X-ray absorption spectroscopy of selenium-containing amino acids

Received: 2 April 1999 / Accepted: 17 September 1999     

Application of X-ray absorption and X-ray fluorescence techniques to the study of metallodrug action

X-ray absorption spectroscopy and X-ray fluorescence microscopy are two synchrotron-based techniques frequently deployed either individually or in tandem to investigate the fates of metallodrugs and their biotransformation products in physiologically relevant sample material. These X-ray methods confer advantages over other analytical techniques in that they are nondestructive and require minimal chemical or physical manipulation of the sample before analysis, conserving both chemical and spatial information of the element(s) under investigation. In this review, we present selected examples of the use of X-ray absorption spectroscopy and X-ray fluorescence microscopy in studies of metallodrug speciation and localisation in vivo, in cell spheroids and in intact tissues and organs, and offer recent highlights in the advances of these techniques as they pertain to research on metallodrug action.     

X-ray spectroscopy of the Mn4Ca cluster in the water-oxidation complex of Photosystem II

The water-oxidation complex of Photosystem II (PS II) contains a heteronuclear cluster of 4 Mn atoms and a Ca atom. Ligands to the metal cluster involve bridging O atoms, and O and N atoms from amino acid side-chains of the D1 polypeptide of PS II, with likely additional contributions from water and CP43. Although moderate resolution X-ray diffraction-based structures of PS II have been reported recently, and the location of the Mn₄Ca cluster has been identified, the structures are not resolved at the atomic level. X-ray absorption (XAS), emission (XES), resonant inelastic X-ray scattering (RIXS) and extended X-ray absorption fine structure (EXAFS) provide independent and potentially highly accurate sources of structural and oxidation-state information. When combined with polarized X-ray studies of oriented membranes or single-crystals of PS II, a more detailed picture of the cluster and its disposition in PS II is obtained.     

Synchrotron-based X-ray fluorescence imaging of human cells labeled with CdSe quantum dots

Synchrotron-based X-ray fluorescence (S-XRF) is a powerful technique for imaging the distribution of many biologically relevant elements as well as of “artificial” elements deliberately introduced into tissues and cells, for example, through functionalized nanoparticles. In this study, we explored the potential of S-XRF for chemical nanoimaging (100 nm spatial resolution, nanoXRF) of human cells through the use of functionalized CdSe/ZnS quantum dots (QDs). We used a commercially available QD-secondary antibody conjugate to label the cancer marker HER2 (human epidermal growth factor receptor 2) on the surface of SKOV3 cancer cells and β-tubulin, a protein associated with cytoskeleton microtubules. We set up samples with epoxy inclusion and intracellular labeling as well as samples without epoxy inclusion and with surface labeling. Epoxy inclusion, also used in electron microscopy, has the advantage of preserving cell morphology and guaranteeing long-term stability. QDs proved to be suitable probes for nanoXRF due to the Se emission band, which is not in close proximity to any other emission band, and the signal specificity, which is preserved in both types of labeling. Therefore, nanoXRF using QD-based markers can be very effective at colocalizing specific intracellular targets with elements naturally present in the cell and may complement confocal fluorescence microscopy in a synergistic fashion.     

A possible molecular link between the toxicological effects of arsenic,selenium and methylmercury: methylmercury(II) seleno bis(<Emphasis Type="Italic">S</Emphasis>-glutathionyl) arsenic(III)

Using a combination of As and Se K-edge and Hg L_III-edge X-ray absorption spectroscopy, ⁷⁷Se nuclear magnetic resonance spectroscopy, electrospray ionization mass spectrometry and molecular modeling, we have structurally characterized the novel species methylmercury(II) seleno bis(S-glutathionyl) arsenic(III). This species is formed in aqueous solution from CH₃HgOH and the seleno bis(S-glutathionyl) arsinium ion and constitutes an important first step towards characterizing the observed toxicologically relevant interaction between arsenite, selenite and methylmercury which has been previously reported in mammals.     

Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging

Although methods for light microscopy of chromatin are well established, there are no quantitative data for nucleosome concentrations in vivo. To establish such a method we used a HeLa clone expressing the core histone H2B fused to the enhanced yellow fluorescent protein (H2B-EYFP). Quantitative gel electrophoresis and fluorescence correlation spectroscopy (FCS) of isolated oligonucleosomes show that 5% of the total H2Bs carry the fluorescent tag and an increased nucleosome repeat length of 204 bp for the fluorescent cells. In vivo, the mobility and distribution of H2B-EYFP were studied with a combination of FCS and confocal imaging. With FCS, concentration and brightness of nascent molecules were measured in the cytoplasm, while in the nucleoplasm a background of mobile fluorescent histones was determined by continuous photobleaching. Combining these results allows converting confocal fluorescence images of nuclei into calibrated nucleosome density maps. Absolute nucleosome concentrations in interphase amount up to 250 microM locally, with mean values of 140(+/-28)microM, suggesting that a condensation-controlled regulation of site accessibility takes place at length scales well below 200 nm.     

