant species at higherpH values. It confirms the results from potentiometric titration.

The calculated difference IR spectrum pH 8 minus pH 4 (pH 8−4,Fig. 1c) provides further information on the spectral changes occurring

Fig. 1. FT-IR spectra of phosvitin in aqueous solution (0.1 M NaCl). Spectra recorded atpH 4 (a) and pH 8 (b). Difference spectrum pH 8 minus pH 4 (c).

721B. Li et al. / Journal of Inorganic Biochemistry 104 (2010) 718–725

uponpHchange. In thedifference spectrumpositive andnegative bandsrepresent the phosvitin molecule at pH 8 and 4, respectively. From thetitration experiment, mainly contributions from phosphoryl groups areexpected, since the pKa values of other functional groups, e.g. carboxyland amino groups, were determined to be beyond this pH range.Contributions from vibrational modes of the sugar rings can beneglected since they do not change significantly in this pH range [40].In the region below 1200 cm−1, the distinctive spectral features at1192 cm−1 (negative), 1110 cm−1, 1046 cm−1 (negative), 984 cm−1,and 931 cm−1 (negative) represent the deprotonation of the phosphategroups, which set up an important spectral reference for the furtherstudy of U(VI) complexation.

However, there are small spectral features indicating a change of theprotonation state of carboxyl groups (Fig. 1c). The small negative bandat 1724 cm−1 and the positive bands at 1569 cm−1 and 1402 cm−1 canbe assigned to the ν(C O) mode of protonated carboxyl groups and tothe νas(COO−) and νs(COO−) modes of carboxylate groups, respective-ly. The appearance of these bands indicate that at pHN4 protons are stillgradually released from carboxyl groups, although the pKa value wasdetermined to be around 2.6.

From the splitting of the ν(COO−) modes in infrared spectra, thetype of complexation with metal ions can be derived [41]. With respectto the prevailing conditions, the observed splitting of these two modesin the difference spectrum of approximately 170 cm−1 representsuncomplexed carboxylate groups. And it is in good agreement withprevious studies [31,42].

3.3. IR spectra of batch samples

The performance of spectroscopic investigation of U(VI) complex-ation with the protein phosvitin requires the knowledge about thebinding capacity of U(VI) of the protein. For this reason, batchexperiments were carried with initial U(VI) concentrations of 10−3

and 10−4 Mwhichwere sufficient to provide denaturing conditions atthe prevailing protein concentrations. From the batch experimentwith an initial U(VI) concentration of 10−3 M, it was found that83.3 mol U(VI) per mol protein was bound (Table 2). Assuming thatonly phosphate and carboxylate groups (Table 1) are available forcomplexation of U(VI) under the prevailing conditions, the bindingcapacity of phosvitin can be calculated to be 63%. At an initial U(VI)concentration of 10−4 M, the amount of U(VI) bound to phosvitindecreased to 9.7 mol U(VI) per mol protein corresponding to abinding capacity of about 7% which approximately correlates with thedecreasing of the initial U(VI) concentration by a factor of ten.

From these data, an identification of the functional groups whichpreferably bind the uranyl ions remains difficult because there is no

Table 2Overview of the U(VI) batch experiments, and the stoichiometry of the aqueousphosvitin–U(VI) solutions.

U(VI) batch experimentsa

Fig. 2d Fig. 2e

Initial U(VI) concentration [mol/L] 10−3 10−4

Initial ratio phosphate/U(VI) 0.3:1 3.3:1Initial ratio carboxyl/U(VI) 0.09:1 1.2:1Amount of complexed U(VI) [mol/mol protein] 83.3 9.7U(VI) binding capacity (%) 63.1b 7.3b

Rel. amount of complexed U(VI) (%) 25 29

Stoichiometries of the aqueous phosvitin–U(VI) solutionsc

Fig. 3a Fig. 3b Fig. 3c Fig. 3d

Initial ratio phosphate/U(VI) 0.3:1 1:1 4:1 30:1Initial ratio carboxyl/U(VI) 0.09:1 0.36:1 1.5:1 11:1

a Denaturing conditions.b Normalized to available phosphate and carboxyl groups.c Initial U(VI) concentration: 10−5 M.

correlation between the number of the potentially available bindingfunctional groups, that is phosphoryl and carboxyl groups, and theamount of U(VI) bound to phosvitin. Therefore, the precipitated U(VI)complexes were analyzed by FT-IR spectroscopy. From the spectra, itis expected to gain a first insight in the complexation behavior of theuranyl ions to phosvitin by the characteristic frequencies of thecarboxyl, phosphoryl and uranyl functional groups.

