Nanostructure and dynamics of N-truncated copper amyloid-ß peptides from advanced X-ray absorption fine structure.

An X-ray absorption spectroscopy (XAS) electrochemical cell was used to collect high-quality XAS measurements of N-truncated Cu:amyloid-ß (Cu:Aß) samples under near-physiological conditions. N-truncated Cu:Aß peptide complexes contribute to oxidative stress and neurotoxicity in Alzheimer's patients' brains. However, the redox properties of copper in different Aß peptide sequences are inconsistent. Therefore, the geometry of binding sites for the copper binding in Aß4-8/12/16 was determined using novel advanced extended X-ray absorption fine structure (EXAFS) analysis. This enables these peptides to perform redox cycles in a manner that might produce toxicity in human brains. Fluorescence XAS measurements were corrected for systematic errors including defective-pixel data, monochromator glitches and dispersion of pixel spectra. Experimental uncertainties at each data point were measured explicitly from the point-wise variance of corrected pixel measurements. The copper-binding environments of Aß4-8/12/16 were precisely determined by fitting XAS measurements with propagated experimental uncertainties, advanced analysis and hypothesis testing, providing a mechanism to pursue many similarly complex questions in bioscience. The low-temperature XAS measurements here determine that CuII is bound to the first amino acids in the high-affinity amino-terminal copper and nickel (ATCUN) binding motif with an oxygen in a tetragonal pyramid geometry in the Aß4-8/12/16 peptides. Room-temperature XAS electrochemical-cell measurements observe metal reduction in the Aß4-16 peptide. Robust investigations of XAS provide structural details of CuII binding with a very different bis-His motif and a water oxygen in a quasi-tetrahedral geometry. Oxidized XAS measurements of Aß4-12/16 imply that both CuII and CuIII are accommodated in an ATCUN-like binding site. Hypotheses for these CuI, CuII and CuIII geometries were proven and disproven using the novel data and statistical analysis including F tests. Structural parameters were determined with an accuracy some tenfold better than literature claims of past work. A new protocol was also developed using EXAFS data analysis for monitoring radiation damage. This gives a template for advanced analysis of complex biosystems.

C-H▪▪▪S Hydrogen Bonds in Ampicillin and Amoxicillin Crystals Investigated by Sulfur K-Edge X-Ray Absorption Near-Edge Structure Spectroscopy and Single-Crystal X-Ray Structure Analysis.

Sulfur K-edge X-ray absorption near-edge structure (XANES) spectroscopy was evaluated for its ability to detect non-conventional C-H▪▪▪S hydrogen bonds in crystals of the sulfur-containing penam antibiotics ampicillin and amoxicillin. The XANES spectra of the nearly isomorphous crystals of ampicillin trihydrate and amoxicillin trihydrate were very similar, whereas that of ampicillin anhydrate displayed unique features. Single-crystal X-ray structure analyses revealed that the C-H▪▪▪S hydrogen bond geometries and the chemical types of the hydrogen donors differed between the isomorphous trihydrate crystals and ampicillin anhydrate crystal. These observations demonstrate that the shapes of the sulfur K-edge XANES spectra are dependent on the nature of the C-H▪▪▪S hydrogen bonds. Sulfur K-edge XANES spectroscopy shows promise for use in the detection and analysis of non-covalent interactions, including hydrogen bonds to sulfur atoms, within active pharmaceutical ingredients.

Improved precision in As speciation analysis with HERFD-XANES at the As K-edge: the case of As speciation in mine waste.

High-energy-resolution fluorescence-detected (HERFD) X-ray absorption near-edge spectroscopy (XANES) is a spectroscopic method that allows for increased spectral feature resolution, and greater selectivity to decrease complex matrix effects compared with conventional XANES. XANES is an ideal tool for speciation of elements in solid-phase environmental samples. Accurate speciation of As in mine waste materials is important for understanding the mobility and toxicity of As in near-surface environments. In this study, linear combination fitting (LCF) was performed on synthetic spectra generated from mixtures of eight measured reference compounds for both HERFD-XANES and transmission-detected XANES to evaluate the improvement in quantitative speciation with HERFD-XANES spectra. The reference compounds arsenolite (As2O3), orpiment (As2S3), getchellite (AsSbS3), arsenopyrite (FeAsS), kankite (FeAsO4·3.5H2O), scorodite (FeAsO4·2H2O), sodium arsenate (Na3AsO4), and realgar (As4S4) were selected for their importance in mine waste systems. Statistical methods of principal component analysis and target transformation were employed to determine whether HERFD improves identification of the components in a dataset of mixtures of reference compounds. LCF was performed on HERFD- and total fluorescence yield (TFY)-XANES spectra collected from mine waste samples. Arsenopyrite, arsenolite, orpiment, and sodium arsenate were more accurately identified in the synthetic HERFD-XANES spectra compared with the transmission-XANES spectra. In mine waste samples containing arsenopyrite and either scorodite or kankite, LCF with HERFD-XANES measurements resulted in fits with smaller R-factors than concurrently collected TFY measurements. The improved accuracy of HERFD-XANES analysis may provide enhanced delineation of As phases controlling biogeochemical reactions in mine wastes, contaminated soils, and remediation systems.

