Strong Uranium(VI) Binding onto Bovine Milk Proteins, Selected Protein Sequences, and Model Peptides.

Hexavalent uranium is ubiquitous in the environment. In view of the chemical and radiochemical toxicity of uranium(VI), a good knowledge of its possible interactions in the environment is crucial. The aim of this work was to identify typical binding and sorption characteristics of uranium(VI) with both the pure bovine milk protein ß-casein and diverse related protein mixtures (caseins, whey proteins). For comparison, selected model peptides representing the amino acid sequence 13-16 of ß-casein and dephosphorylated ß-casein were also studied. Complexation studies using potentiometric titration and time-resolved laser-induced fluorescence spectroscopy revealed that the phosphoryl-containing proteins form uranium(VI) complexes of higher stability than the structure-analog phosphoryl-free proteins. That is in agreement with the sorption experiments showing a significantly higher affinity of caseins toward uranium(VI) in comparison to whey proteins. On the other hand, the total sorption capacity of caseins is lower than that of whey proteins. The discussed binding behavior of milk proteins to uranium(VI) might open up interesting perspectives for sustainable techniques of uranium(VI) removal from aqueous solutions. This was further demonstrated by batch experiments on the removal of uranium(VI) from mineral water samples.

Uranium(VI) Binding Forms in Selected Human Body Fluids: Thermodynamic Calculations versus Spectroscopic Measurements.

Human exposure to uranium increasingly becomes a subject of interest in many scientific disciplines such as environmental medicine, toxicology, and radiation protection. Knowledge about uranium chemical binding forms(speciation) in human body fluids can be of great importance to understand not only its biokinetics but also its relevance in risk assessment and in designing decorporation therapy in the case of accidental overexposure. In this study, thermodynamic calculations of uranium speciation in relevant simulated and original body fluids were compared with spectroscopic data after ex-situ uranium addition. For the first time, experimental data on U(VI) speciation in body fluids (saliva, sweat, urine) was obtained by means of cryogenic time-resolved laser-induced fluorescence spectroscopy (cryo-TRLFS) at 153 K. By using the time dependency of fluorescence decay and the band positions of the emission spectra, various uranyl complexes were demonstrated in the studied samples. The variations of the body fluids in terms of chemical composition, pH, and ionic strength resulted in different binding forms of U(VI). The speciation of U(VI) in saliva and in urine was affected by the presence of bioorganic ligands, whereas in sweat, the distribution depends mainly on inorganic ligands. We also elucidated the role of biological buffers, i.e., phosphate (H(2)PO(4−)/HPO(4)(2−)) on U(VI) distribution, and the system Ca(2+)/UO(2)(2+)/PO(4)(3−) was discussed in detail in both saliva and urine. The theoretical speciation calculations of the main U(VI) species in the investigated body fluids were significantly consistent with the spectroscopic data. Laser fluorescence spectroscopy showed success and reliability for direct determination of U(VI) in such biological matrices with the possibility for further improvement.

Interaction of U(VI) with Schizophyllum commune studied by microscopic and spectroscopic methods.

Biosorption of actinides like uranium by fungal cells can play an important role in the mobilization or immobilization of these elements in nature. Sorption experiments of U(VI) with Schizophyllum commune at different initial uranium concentrations and varying metal speciation showed high uranium sorption capacities in the pH range of 4­7. A combination of high angle annular dark-field and scanning transmission electron microscopy analysis (HAADF-STEM) showed that living mycelium cells accumulate uranium at the cell wall and intracellular. For the first time the fluorescence properties of uranium accumulates were investigated by means of time-resolved laser-induced fluorescence spectroscopy (TRLFS) beside the determination of corresponding structural parameters using X-ray absorption fine structure spectroscopy (EXAFS). While the oxidation state of uranium remained unchanged during sorption, uranium speciation changed significantly. Extra and intracellular phosphate groups are mainly responsible for uranium binding. TRLFS spectra clearly show differences between the emission properties of dissolved species in the initial mineral medium and of uranium species on fungi. The latter were proved to be organic and inorganic uranyl phosphates formed depending on the uranyl initial concentration and in some cases on pH.