In analogy to the U(VI) samples, precipitates of phosvitin, whichwere denatured by adding Ba2+ or Pb2+ ions and by thermal treatment,were prepared and analyzed by FT-IR spectroscopy for comparison. Forall these batch samples, the Lowry protein concentration determinationrevealed that no protein was found in the supernatant after the lastcentrifugation step. It means that nearly 100% phosvitin is precipitated.

The respective infrared spectra are shown in Fig. 2a–e in thespectral range between 1600 and 850 cm−1. A spectrum of blanksolution was subtracted from all spectra in the figure as backgroundcorrection. Table 3 presents an overview of the frequencies of themost prominent band maxima observed in the spectra of this work.Additionally, a tentative assignment to vibrational modes is given. Theupper part of the table displays the bands of the absorption spectra,that is the spectra of the aqueous phosvitin solutions at pH 4 and 8 andthe spectra of the batch samples. In the lower part of the table thefrequencies of the difference bands of the aqueous phosvitin–U(VI)complexes and the difference spectrum of Fig. 1c are listed.

The overall shape of the spectra of the samples without U(VI) arevery similar (Fig. 2a–c). In these spectra, the most significant bandsare observed around 1550, 1400, 1084, and 1005 cm−1. The first bandcan be assigned to the amide-II mode of the protein backbonewhereas the band at 1400 cm−1 is attributed to the νs(COO−) modeof the carboxylic side chains according to the assignment derived fromthe difference spectrum shown in Fig. 1c.

The broad bands around 1084 cm−1 each showing a band withreduced intensity around 1005 cm−1 are most likely to represent thenumerous phosphate groups of the phosvitin protein. A similar pattern ofbands is observed in the spectrum of the aqueous phosvitin samplerecorded at pH8 (Fig. 1b) andwere assigned to theνas(P–O) andνs(P–O)modes of the phosphoryl groups, suggesting a similar symmetry ofdianionic phosphate ester in theses samples. It has to be noted that theappearance of vibrational modes of functional groups in infrared spectraismainly due to reasons ofmolecular symmetrywhereas the frequenciesof their maxima might be significantly affected by the molecularenvironment. Therefore, the spectral analogy of the spectra indicates

Fig. 2. FT-IR spectra of denatured phosvitin samples. Phosvitin after denaturation bythermal treatment (a), adding 0.1 mM Ba2+ (b), 0.1 mM Pb2+ (c), 1 mM UO2

2+ (d), and0.1 mM UO2

2+ (e).

Table 3Overview and tentative assignment of the infrared bands observed in the infrared spectra. Positive and negative bands of difference spectra are marked as (+) and (−), respectively.Indicated values are given in cm−1.

Absorption spectra Tentativeassignment

Phosvitin aq. Phosvitin batch samples

pH 4 pH 8 Therm.denat.a

Ba(II)a Pb(II)a [U(VI)]init10−3 Ma

[U(VI)]init10−4 Ma

Fig. 1a Fig. 1b Fig. 2a Fig. 2b Fig. 2c Fig. 2d Fig. 2e

1658 1658 Amide-I1546 1546 1550 1550 1550 1525 1536 Amide-II/νas(COO−)

1400 1400 1400 1463 1463 νs(COO−)1230 ν(P O)1180 νas(P–O)

1094 1084 1084 1084 1084 1084 νas(P–O)1080 νs(P–O)

982 ∼1005 1005 1005 1020 1020 νs(P–O)936 νs(P–OH)– – – – – 925 916 νas(UO2)

Difference spectra of aqueous solutions

Phosvitin Phosvitin-U(VI) minus phosvitin aq.; pH 4

pH 8−pH 4 0.3:1b 1:1b 4:1b 30:1b

Fig. 1c Fig. 3a Fig. 3b Fig. 3c Fig. 3d

1724(−) ν(COOH)1569(+) νas(COO−)

1530(+) 1530(+) 1530(+) Amide-II/νas(COO−)1402(+) 1458(+) 1458(+) 1458(+) 1458(+) νs(COO−)1192(−) 1198(−) 1198(−) 1198(−) 1198(−) νas(P–O)1110(+) 1102(+) 1115(+) 1115(+) 1102/1130(+) νas(P–O)1046(−) νs(P–O)984(+) 1020(+) 1020(+) 1020(+) – νs(P–O)931(−) νs(P–O)– 925(+) 925/918(+) 918(+) 905(+) νas(UO2)

a Phosvitin after denaturation by thermal treatment, adding Ba(II), Pb(II) and U(VI) as given in caption of Fig. 2.b U(VI)–phosvitin complexes at different initial phosphate/U(VI) ratios; [U(VI)]=10−5 M.