A contribution to the world selenium map

Atomic absorption spectrophotometric method was used to determine the serum selenium levels of 86 healthy individuals. Variations in age, sex, and geographically different urban regions of Yugoslavia were investigated. A group of 63 healthy children, ages 8–15 yr, were examined. Mean±standard deviation of the serum selenium concentration was 57±9 μg/L; age and geographic area had no effect on the Se status of children, but the difference between boys and girls was significant (P<0.05). A group of 23 men from Zagreb, ages 22–37 yr, were examined. The group was divided into three age subgroups and no difference was found among these groups. The mean Se concentration was 69±18 μg/L, and a statistically significant difference was found only between the group of adults and the group of children (P<0.05).     

Comparison of distribution and metabolism between tellurium and selenium in rats

Tellurium (Te) has shown recent increase in use as a component of optical magnetic disks having phase-change property, such as digital versatile disk-random access memory (DVD-RAM) and DVD-rewritable (DVD-RW). However, the toxicity and metabolic pathway of Te remain unclear despite its being known as a non-essential and harmful metalloid. This study was performed to gain an insight into Te metabolism in the body. The mechanism for the distinction of Te from selenium (Se), an essential metalloid belonging to the same group as Te, was also clarified. Rats were given drinking water containing tellurite and ⁸²Se-labeled selenite at the same concentration, and the concentrations of these metalloids in organs, body fluid and excreta were determined 2 days later. The results demonstrate that urinary and fecal excretion of Te was, respectively, lower and higher than that of exogenous (labeled) Se, suggesting that Te was less absorbed than Se. The ingested Te was transformed, i.e., methylated in organs and effluxed into bloodstream, and the effluxed Te was highly accumulated in rat red blood cells (RBCs) in the form of dimethylated Te. In contrast, Se was not accumulated in RBCs. Finally, Te was excreted in urine as trimethyltelluronium and might be exhaled as dimethyltelluride. The results suggest that the metabolism of Te was distinct from that of Se in rats.     

Localization of selenium in bacterial cells using TEM and energy dispersive X-ray analysis

Bacteria isolated from lake sediment samples reduced sodium selenite to elemental selenium. Finestructural observations were made on a number of different bacterial species cultured in the presence of sodium selenite. Examination of Escherichia coli and a Pseudomonas species revealed electron-dense deposits of irregular shape, composed of smaller units, within the cytoplasm but not on the cell wall and cell membrane. Cells of Aeromonas and Flavobacterium species exhibited conspicuous intranuclear fibrillary aggregates and different electron-dense inclusions. It appeared that the membrane structures were somewhat more easily stained in some bacterial cells after growth on agar plates containing sodium selenite. The deposits and fibrillary accumulations were interpreted to contain selenium on the basis of energy dispersive X-ray analysis. Control preparations and cells grown in the presence of sodium selenate were void of any fine-structural abnormalities. Alterations in fine structure are discussed in relation to the metabolism of selenium by bacterial cells and possible sites of inhibition.Abbreviations TEM transmission electron microscopy - EDX energy dispersive X-ray     

Human serum albumin-bound selenium (Se-HSA) in serum and its correlation with other selenium species

IntroductionSelenium (Se) is a trace element with different toxicological and nutritional properties according to its chemical forms. Among the wide range of selenium species, human serum albumin-bound selenium (Se-HSA) has still uncertain composition in terms of organic or inorganic selenium species. This study aimed at investigating the relation between Se-HSA levels with total selenium and the specific organic and inorganic selenium species.MethodsWe determined levels of total selenium and selenium species in serum of participants enrolled in two populations of the Emilia-Romagna region, in Northern Italy. Anion exchange chromatography coupled with inductively coupled plasma dynamic reaction cell mass spectrometry was used as quantification method. Correlations between Se-HSA and the other selenium compounds were analyzed using linear regression and restricted cubic spline regression models, adjusted for potential confounders.ResultsThe first cohort comprised 50 participants (men/women: 26/24) with median (interquartile range, IQR) age 50 (55−62) years, while the second was composed of 104 participants (M/W: 50/54), median (IQR) age 48 (44−53) years. Median (IQR) levels of total selenium were 118.5 (109−136) µg/L and 116.5 (106−128) µg/L, respectively, while Se-HSA was 25.5 µg/L (16.2–51.5) and 1.1 (0.03–3.1) µg/L, respectively. In both populations, Se-HSA was positively associated with inorganic selenium species. Conversely, Se-HSA was inversely associated with organic selenium, especially with selenoprotein P-bound-Se (Se-SELENOP) and less strongly with selenomethionine-bound-Se (Se-Met), while the relation was null or even positive with other organic species. Evaluation of non-linear trends showed a substantially positive association with inorganic selenium, particularly selenite, until a concentration of 30 µg/L, above which a plateau was reached. The association with Se-SELENOP was inverse and strong until 100 µg/L, while it was almost null at higher levels.ConclusionsOur findings seem to indicate that Se-HSA incorporates more selenium when circulating levels of inorganic compounds are higher, thus supporting its mainly inorganic nature, particularly at high circulating levels of selenite.     