Formic acid interaction with the uranyl(VI) ion: structural and photochemical characterization.

Complex formation between the uranyl(VI) ion and formic acid was studied by infrared absorption (IR) and X-ray absorption (EXAFS) spectroscopy as well as density functional theory (DFT) calculations. In contrast to the acetate ion which forms exclusively a bidentate complex with uranyl(VI), the formate ion binds to uranyl(VI) in a unidentate fashion. The photochemistry of the uranyl(VI)-formic acid system was explored by DFT calculations and photoreduction of uranyl(VI) in the presence of formic acid was found to occur via an intermolecular process, that is, hydrogen abstraction from hydrogenformate by the photo-excited uranyl(VI). There is no photo-induced decarboxylation of uranyl(VI) formate via an intramolecular process, presumably due to lack of a C=C double bond.

Investigation of uranium binding forms in selected German mineral waters.

Cryogenic time-resolved laser-induced fluorescence spectroscopy was successfully used to identify uranium binding forms in selected German mineral waters of extremely low uranium concentrations (<2.0 µg/L). The measurements were performed at a low temperature of 153 K. The spectroscopic data showed a prevalence of aquatic species Ca2UO2(CO3)3 in all investigated waters, while other uranyl-carbonate complexes, viz, UO2CO3(aq) and UO2(CO3)2 (2-), only existed as minor species. The pH value, alkalinity (CO3 (2-)), and the main water inorganic constituents, specifically the Ca(2+) concentration, showed a clear influence on uranium speciation. Speciation modeling was performed using the most recent thermodynamic data for aqueous complexes of uranium. The modeling results for the main uranium binding form in the investigated waters indicated a good agreement with the spectroscopy measurements.

The U(VI) speciation influenced by a novel Paenibacillus isolate from Mont Terri Opalinus clay.

Bacterial cell walls have a high density of ionizable functional groups available for U(VI) binding, hence have a great potential to affect the speciation of this contaminant in the environment. The studied strain of the genus Paenibacillus is a novel isolate originating from the Mont Terri Opalinus clay formations (Switzerland) which are currently investigated as a potential host rock for future nuclear waste storage. U(VI) binding to the cell surface functional groups was studied by potentiometry combined with time-resolved laser-induced fluorescence spectroscopy (TRLFS). Four bacterial U(VI) surface complexes were identified: R-COO-UO2(+), R-O-PO3-UO2, R-O-PO3H-UO2(+), and (R-O-PO3)2-UO2(2-). The corresponding complex stability constants were calculated to be 5.33 ± 0.08, 8.89 ± 0.04, 12.92 ± 0.05, and 13.62 ± 0.08, respectively. Hence UO2(2+) displays a moderate to strong interaction with the bacterial surface functional groups. In the acidic pH range (pH 3) UO2(2+) binding onto the cell envelope is governed by coordination to hydrogen phosphoryl sites. Upon increasing the pH an increasing coordination of UO2(2+) to carboxylic and deprotonated phosphoryl sites was found. At a pH greater than 7 uranyl hydroxides dominate the speciation. Additionally the bacteria-mediated release of inorganic phosphate in dependence on [U(VI)] at different pH values was studied to assess the influence of phosphate release on U(VI) mobilization.

Chitin-based renewable materials from marine sponges for uranium adsorption.

Marine sponges of the order Verongida form three-dimensional networks of fibrous chitin, which can easily be extracted. In the hydrated state, these networks are flexible, mechanically stable and can be cut or pressed into any desired form. Here, we show for the first time that chitin-based networks of sponge origin are useful for effective uranium adsorption. They adsorb uranium from solution with a higher adsorption capacity than many other chitinous sorbents. Up to 288 mg/g could be achieved. Solid-state NMR, infrared, and Raman spectroscopy indicated that the uranyl is bound to the chitin by weak interactions. 90% of the uranyl could be desorbed using diluted hydrochloric acid. Uranium adsorption and desorption did not result in any destruction of the chitin-based material.