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution X-Ray Absorption Spectroscopy.

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.

Paving the way for examination of coupled redox/solid-liquid interface reactions: 1 ppm Np adsorbed on clay studied by Np M5-edge HR-XANES spectroscopy.

The recently emerged actinide (An) M4,5-edge high-energy resolution X-ray absorption near-edge structure (HR-XANES) technique has proven to be very powerful for oxidation state studies of actinides. In this work, for the first time, Np M5-edge HR-XANES was applied to study Np sorption on illite. By improving the experimental conditions, notably by operation of the spectrometer under He atmosphere, it was possible to measure Np M5-edge HR-XANES spectra of a sample with ≈ 1 µg Np/g illite (1 ppm). This is 30-2000 times lower than Np loadings on mineral surfaces usually investigated by X-ray absorption spectroscopy. A newly designed cryogenic configuration enabled sample temperatures of 141.2 ± 1.5 K and successfully prevented beam-induced changes of the Np oxidation state. The described approach paves the way for the examination of coupled redox/solid-liquid interface reactions of actinide ions via An M4,5-edge HR-XANES spectroscopy at low metal ion concentrations, which are of specific relevance for contaminated sites and nuclear waste disposal studies.

Layer thickness prediction and tissue classification in two-layered tissue structures using diffuse reflectance spectroscopy.

During oncological surgery, it can be challenging to identify the tumor and establish adequate resection margins. This study proposes a new two-layer approach in which diffuse reflectance spectroscopy (DRS) is used to predict the top layer thickness and classify the layers in two-layered phantom and animal tissue. Using wavelet-based and peak-based DRS spectral features, the proposed method could predict the top layer thickness with an accuracy of up to 0.35 mm. In addition, the tissue types of the first and second layers were classified with an accuracy of 0.95 and 0.99. Distinguishing multiple tissue layers during spectral analyses results in a better understanding of more complex tissue structures encountered in surgical practice.

Towards Understanding Excited-State Properties of Organic Molecules Using Time-Resolved Soft X-ray Absorption Spectroscopy.

The extension of the pump-probe approach known from UV/VIS spectroscopy to very short wavelengths together with advanced simulation techniques allows a detailed analysis of excited-state dynamics in organic molecules or biomolecular structures on a nanosecond to femtosecond time level. Optical pump soft X-ray probe spectroscopy is a relatively new approach to detect and characterize optically dark states in organic molecules, exciton dynamics or transient ligand-to-metal charge transfer states. In this paper, we describe two experimental setups for transient soft X-ray absorption spectroscopy based on an LPP emitting picosecond and sub-nanosecond soft X-ray pulses in the photon energy range between 50 and 1500 eV. We apply these setups for near-edge X-ray absorption fine structure (NEXAFS) investigations of thin films of a metal-free porphyrin, an aggregate forming carbocyanine and a nickel oxide molecule. NEXAFS investigations have been carried out at the carbon, nitrogen and oxygen K-edge as well as on the Ni L-edge. From time-resolved NEXAFS carbon, K-edge measurements of the metal-free porphyrin first insights into a long-lived trap state are gained. Our findings are discussed and compared with density functional theory calculations.

NRVS investigation of ascorbate peroxidase compound II: Observation of Iron(IV)oxo stretching.