Structure of bovine milk calcium phosphate determined by x-ray absorption spectroscopy

Calcium in cow's milk is mainly in the form of calcium phosphate-phosphoprotein complexes known as casein micelles. These micelles, in contrast to other phosphoprotein complexes in bone and other tissues, can be readily isolated and studied, but conventional techniques have given ambiguous and conflicting evidence on the structure of milk calcium phosphate. Extended X-ray absorption fine structure and near-edge structure measurements at the newly commissioned Synchrotron Radiation Source at Daresbury indicate that it closely resembles brushite, CaHPO₄·2H₂O. This result, and chemical analysis, requires that phosphate groups from the matrix phosphoproteins be incorporated in the brushite lattice, probably in the surface, suggesting that these organic phosphate groups act as heterogeneous nucleation sites for phase separation of the calcium phosphate from solution.     

Structural characterization of the mononuclear iron site in Pseudomonas cepacia phthalate DB01 dioxygenase using X-ray absorption spectroscopy

Phthalate dioxygenase (PDO) from Pseudomonas cepacia contains a Rieske-like [2Fe-2S] cluster and a mononuclear non-heme Fe(II) site. The mononuclear iron can be replaced by a variety of divalent metal ions, although only Fe(II) permits catalytic activity. We used X-ray absorption spectroscopy to characterize the structural properties of the mononuclear iron site and to follow the structural changes in this site as a function both of Rieske site oxidation state and of phthalate binding. Data for the mononuclear site have been measured directly for PDO substituted with Co or Zn in the mononuclear site, and by difference for the native 3-Fe protein. The mononuclear site was modeled well by low Z-ligation (oxygen or nitrogen) and showed no evidence for high-Z ligands (e.g., sulfur). The relatively short average first shell bond lengths and the absence of significant outer shell scattering suggest that the mononuclear site has several oxygen ligands. With Zn in the mononuclear site, the average bond length (2.00?Å) suggests a 5-coordinate site under all conditions. In contrast, the Co- or Fe-containing mononuclear site appeared to be 6-coordinate and changed to 5-coordinate when substrate was bound, since the first shell bond length changed from 2.08 to 2.02?Å (Co) or 2.10 to 2.06?Å (Fe). The implications of these findings for the PDO mechanism are discussed.     

Chloride ligation in inorganic manganese model compounds relevant to Photosystem II studied using X-ray absorption spectroscopy

Chloride ions are essential for proper function of the photosynthetic oxygen-evolving complex (OEC) of Photosystem II (PS II). Although proposed to be directly ligated to the Mn cluster of the OEC, the specific structural and mechanistic roles of chloride remain unresolved. This study utilizes X-ray absorption spectroscopy (XAS) to characterize the Mn–Cl interaction in inorganic compounds that contain structural motifs similar to those proposed for the OEC. Three sets of model compounds are examined; they possess core structures Mn^IV₃O₄X (X=Cl, F, or OH) that contain a di--oxo and two mono--oxo bridges or Mn^IV₂O₂X (X=Cl, F, OH, OAc) that contain a di--oxo bridge. Each set of compounds is examined for changes in the XAS spectra that are attributable to the replacement of a terminal OH or F ligand, or bridging OAc ligand, by a terminal Cl ligand. The X-ray absorption near edge structure (XANES) shows changes in the spectra on replacement of OH, OAc, or F by Cl ligands that are indicative of the overall charge of the metal atom and are consistent with the electronegativity of the ligand atom. Fourier transforms (FTs) of the extended X-ray absorption fine structure (EXAFS) spectra reveal a feature that is present only in compounds where chloride is directly ligated to Mn. These FT features were simulated using various calculated Mn–X interactions (X=O, N, Cl, F), and the best fits were found when a Mn–Cl interaction at a 2.2–2.3 Å bond distance was included. There are very few high-valent Mn halide complexes that have been synthesized, and it is important to make such a comparative study of the XANES and EXAFS spectra because they have the potential for providing information about the possible presence or absence of halide ligation to the Mn cluster in PS II.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-003-0520-1Abbreviations bpea N,N-bis(2-pyridylmethyl)ethylamine - EXAFS extended X-ray absorption fine structure - FT Fourier transform - IPE inflection point energy - OEC oxygen evolving complex - PS II Photosystem II - tacn 1,4,7-triazacyclononane - XANES X-ray absorption near edge structure - XAS X-ray absorption spectroscopy - XRD X-ray diffraction     