Insights into the uranium(VI) speciation with Pseudomonas fluorescens on a molecular level.

Microorganisms have great potential to bind and thus transport actinides in the environment. Thus microbes indigenous to designated nuclear waste disposal sites have to be investigated regarding their interaction mechanisms with soluble actinyl ions when assessing the safety of a planned repository. This paper presents results on the pH-dependent sorption of U(VI) onto Pseudomonas fluorescens isolated from the granitic rock aquifers at Äspö Hard Rock Laboratory, Sweden. To characterize the U(VI) interaction on a molecular level, potentiometric titration in combination with time-resolved laser-induced fluorescence spectroscopy (TRLFS) were applied. This paper as a result is one of the very few sources which provide stability constants of U(VI) complexed by cell surface functional groups. In addition the bacteria-mediated liberation of inorganic phosphate in dependence on [U(VI)] at different pHs was studied to judge the influence of phosphate release on U(VI) mobilization. The results demonstrate that in the acidic pH range U(VI) is bound by the cells mainly via protonated phosphoryl and carboxylic sites. The complexation by carboxylic groups can be observed over a wide pH range up to around pH 7. At neutral pH fully deprotonated phosphoryl groups are mainly responsible for U(VI) binding. U(VI) can be bound by P. fluorescens with relatively high thermodynamic stability.

Coordination of uranium(VI) with functional groups of bacterial lipopolysaccharide studied by EXAFS and FT-IR spectroscopy.

The complexation of uranyl ions with lipopolysaccharide (LPS), the main component of the cell wall of Gram-negative bacteria, was investigated on a molecular level with U L(III)-edge extended X-ray absorption fine structure (EXAFS) and attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy over a wide pH range (2.6 to 7.0). For the first time, structural determinations of uranyl complexes with cell wall compounds were extended from acidic up to neutral pH. The main functionalities responsible for uranyl binding are phosphoryl and carboxyl groups. At an excess of LPS, related to environmental conditions, the uranyl ion is mainly complexed by phosphoryl groups four-fold monodentately coordinated in the equatorial plane of the uranyl dioxo cation UO(2)(2+) showing great homologies to the uranyl mineral phase meta-autunite in the EXAFS spectra. At equimolar ratios of uranyl and functional groups of LPS, according to a slight deficit of phosphoryl groups, additional carboxyl coordination in a bidentate manner becomes important as it is shown by IR spectroscopy. From the vibrational spectra, a mixed coordination of UO(2)(2+) with both phosphoryl and carboxyl groups is derived. The coordination of uranyl ions to the LPS molecule is obviously mainly controlled by the U/LPS concentration ratio, and the influence of pH is only of minor significance at the investigated range.

Impact of uranium (U) on the cellular glutathione pool and resultant consequences for the redox status of U.

Uranium (U) as a redox-active heavy metal can cause various redox imbalances in plant cells. Measurements of the cellular glutathione/glutathione disulfide (GSH/GSSG) by HPLC after cellular U contact revealed an interference with this essential redox couple. The GSH content remained unaffected by 10 µM U whereas the GSSG level immediately increased. In contrast, higher U concentrations (50 µM) drastically raised both forms. Using the Nernst equation, it was possible to calculate the half-cell reduction potential of 2GSH/GSSG. In case of lower U contents the cellular redox environment shifted towards more oxidizing conditions whereas the opposite effect was obtained by higher U contents. This indicates that U contact causes a consumption of reduced redox equivalents. Artificial depletion of GSH by chlorodinitrobenzene and measuring the cellular reducing capacity by tetrazolium salt reduction underlined the strong requirement of reduced redox equivalents. An additional element of cellular U detoxification mechanisms is the complex formation between the heavy metal and carboxylic functionalities of GSH. Because two GSH molecules catalyze electron transfers each with one electron forming a dimer (GSSG) two UO(2) (2+) are reduced to each UO(2) (+) by unbound redox sensitive sulfhydryl moieties. UO(2) (+) subsequently disproportionates to UO(2) (2+) and U(4+). This explains that in vitro experiments revealed a reduction to U(IV) of only around 33% of initial U(VI). Cellular U(IV) was transiently detected with the highest level after 2 h of U contact. Hence, it can be proposed that these reducing processes are an important element of defense reactions induced by this heavy metal.