722 B. Li et al. / Journal of Inorganic Biochemistry 104 (2010) 718–725

that the phosphate groups are deprotonated upon the denaturationprocess.

The spectra of the U(VI) containing samples (Fig. 2d,e) show smallbut distinct deviations from the previous spectra. The band at1005 cm−1 has shifted to higher wavenumber and is observed around1020 cm−1 in the spectra of the U(VI)–phosvitin precipitatesindicating the interactions of the uranyl ion with phosphoryl groups.

Furthermore, there are additional bands around 920 cm−1 (Fig. 2d,e)which are attributed to the antisymmetric stretching vibration of theuranyl ion, ν3(UO2) [42–44]. The absence of similar bands in the spectraof the other denatured protein samples (Fig. 2a–c) provides strongevidence for the assignment of these bands to the νas(UO2) mode.

The frequency of themaximumof this band is observed at 925 cm−1

(Fig. 2d) and is slightly bathochromically shifted to 916 cm−1 upondecreasing the initial U(VI) concentration used for denaturation by afactor of ten (Fig. 2e). Since this band shift was highly reproducible aftertheperformanceof several batch reactionsunder identical experimentalconditions, a change of the complexed actinide species with decreasingthe initial U(VI) concentration can be derived. The most prominentfunctional groups of the phosvitin, which can be regarded as relevantbinding sites for the actinide cations, aremainly carboxyl andphosphateresidues in the investigated pH range. Therefore, the shifting of the bandat different U(VI)/phosphate ratios can be explained by the formation ofdifferent complex species.

It has to be noted that the characteristic vibrational frequency of theuranyl band in a binary complex where the uranyl ion is coordinated toan organic phosphate group is difficult to be estimated, since there areno vibrational spectroscopic data available from vibrational spectro-scopicmodel investigations. In contrast, the complexationof uranyl ionswith monocarboxylic ligands was shown to generate uranyl bands atfrequencies above 920 cm−1 [31,42,45]. Consequently, from the slight

red-shift of the uranyl band to 916 cm−1 at lower U(VI)/phosphateratios observed in the spectrum of the lower initial U(VI) concentration(Fig. 2e), it can be suggested that contributions from U(VI)–phosphatespecies might become predominant.

Furthermore, there is an additional feature in the spectra of thebatch samples demonstrating the affinity of the uranyl ions tocarboxyl groups in general. The spectra of the thermally denaturedsample and of the samples containing Ba2+ and Pb2+ ions show aband at 1400 cm−1 (Fig. 2a–c), whereas this band is hardly observedin the spectra of the UO2

2+ complexes (Fig. 2d,e). In the previoussection, a band is assigned at nearly the same frequency to the υsmode of carboxylate groups. From model investigations, it is wellknown that bidendate UO2

2+ complexes with carboxylic functionalgroups generally show a band of the υs(COO−) mode which is shiftedabout 60 cm−1 to higher frequencies [31,42]. Such a band is obviouslyobserved at 1463 cm−1 in the spectrum of the batch sampledenatured with 0.1 mM UO2

2+. The corresponding band of the υas(COO−) mode, which was identified at 1569 cm−1 in the spectrum ofthe dissolved protein (Fig. 1c), cannot be observed because it ishidden under the broad amide-II band around 1550 cm−1 in therespective spectra of the batch samples (Fig. 2).

From the spectra of the batch samples, a different complexationbehavior of Ba2+, Pb2+andUO2

2+ tophosvitin canbederived. Thoughallmetals provoke an unfolding of the phosvitin molecule, only the uranylions are coordinated to carboxyl groups to a significant extent. From thecomparison of the spectra of the batch samples exposed to elevatedtemperature, Ba(II) andPb(II), a significant complexation of the bivalentmetals to carboxyl groups can be ruled out. Although interactions ofthese metal ions with phosphate groups might become of somerelevance for the denaturation process, the breakup of intramolecularinteractions, such as H-bonds, hydrophobic interactions and/or van der

723B. Li et al. / Journal of Inorganic Biochemistry 104 (2010) 718–725

Waals interactions, is suggested to be decisive for the denaturationprocess.