The protonation state of ascorbate peroxidase compound II (APX-II) has been a subject of debate. A combined X-ray/neutron crystallographic study reported that APX-II is best described as an iron(IV)hydroxide species with an FeO distance of 1.88 Å (Kwon, et al. Nat Commun2016, 7, 13,445), while X-ray absorption spectroscopy (XAS) experiments (utilizing extended X-ray absorption fine structure (EXAFS) and pre-edge analyses) indicate APX-II is an authentic iron(IV)oxo species with an FeO distance 1.68 Å (Ledray, et al. Journal of the American Chemical Society2020,142, 20,419). Previous debates concerning ferryl protonation states have been resolved through the application of Badger's rule, which correlates FeO bond distances with FeO vibrational frequencies. To obtain the required vibrational data, we have collected Nuclear Resonance Vibrational Spectroscopy (NRVS) data for APX-II. We observe a broad vibrational feature in the range associated with iron(IV)oxo stretching (700-800 cm-1). This feature appears to have two peaks at 732 cm-1 and 770 cm-1, corresponding to FeO distances of 1.69 and 1.67 Å, respectively. The broad vibrational envelope and the presence of multiple resonances could reflect a distribution of hydrogen bonding interactions within the active-site pocket.

XANES reflects coordination change and underlying surface disorder of zinc adsorbed to silica.

Zinc K-edge X-ray absorption near-edge structure (XANES) spectroscopy of Zn adsorbed to silica and Zn-bearing minerals, salts and solutions was conducted to explore how XANES spectra reflect coordination environment and disorder in the surface to which a metal ion is sorbed. Specifically, XANES spectra for five distinct Zn adsorption complexes (Znads) on quartz and amorphous silica [SiO2(am)] are presented from the Zn-water-silica surface system: outer-sphere octahedral Znads on quartz, inner-sphere octahedral Znads on quartz, inner-sphere tetrahedral Znads on quartz, inner-sphere octahedral Znads on SiO2(am) and inner-sphere tetrahedral Znads on SiO2(am). XANES spectral analysis of these complexes on quartz versus SiO2(am) reveals that normalized peak absorbance and K-edge energy position generally decrease with increasing surface disorder and decreasing Zn-O coordination. On quartz, the absorption-edge energy of Znads ranges from 9663.0 to 9664.1 eV for samples dominated by tetrahedrally versus octahedrally coordinated species, respectively. On SiO2(am), the absorption-edge energy of Znads ranges from 9662.3 to 9663.4 eV for samples dominated by tetrahedrally versus octahedrally coordinated species, respectively. On both silica substrates, octahedral Znads presents a single K-edge peak feature, whereas tetrahedral Znads presents two absorbance features. The energy space between the two absorbance peak features of the XANES K-edge of tetrahedral Znads is 2.4 eV for Zn on quartz and 3.2 eV for Zn on SiO2(am). Linear combination fitting of samples with a mixture of Znads complex types demonstrates that the XANES spectra of octahedral and tetrahedral Znads on silica are distinct enough for quantitative identification. These results suggest caution when deciphering Zn speciation in natural samples via linear combination approaches using a single Znads standard to represent sorption on a particular mineral surface. Correlation between XANES spectral features and prior extended X-ray absorption fine structure (EXAFS) derived coordination environments for these Znads on silica samples provides insight into Zn speciation in natural systems with XANES compatible Zn concentrations too low for EXAFS analysis.

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.

Probing Structural Information of Gas-Phase Peptides by Near-Edge X-ray Absorption Mass Spectrometry.

Near-edge X-ray absorption mass spectrometry (NEXAMS) is an action-spectroscopy technique of growing interest for investigations into the spatial and electronic structure of biomolecules. It has been used successfully to give insights into different aspects of the photodissociation of peptides and to probe the conformation of proteins. It is a current question whether the fragmentation pathways are sensitive toward effects of conformational isomerism, tautomerism, and intramolecular interactions in gas-phase peptides. To address this issue, we studied the cationic fragments of cryogenically cooled gas-phase leucine enkephalin ([LeuEnk+H]+) and methionine enkephalin ([MetEnk+H]+) produced upon soft X-ray photon absorption at the carbon, nitrogen, and oxygen K-edges. The interpretation of the experimental ion yield spectra was supported by density-functional theory and restricted-open-shell configuration interaction with singles (DFT/ROCIS) calculations. The analysis revealed several effects that could not be rationalized based on the peptide's amino acid sequences alone. Clear differences between the partial ion yields measured for both peptides upon C 1s → π*(C═C) excitations in the aromatic amino acid side chains give evidence for a sulfur-aromatic interaction between the methionine and phenylalanine side chain of [MetEnk+H]+. Furthermore, a peak associated with N 1s → π*(C═N) transitions, linked to a tautomeric keto-to-enol conversion of peptide bonds, was only present in the photon energy resolved ion yield spectra of [MetEnk+H]+.