Complexation of U(VI) with highly phosphorylated protein, phosvitin A vibrational spectroscopic approach.

The complexation of uranium(VI) to variant functional groups of the highly phosphorylated protein phosvitin in aqueous solution was investigated by attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy. For the verification of the affinity of the actinyl ions to carboxyl and phosphate groups of the amino acid side chains, samples with different phosphate to uranium(VI) (P/U) ratios were investigated under denaturing conditions as well as in aqueous medium. From a comparative study with other heavy metal ions, i.e. Ba(2+) and Pb(2+), a strong coordination of U(VI) to carboxyl and phosphoryl groups can be derived. Furthermore, with increasing P/U ratios, a preferential binding of U(VI) to phosphoryl groups is indicated 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/U ratio. 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.

Complexation of uranium(VI) with peptidoglycan.

We investigated the interaction of UO(2)(2+) with peptidoglycan (PG), the main part of the outer membrane of Gram-positive bacteria, by potentiometric titration and time-resolved laser-induced fluorescence spectroscopy (TRLFS) over a wide pH (2.0 to 9.0) and concentration range (10(-5) to 10(-4) M U(vi), 0.01 to 0.2 g L(-1) PG). With potentiometry two different dissociation constants for the carboxyl sites of glutamic acid and diaminopimelic acid (pK(a) = 4.55 +/- 0.02 and 6.31 +/- 0.01), and one averaged pK(a) for hydroxyl and amino groups (which are not distinguishable) (9.56 +/- 0.03) and the site densities could be identified. With potentiometry three different uranyl PG complexes were ascertained: two 1 : 1 uranyl carboxyl complexes R-COO-UO(2)(+), one with the glutamic acid carboxyl group (log beta(110) = 4.02 +/- 0.03), which has a very small formation ratio, and one with the diaminopimelic acid carboxyl group (log beta(110) = 7.28 +/- 0.03), and a mixed 1 : 1 : 1 complex with additional hydroxyl or amino coordination, R-COO-UO(2)((+))-A(i)-R (A(i) = NH(2) or O(-)) (log beta(1110) = 14.95 +/- 0.02). With TRLFS, also three, but different, species could be identified: a 1 : 1 uranyl carboxyl complex R-COO-UO(2)(+) (log beta(110) = 6.9 +/- 0.2), additionally a 1 : 2 uranyl carboxyl complex (R-COO)(2)-UO(2) (log beta(120) = 12.1 +/- 0.2), both with diaminopimelic acid carboxyl groups, and the mixed species R-COO-UO(2)((+))-A(i)-R (A(i) = NH(2) or O(-)) (log beta(1110) = 14.5 +/- 0.1). The results are in accordance within the errors of determination.

Visualizing different uranium oxidation states during the surface alteration of uraninite and uranium tetrachloride.

Low-temperature alteration reactions on uranium phases may lead to the mobilization of uranium and thereby poses a potential threat to humans living close to uranium-contaminated sites. In this study, the surface alteration of uraninite (UO(2)) and uranium tetrachloride (UCl(4)) in air atmosphere was studied by confocal laser scanning microscopy (CLSM) and laser-induced fluorescence spectroscopy using an excitation wavelength of 408 nm. It was found that within minutes the oxidation state on the surface of the uraninite and the uranium tetrachloride changed. During the surface alteration process U(IV) atoms on the uraninite and uranium tetrachloride surface became stepwise oxidized by a one-electron step at first to U(V) and then further to U(VI). These observed changes in the oxidation states of the uraninite surface were microscopically visualized and spectroscopically identified on the basis of their fluorescence emission signal. A fluorescence signal in the wavelength range of 415-475 nm was indicative for metastable uranium(V), and a fluorescence signal in the range of 480-560 nm was identified as uranium(VI). In addition, the oxidation process of tetravalent uranium in aqueous solution at pH 0.3 was visualized by CLSM and U(V) was fluorescence spectroscopically identified. The combination of microscopy and fluorescence spectroscopy provided a very convincing visualization of the brief presence of U(V) as a metastable reaction intermediate and of the simultaneous coexistence of the three states U(IV), U(V), and U(VI). These results have a significant importance for fundamental uranium redox chemistry and should contribute to a better understanding of the geochemical behavior of uranium in nature.