In addition, first indications for a preferential affinity of the uranylions to phosphate groups of the protein's side chains can be derivedfrom the spectra of the batch samples with different initial U(VI)concentrations which will be verified by the series of experiments ofaqueous complexes in the following section.

3.4. IR spectra of the phosvitin–uranyl complexes in aqueous solution

For a detailed understanding of the chemotoxicity of U(VI)compounds under physiologically relevant conditions, it is mandatoryto investigate the molecular mechanisms of the interaction of theuranyl ions in homogeneous aqueous phases. This comprises theverification of the hypothesis of a preferential binding of UO2

2+ ions toorganic phosphate groups on a protein. The alteration of the nativeprotein conformation in solution during the denaturation processes,as it was presented in the previous section, can solely be considered asan exemplary investigation with extreme consequences for theprotein molecule. Consequently, in this section we aim at thespectroscopic characterization of stable phosvitin–U(VI) complexesin aqueous solution, which can only be achieved by a significantreduction of the U(VI) concentration down to 10−5 M. Furthermore,to investigate the complexation at different phosphate/U(VI) ratios,the phosvitin concentration has to be adjusted. From the results, it canbe expected that spectral features are eliminated, which are due to thedenaturation processes, and, thus, do not accurately represent themolecular interaction of the uranyl ion with the functional groups.

Fig. 3 presents the respective spectra of the aqueous complexes atU(VI) concentration of 10−5 M and the resulting P/U ratios. Thepositive and negative bands in the difference spectra represent thephosvitin–U(VI) complexes and the native phosvitin in solution,respectively.

The spectra show bands in three spectral regions above 1450 cm−1,between 1200 and 1000 cm−1, and below 950 cm−1 representing thechanges of the carboxyl and phosphate groups, and the presence of theUO2

2+ ion in the protein complexes, respectively. The abscissa of thelatter spectral region is enlarged for clarity.

In analogy to the batch experiments, the maximum of the bandrepresenting the υ3(UO2) mode is shifted to lower wavenumbers withincreasing P/U ratio. In the spectrum of the complex with lowest P/Uratio (Fig. 3a), the band is observed at 925 cm−1. This is in excellentagreement with the corresponding spectrum of the batch sample,which contains an initial U(VI) concentration of 10−3 M, and sharethe same P/U ratio of 0.3:1 (Fig. 2d and Table 2). The band is slightlyshifted to 918 cm−1 at higher P/U ratios (Fig. 3b,c). Simultaneously,

Fig. 3. FT-IR spectra of aqueous U(VI)–phosvitin complexes at different initialphosphate/U(VI) ratios. 0.3:1 (a), 1:1 (b), 4:1 (c), and 30:1 (d). U(VI) concentrationin all the samples is 10−5 M.

the band shows decreasing intensity at 925 cm−1 whereas a shoulderaround 905 cm−1 appears in the spectrum of the P/U ratio 4:1(Fig. 3c). At a higher P/U ratio of 30, the band has shifted to 905 cm−1

(Fig. 3d).In aqueous solution, the absorption maximum of the fully hydrated

UO22+ ion is generally observed at 961 cm−1 [43,44]. This frequency is

shifted to lowerwavenumbers upon formationof hydrolysis products orbecause of complexation of the actinyl ion with appropriate organic orinorganic ligands [41,46–48]. The electronegative ligands in theequatorial plane of the linear actinyl ionweaken theU–Obond resultingin the bathochromic shift in the infrared spectra. Therefore, thecontinuous shift of the uranyl band with increasing P/U ratio obviouslyreflects an advanced complexation of the uranyl ion.