Arsenic speciation in cooked food and its bioaccessible fraction using X-ray absorption spectroscopy.

Thermal processing or the digestion process can alter the forms of arsenic (As) present in food. Identification of As species is necessary to accurately determine the risk associated with food consumption. X-ray absorption near-edge structure (XANES) was used to investigate As species in rice, asparagus, and garlic boiled in water containing As(V), and in their bioaccessible fractions (solubilized As after gastrointestinal digestion). The XANES analysis revealed the presence of As(III) (11871.5 eV) or As(III)-S [As(III)-Cys, 11869.6 eV] solution in the cooked foods and in their bioaccessible fractions. The percentage of trivalent species (12-55%) followed the order asparagus ≫ rice ≈ garlic. In the asparagus and garlic samples, part of the As(V) (tetrahedral form) [11875 eV] that had been added appeared in the form of an octahedral As(V) compound [As(V)-glycerol, 11876 eV]. All these changes could considerably modify the risk associated with ingestion of As-contaminated food.

In Situ/Operando Electrocatalyst Characterization by X-ray Absorption Spectroscopy.

During the last decades, X-ray absorption spectroscopy (XAS) has become an indispensable method for probing the structure and composition of heterogeneous catalysts, revealing the nature of the active sites and establishing links between structural motifs in a catalyst, local electronic structure, and catalytic properties. Here we discuss the fundamental principles of the XAS method and describe the progress in the instrumentation and data analysis approaches undertaken for deciphering X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectra. Recent usages of XAS in the field of heterogeneous catalysis, with emphasis on examples concerning electrocatalysis, will be presented. The latter is a rapidly developing field with immense industrial applications but also unique challenges in terms of the experimental characterization restrictions and advanced modeling approaches required. This review will highlight the new insight that can be gained with XAS on complex real-world electrocatalysts including their working mechanisms and the dynamic processes taking place in the course of a chemical reaction. More specifically, we will discuss applications of in situ and operando XAS to probe the catalyst's interactions with the environment (support, electrolyte, ligands, adsorbates, reaction products, and intermediates) and its structural, chemical, and electronic transformations as it adapts to the reaction conditions.

Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Archon2.

Archon2 is a fluorescent voltage sensor derived from Archaerhodopsin 3 (Arch) of Halorubrum sodomense using robotic multidimensional directed evolution approach. Here we report absorption and emission spectroscopic studies of Archon2 in Tris buffer at pH 8. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation spectra were determined. The thermal stability of Archon2 was studied by long-time attenuation coefficient measurements at room temperature (21 ± 1 °C) and at refrigerator temperature (3 ± 1 °C). The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 63 ± 3 °C). In the protein melting process protonated retinal Schiff base (PRSB) with absorption maximum at 586 nm converted to de-protonated retinal Schiff base (RSB) with absorption maximum at 380 nm. Storage of Archon2 at room temperature and refrigerator temperature caused absorption coefficient decrease because of partial protein clustering to aggregates at condensation nuclei and sedimentation. At room temperature an onset of light scattering was observed after two days because of the beginning of protein unfolding. During the period of observation (18 days at 21 °C, 22 days at 3 °C) no change of retinal isomer composition was observed indicating a high potential energy barrier of S0 ground-state isomerization.

Probing a Silent Metal: A Combined X-ray Absorption and Emission Spectroscopic Study of Biologically Relevant Zinc Complexes.

As the second most common transition metal in the human body, zinc is of great interest to research but has few viable routes for its direct structural study in biological systems. Herein, Zn valence-to-core X-ray emission spectroscopy (VtC XES) and Zn K-edge X-ray absorption spectroscopy (XAS) are presented as a means to understand the local structure of zinc in biological systems through the application of these methods to a series of biologically relevant molecular model complexes. Taken together, the Zn K-edge XAS and VtC XES provide a means to establish the ligand identity, local geometry, and metal-ligand bond lengths. Experimental results are supported by correlation with density-functional-theory-based calculations. Combining these theoretical and experimental approaches will enable future applications to protein systems in a predictive manner.

Crystalline and amorphous calcium carbonate as structural components of the Calappa granulata exoskeleton.