Fluorescence properties of a uranyl(V)-carbonate species [U(V)O(2)(CO(3))(3)](5-) at low temperature.

Fluorescence properties of a uranyl(V)-carbonate species in solution are reported for the first time. The fluorescence characteristics of the stable aqueous uranyl(V)-carbonate complex [U(V)O(2)(CO(3))(3)](5-) was determined in a frozen solution (T=153K) of 0.5mM uranium and 1.5M Na(2)CO(3) at pH 11.8 by time resolved laser-induced fluorescence spectroscopy (TRLFS). Two different wavelengths of 255nm and 408nm, respectively were used to independently of each other excite the uranyl(V)-carbonate species. The resulting U(V) fluorescence emission bands were detected between 380nm and 440nm, with a maxima at 404.7nm (excitation with 255nm) and 413.3nm (excitation with 408nm), respectively. It was found that by using an excitation wavelength of 255nm the corresponding extinction coefficient was much higher and the fluorescence spectrum better structured than the ones excited at 408nm. The fluorescence lifetime of the uranyl(V)-carbonate species was determined at 153K as 120micros. TRLFS investigations at room temperature, however, showed no fluorescence signal at all.

Complex formation of uranium(VI) with L-phenylalanine and 3-phenylpropionic acid studied by attenuated total reflection Fourier transform infrared spectroscopy.

Uranyl complexes with phenylalanine and the analogous ligand phenylpropionate were investigated in aqueous solution by attenuated total reflection (ATR) Fourier transform infrared (FT-IR) spectroscopy. The assignment of the observed bands to vibrational modes was accomplished using spectra of the pure ligands recorded at different pH values and spectra of the 15N labeled analogous compounds of the amino acid. The results presented in this work provide a detailed description of the binding states of the uranyl complexes in solution. A bidentate binding of the carboxylate group to the actinide ion was observed by the characteristic shifts of the carboxylate modes. From the spectra the presence of the protonated amino group in the actinide complex can be derived. Due to these findings, contributions of the amino group to the binding to the uranyl ion in the amino acid complex can be ruled out.

Interaction of uranium(VI) with lipopolysaccharide.

Bacteria have a great influence on the migration behaviour of heavy metals in the environment. Lipopolysaccharides form the main part of the outer membrane of Gram-negative bacteria. We investigated the interaction of the uranyl cation (UO2(2+)) with lipopolysaccharide (LPS) from Pseudomonas aeruginosa by using potentiometric titration and time-resolved laser-induced fluorescence spectroscopy (TRLFS) over a wide pH and concentration range. Generally, LPS consists of a high density of different functionalities for metal binding such as carboxyl, phosphoryl, amino and hydroxyl groups. The dissociation constants and corresponding site densities of these functional groups were determined using potentiometric titration. The combination of both methods, potentiometry and TRLFS, show that at an excess of LPS uranyl phosphoryl coordination dominates, whereas at a slight deficit on LPS compared to uranyl, carboxyl groups also become important for uranyl coordination. The stability constants of one uranyl carboxyl complex and three different uranyl phosphoryl complexes and the luminescence properties of the phosphoryl complexes are reported.

Complexation of uranium(VI) with aromatic acids in aqueous solution: a combined computational and experimental study.