With respect to the excess of UO22+ ions in the sample of the lowest

P/U ratio (Fig. 3a), the spectral properties of the aqueous species of theuranyl ions under the given conditions have to be considered. Fromour recent investigation of the hydrolysis reaction of U(VI), thespectra of the pure uranyl solution recorded in the pH range betweenpH 3.2 and 5.6 show a band at 923 cm−1 which was tentativelyassigned to the monomeric UO2(OH)20 species [43]. In the spectrum ofthe phosvitin–U(VI) complex at low P/U ratio, the band of the νas(UO2) mode is observed at nearly the same frequencies which makesit difficult to discriminate between the complexed and aqueousspecies in this sample. However, in this spectrum significant bands areobserved representing strong alterations of phosphate groups. Thepositive bands around 1115 and 1020 cm−1 obviously representdeprotonated phosphate groups. From the spectra of the nativephosvitin solutions (Fig. 1), it is demonstrated that the seconddeprotonation step of the phosphate groups generates two differencebands with maxima at 1110 and 984 cm−1 (Fig. 1c) which areassigned to the antisymmetric and symmetric stretching vibrations ofthe phosphoryl groups, respectively. It has to be noted that thesefrequencies are expected in the absence of strong complexing cations.Consequently, the different frequencies of the bands of the phosphategroups observed in the spectrum of the low P/U ratio obviously reflectstrong contributions from the complexation of the uranyl ions withphosphate groups. Moreover, the uranyl band at 925 cm−1 shows aweak shoulder around 905 cm−1 indicating the presence of U(VI)–phosphate interactions which will be discussed in detail later on.

The spectra of the P/U ratios 1:1 and 4:1 evidence the complexationof the uranyl ions to the functional groups of phosvitin without theinterference of the aqueous hydrolysis species of U(VI) (Fig. 3b,c). Inthese spectra, the slight shift to 918 cm−1 and the different shape of theuranyl band indicate a different composition of the uranyl species inthese samples. In particular, at a P/U ratio of 4:1, a shoulder at 905 cm−1

becomes more evident whereas the intensity at 925 cm−1 is signifi-cantly decreased (Fig. 3c). Furthermore, the bands representing thephosphoryl groups are now observed at 1198 cm−1 (negative) and at1115 and 1020 cm−1 (positive). The negative band is assigned to theantisymmetric stretching vibration of the P O bond in protonatedphosphoryl groups, whereas the positive bands again represent theantisymmetric and symmetric stretching vibrations of the complexedphosphoryl groups, respectively. From the difference spectra of nativephosvitin in aqueous solution a similar pattern of the phosphate bandswas observed. The negative band at 1198 cm−1 is present in bothspectra (comp. Figs. 1c and 3b,c) representing a characteristic spectralfeature of the last deprotonation step of the phosphate groups. Thereduced splitting of the positive bands in the difference spectra of theaqueous U(VI) complexes demonstrate the coordination of thephosphate groups to the actinyl ions.

In the spectral region of the carboxyl groups (N1400 cm−1), thespectra show bands at 1530 and 1458 cm−1 which can be assigned tothe υas and υs modes of the complexed carboxylate groups, respectively.The frequencies of these modes shift upon complexation from 1569 to1530 cm−1 for the υas(COO−) mode and from 1402 to 1458 cm−1 forthe υs(COO−) mode. This is in excellent agreement with the results of

Fig. 4. Scheme of possible U(VI) complexes with carboxylic and/or phosphate groups.(a) Bidentate carboxyl U(VI) coordination, (b) monodentate phosphate U(VI)coordination, (c) bidentate phosphate U(VI) coordination, (d) 1:2 monodentatephosphate U(VI) coordination, and (e) complex U(VI) coordination to both carboxyland phosphate group.

724 B. Li et al. / Journal of Inorganic Biochemistry 104 (2010) 718–725

recent model investigations of uranyl complexes with carboxylic acidligands [31,42,45]. However, it has to be noted that the amide-IImodeofthe protein backbone might contribute to the band at 1530 cm−1. Thiscan obviously be observed in the spectrum of the P/U ratio 1:1 (Fig. 3b)where this band is badly resolved and can hardly be observed.

The spectrum of the highest P/U ratio shows great differences in allspectral regions. The maximum of the uranyl band has shifted to905 cm−1 and the band of the carboxylate groups around 1460 cm−1

is not present any more. Consequently, the relatively broad bandaround 1530 cm−1 can be assigned to the amide-II mode of the protein.Furthermore, the band at 1115 cm−1 is considerably broadened and theband of the υs(PO) mode, which showed up at 1020 cm−1 in theprevious spectra, is not observed.