Rapid reduction of iron in horse spleen ferritin by thioglycolic acid measured by dispersive X-ray absorption spectroscopy

Summary The release of iron from ferritin is important in the formation of iron proteins and for the management of diseases in both animals and plants associated with abnormal accumulations of ferritin iron. Much more iron can be released experimentally by reduction of the ferric hydrous oxide core than by chelation of Fe³⁺ which has led to the notion that reduction is also the major aspect of iron release in vivo. Variations in the kinetics of reduction of the mineral core of ferritin have been attributed to the redox potential of the reductant, redox properties of the iron core, the structure of the protein coat, the analytical method used to detect Fe²⁺ and reactions at the surface of the mineral. Direct measurements of the oxidation state of the iron during reduction has never been used to analyze the kinetics of reduction, although Mössbauer spectroscopy has been used to confirm the extent of reduction after electrochemical reduction using dispersive X-ray absorption spectroscopy (DXAS). We show that the near edge of X-ray absorption spectra (XANES) can be used to quantify the relative amounts of Fe²⁺ and Fe³⁺ in mixtures of the hydrated ions. Since the nearest neighbors of iron in the ferritin iron core do not change during reduction, XANES can be used to monitor directly the reduction of the ferritin iron core. Previous studies of iron core reduction which measured by Fe²⁺ · bipyridyl formation, or coulometric reduction with different mediators, suggested that rates depended mainly on the redox potential of the electron donor. When DXAS was used to measure the rate of reduction directly, the initial rate was faster than previously measured. Thus, previously measured differences in reduction rates appear to be influenced by the accessibility of Fe²⁺ to the complexing reagent or by the electrochemical mediator. In the later stages of ferritin iron core dissolution, reduction rates drop dramatically whether measured by DXAS or formation of Fe²⁺ complexes. Such results emphasize the heterogeneity of ferritin core structure.     

Molybdenum X-ray absorption edges from 200 to 20,000 eV: The benefits of soft X-ray spectroscopy for chemical speciation

We have surveyed the chemical utility of the near-edge structure of molybdenum X-ray absorption edges from the hard X-ray K-edge at 20,000 eV down to the soft X-ray M_4,5-edges at ∼230 eV. We compared, for each edge, the spectra of two tetrahedral anions, and . We used three criteria for assessing near-edge structure of each edge: (i) the ratio of the observed chemical shift between and and the linewidth, (ii) the chemical information from analysis of the near-edge structure and (iii) the ease of measurement using fluorescence detection. Not surprisingly, the K-edge was by far the easiest to measure, but it contained the least information. The L_2,3-edges, although harder to measure, had benefits with regard to selection rules and chemical speciation in that they had both a greater chemical shift as well as detailed lineshapes which could be theoretically analyzed in terms of Mo ligand field, symmetry, and covalency. The soft X-ray M_2,3-edges were perhaps the least useful, in that they were difficult to measure using fluorescence detection and had very similar information content to the corresponding L_2,3-edges.Interestingly, the soft X-ray, low energy (∼230 eV) M_4,5-edges had greatest potential chemical sensitivity and using our high-resolution superconducting tunnel junction (STJ) fluorescence detector they appear to be straightforward to measure. The spectra were amenable to analysis using both the TT-multiplet approach and FEFF. The results using FEFF indicate that the sharp near-edge peaks arise from 3d → 5p transitions, while the broad edge structure has predominately 3d → 4f character. A proper understanding of the dependence of these soft X-ray spectra on ligand field and site geometry is necessary before a complete assessment of the utility of the Mo M_4,5-edges can be made. This work includes crystallographic characterization of sodium tetrathiomolybdate.     