The complexes of uranium(VI) with salicylhydroxamate, benzohydroxamate, and benzoate have been investigated in a combined computational and experimental study using density functional theory methods and extended X-ray absorption fine structure spectroscopy, respectively. The calculated molecular structures, relative stabilities, as well as excitation spectra from time-dependent density functional theory calculations are in good agreement with experimental data. Furthermore, these calculations allow the identification of the coordinating atoms in the uranium(VI)-salicylhydroxamate complex, i.e. salicylhydroxamate binds to the uranyl ion via the hydroxamic acid oxygen atoms and not via the phenolic oxygen and the nitrogen atom. Carefully addressing solvation effects has been found to be necessary to bring in line computational and experimental structures, as well as excitation spectra.

Spectroscopic investigations of U(VI) species sorbed by the green algae Chlorella vulgaris.

The green alga Chlorella vulgaris has the ability to bind high amounts of uranium(VI) in the pH range from 3 to 6. At pH 3 up to 40% of the uranium are bound by the algal cells. The uranium removal is almost complete at pH 5 and 6 under the given experimental conditions. Scanning electron microscopy and laser-induced fluorescence spectroscopy were used to characterize uranyl species formed in the selected pH range. The micrographs show a regular distribution of U(VI) on the cell surface. Fluorescence spectroscopic investigations of formed algal uranyl complexes indicate that the binding of U(VI) to carboxyl groups plays a dominating role at pH 3, whereas a minor impact of organic phosphate compounds on the U(VI) sorption cannot be excluded. In contrast, at pH 5 and 6 the phosphate groups are mainly responsible for the removal and binding of U(VI) by formation of organic and/or inorganic uranyl phosphates.

Identification of fluorescent U(V) and U(VI) microparticles in a multispecies biofilm by confocal laser scanning microscopy and fluorescence spectroscopy.

Fluorescent uranium(V) and uranium(VI) particles were observed for the first time in vivo by a combined laser fluorescence spectroscopy and confocal laser scanning microscopy approach in a living multispecies biofilm grown on biotite plates. These particles ranged between 1 and 7 um in width and up to 20 microm in length and were located at the bottom and at the edges of biofilms colonies. Analysis of amplified 16S rRNA fragments and fluorescence in situ hybridization were used to characterize the biofilm communities. Laser fluorescence spectroscopy was used to identify these particles. The particles showed either a characteristic fluorescence spectrum in the wavelength range of 415-475 nm, indicative for uranium(V), or in the range of 480-560 nm, which is typical for uranium(VI). Particles of uranium(V) as well as uranium(VI) were simultaneously observed in the biofilms. These uranium particles were attributed for uranium(VI) to biologically mediated precipitation and for uranium(V) to redox processes taking place within the biofilm. The detection of uranium(V) in a multispecies biofilm was interpreted as a short-lived intermediate of the uranium(VI) to uranium(IV) redox reaction. Its presence clearly documents that the uranium(VI) reduction is not a two electron step but that only one electron is involved.

U(VI)-kaolinite surface complexation in absence and presence of humic acid studied by TRLFS.

Time-resolved laser-induced fluorescence spectroscopy (TRLFS) was applied to study the U(VI) surface complexes on kaolinite in the presence and absence of humic acid (HA). Two uranyl surface species with fluorescence lifetimes of 5.9 +/- 1.4 and 42.5 +/- 3.4 micros and 4.4 +/- 1.2 and 30.9 +/- 7.2 micros were identified in the binary (U(VI)-kaolinite) and ternary system (U(VI)-HA-kaolinite), respectively. The fluorescence spectra of adsorbed uranyl surface species are described with six and five fluorescence emission bands in the binary and ternary system, respectively. The positions of peak maxima are shifted significantly to higher wavelengths compared to the free uranyl ion in perchlorate medium. HA has no influence on positions of the fluorescence emission bands. In the binary system, both surface species can be attributed to adsorbed bidentate mononuclear surface complexes, which differ in the number of water molecules in their coordination environment. In the ternary system, U(VI) prefers direct binding on kaolinite rather than via HA, but it is sorbed as a uranyl-humate complex. Consequently, the hydration shell of the U(VI) surface complexes is displaced with complexed HA, which is simultaneously distributed between kaolinite particles. Aluminol binding sites are assumed to control the sorption of U(VI) onto kaolinite.