At this high P/U ratio, complexation of the UO22+ ions mostly to

phosphate groups is derived from the red-shifted maximum of theuranyl band at 905 cm−1. Such a strong shift, which is about 60 cm−1

bathochromically to the uncomplexed ion, was not yet reported for auranyl complex with carboxylate ligands. Additionally, the bandrepresenting the υs(COO−) mode is no longer observed indicatingthat no complexation with these type of functional groups occur.Furthermore, since all spectra of the lower P/U ratios show a shoulderat this frequency, the presence of U(VI)–phosphate complexes can bealready assumed under these conditions. However, the pattern of thebands in the spectral phosphate region is different in the spectrum ofthe high P/U ratio. Although only a tentative interpretation of thesefindings can be given according to the knowledge up to date, it isobvious that this different spectral feature represents a different U(VI)complexation as it is found at lower P/U ratios.

Upon complexation with the actinyl ion, the splitting of the phos-phate modes in the difference spectra is decreased from 126 cm−1

(Fig. 1c) to 95 cm−1 (Fig. 3b,c). At high P/U ratio, the splitting seemsto be further decreased resulting in overlapping bands above∼1040 cm−1. The formation of U(VI)–phosphate complexes witha higher coordination number might cause the further decrease ofthis splitting. Another explanation for the spectral alterations is achange of the type of complexes. It can be assumed that the uranyl

ion forms more stable complexes possibly with a bidentatecharacter at a deficient concentration. When the U(VI) concentra-tion increases, the formation of complexes with phosphate groupsshowing a monodentate character and bidentate complexes withcarboxyl groups might become predominant later. Possible func-tional group–U(VI) complexes are proposed in Fig. 4.

3.5. Summary and outlook

The aim of this study is the vibrational spectroscopic investigation ofthe U(VI) complexation to a highly phosphorylated protein. Inparticular, the spectral differentiation of U(VI)–carboxyl and –phos-phate complexes is the major goal of this work.

From the determination of the pKa values of the functional groups,pH 4 was chosen as an adequate experimental parameter for the U(VI)complexation where the phosphate groups appear as hydrogenphosphate ester on the phosvitin. Furthermore, at this pH level aqueousU(VI) solutions are sufficiently stable and the formation of colloids isprevented.

The IR spectra of the batch samples demonstrate that phosphatedeprotonation fromhydrogenphosphate ester to phosphate ester at pH4occurs during the denaturation process of the protein. Moreover, thespectra clearly show that U(VI) is coordinated to carboxyl and phosphategroups of the amino acid side chains,whereas thedenaturationprocessesinduced by heavymetal ions such as Ba(II) and Pb(II) are obviouslymorerelated to the breakup of intramolecular interactions relevant for thenative secondary structure of the protein in aqueous solution.

The spectra of the soluble U(VI) phosvitin complexes clearly showthat U–phosphate complexes become predominant at high P/U ratios.The further shift of the νas(UO2) mode to lower wavenumbers withincreasing P/U ratio suggests the formation of complexes with a highcoordination number of the uranyl ion. Fig. 4 schematically sum-marizes conceivable U(VI) complexes with carboxyl and phosphategroups. The strongest shifts of the νas(UO2) mode are expected to bedue to complexes with a high number of coordinated ligands in theequatorial plane of the UO2

2+ ion (Fig. 4c–e). Consequently, theobserved frequency of the νas(UO2) mode in the spectrum of thehighest P/U ratio (Fig. 3d) obviously represents such U(VI) complexes.With respect to the large number of phosphorylated residues ofphosvitin, interactions of U(VI), where the actinide ion is coordinatedto two phosphate groups as shown in Fig. 4c,d, are most probable.

The formation of oligomeric species as it was found in modelinvestigations with nucleotides such as AMP, namely the so-calledFeldman complex [49], are unlikely because of the low initial U(VI)concentration of 10−5 M where the presence of oligomeric hydrolysisproducts can be ruled out [43]. However, future investigations usingspectroscopic techniques, such as X-ray absorption spectroscopy willprovide complementary structural information of the moleculecomplexes.

The reference spectra provided by phosvitin–U(VI) complex of thisworkwill support the interpretation of the spectroscopic data of furtherinvestigations, and will facilitate the identification of U(VI) molecule inmore complex systems. Moreover, because different U(VI) complexeswere found depending on the functional group/U ratio, futureinvestigations potentially provide additional information of the forma-tion of different U(VI) species under different conditions such asincreased pH value.

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