Interplay between glutathione, Atx1 and copper: X-ray absorption spectroscopy determination of Cu(I) environment in an Atx1 dimer

X-ray absorption techniques have been used to characterise the primary coordination sphere of Cu(I) bound to glutathionate (GS⁻), to Atx1 and in Cu₂^I(GS⁻)₂(Atx1)₂, a complex recently proposed as the major form of Atx1 in the cytosol. In each complex, Cu(I) was shown to be triply coordinated. When only glutathione is provided, each Cu(I) is triply coordinated by sulphur atoms in the binuclear complex Cu^I ₂(GS⁻)₅, involving bridging and terminal thiolates. In the presence of Atx1 and excess of glutathione, under conditions where Cu^I ₂(GS⁻)₂(Atx1)₂ is formed, each Cu(I) is triply coordinated by sulphur atoms. Given these constraints, there are two different ways for Cu(I) to bridge the Atx1 dimer: either both Cu(I) ions contribute to bridging the dimer, or only one Cu(I) ion is responsible for bridging, the other one being coordinated to two glutathione molecules. These two models are discussed as regards Cu(I) transfer to Ccc2a.

X-ray photoelectron spectroscopy and tunable photoluminescence study of gold nanoparticles embedded in PVA films

This article reports the systematic photoluminescence study of the various contents of gold nanocomposites in polyvinyl alcohol (PVA) films. The variations in the gold content in PVA film were 0.2, 0.5, 1.0, and 1.5 wt%. All the samples were excited at two selected wavelengths; those are at 400 nm and 532 nm. On exciting the gold-PVA nanocomposite films at 400 nm the photoluminescence was observed in the region of 430–500 nm in comparison to pure PVA films that show an emission at 400 nm. However, on exciting the gold-PVA nanocomposites at 532 nm, the emission was observed at 560–650 nm with a long tail till 700 nm that is unlike the pure PVA films that do not show any emission peak in this region. This suggests that emission between 430 and 500 nm regions is due to the coordination of PVA with gold nanoparticles because PVA has an emission at 400 nm. However, the emission peak between 560 and 650 nm is entirely due to the gold nanocomposite particle. The peak also shows a smaller red-shift that is usually with the increasing nanoparticles size with the increasing content in the PVA films. The formation of gold nanoparticles was justified by X-ray diffraction (XRD) analysis which is further supported by X-ray photoelectron spectroscopy (XPS) analysis.     

An X-ray diffraction and X-ray absorption spectroscopy joint study of neuroglobin

Neuroglobin (Ngb) is a member of the globin family expressed in the vertebrate brain, involved in neuroprotection. A combined approach of X-ray diffraction (XRD) on single crystal and X-ray absorption spectroscopy (XAS) in solution, allows to determine the oxidation state and the structure of the Fe-heme both in the bis-histidine and the CO-bound (NgbCO) states. The overall data demonstrate that under X-ray the iron is photoreduced fairly rapidly, and that the previously reported X-ray structure of ferric Ngb [B. Vallone, K. Nienhaus, M. Brunori, G.U. Nienhaus, Proteins 56 (2004) 85-92] very likely refers to a photoreduced species indistinguishable from the dithionite reduced protein. Results from the XAS analysis of NgbCO in solution are in good agreement with XRD data on the crystal. However prolonged X-ray exposure at 15 K determines CO release. This preliminary result paves the way to experiments aimed at the characterization of pentacoordinate ferrous Ngb, the only species competent in binding external ligands such as O₂, CO or NO.     

Saccharose solid matrix embedded proteins: a new method for sample preparation for X-ray absorption spectroscopy

In this study, solid samples of hemoglobin and hemocyanin have been prepared by embedding the proteins into a saccharose-based matrix. These materials have been developed specifically for specimens for X-ray absorption spectroscopy (XAS). The preservation of protein conformation and active site organization was tested, making comparisons between the solid and the corresponding liquid samples, using resonance Raman, infra red, fluorescence and XAS. The XAS spectra of irradiated solid and liquid samples were then compared, and the preservation of biological activity of the proteins during both preparation procedure and X-ray irradiation was assessed. In all cases, the measurements clearly demonstrate that protein solid samples are both structurally and functionally quite well preserved, much better than those in the liquid state. The saccharose matrix provides an excellent protection against X-ray damages, allowing for longer exposure to the X-ray beam. Moreover, the demonstrated long-term stability of samples permits their preparation and storage in optimal conditions, allowing for the repetition of data collection with the same sample in several experimental sessions. The very high protein concentration that can be reached results in a significantly better signal-to-noise ratio, particularly useful for high molecular weight proteins with a low metal-to-protein ratio. On the bases of the above-mentioned results, we propose the new method as a standard procedure for the preparation of biological samples to be used for XAS spectroscopy. Received: 3 August 1999 / Revised version: 5 April 2000 / Accepted: 6 April 2000