The exoskeleton of crustaceans consists of chitin biopolymers where the embedded inorganic biominerals, mainly CaCO3, affect strongly its mechanical properties. Raman and Near Edge X-ray Absorption Fine Structure (NEXAFS) spectroscopies and Transmission Electron Microscopy (TEM) are applied to investigate the CaCO3 structure in various parts of the Calappa granulata crab exoskeleton. The shape of the main Raman peak of CaCO3 reveals the presence of two phases which are identified as calcite and amorphous calcium carbonate (ACC). The relative concentration of the two phases in various parts of the exoskeleton is determined from the area ratio under the corresponding peaks. The results of the Ca L3,2-edge NEXAFS analysis are in line with the Raman findings, since the energy separation of peaks that appear in the lower frequency region of the main L2 and L3 peaks due to crystal field splitting, is directly related to the percentage of the ACC phase in the total CaCO3 mineral content. The C K-edge spectra are used for the determination of the extent of calcification of the exoskeleton. Furthermore, dark and bright field TEM images reveal the presence of nanocrystallites with an average size of 20 nm. The structure of the nanocrystallites, as derived from the Selected Area Electron Diffraction patterns, is calcite. The results suggest that ACC plays a structural role in the exoskeleton of Calappa granulata.

Human red blood cell uptake and sequestration of arsenite and selenite: Evidence of seleno-bis(S-glutathionyl) arsinium ion formation in human cells.

Over 200 million people worldwide are exposed to the human carcinogen, arsenic, in contaminated drinking water. In laboratory animals, arsenic and the essential trace element, selenium, can undergo mutual detoxification through the formation of the seleno-bis(S-glutathionyl) arsinium ion [(GS)2AsSe]-, which undergoes biliary and fecal elimination. [(GS)2AsSe]-, formed in animal red blood cells (RBCs), sequesters arsenic and selenium, and slows the distribution of both compounds to peripheral tissues susceptible to toxic effects. In human RBCs, the influence of arsenic on selenium accumulation, and vice versa, is largely unknown. The study aims were to characterize arsenite (AsIII) and selenite (SeIV) uptake by human RBCs, to determine if SeIV and AsIII increase the respective accumulation of the other in human RBCs, and ultimately to determine if this occurs through the formation and sequestration of [(GS)2AsSe]-. 75SeIV accumulation was temperature and Cl--dependent, inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS) (IC50 1 ± 0.2 µM), and approached saturation at 30 µM, suggesting uptake is mediated by the erythrocyte anion-exchanger 1 (AE1 or Band 3, gene SLC4A1). HEK293 cells overexpressing AE1 showed concentration-dependent 75SeIV uptake. 73AsIII uptake by human RBCs was temperature-dependent, partly reduced by aquaglyceroporin 3 inhibitors, and not saturated. AsIII increased 75SeIV accumulation (in the presence of albumin) and SeIV increased 73AsIII accumulation in human RBCs. Near-edge X-ray absorption spectroscopy revealed the formation of [(GS)2AsSe]- in human RBCs exposed to both AsIII and SeIV. The sequestration of [(GS)2AsSe]- in human RBCs potentially slows arsenic distribution to susceptible tissues and could reduce arsenic-induced disease.

Monte Carlo bottom-up evaluation of global instrumental quantification uncertainty: Flexible and user-friendly computational tool.

Many instrumental methods of analysis require the daily collection of calibrator signals to calibrate their response. The quality of quantifications based on these calibrations depends on calibrators quality, instrumental signal performance and regression model fitness. Linear Ordinary Least Squares (LOLS), Linear Weighted Least Squares (LWLS) or Linear Bivariate Least Squares (LBLS) regression models can be used to calibrate and evaluate the uncertainty from instrumental quantifications, but require the fulfilment of some assumptions, namely, constant signal variance (LOLS), high calibrators quality (LOLS and LWLS) and linear variation of instrumental signal with calibrator values. The LBLS is flexible regarding calibrator values uncertainty and correlation but requires the determination of calibrator values and signals covariances. This work developed a computational tool for the bottom-up evaluation of global instrumental quantifications uncertainty which simulates calibrator values correlations from entered calibrators preparation procedure and simulates calibrators and samples signals precision from prior precision data, allowing accurate uncertainty evaluation from a few replicate signals of the daily calibration. The used signal precision models were built from previously observed repeatability variation throughout the calibration interval adjusted to daily precision condition from a residual standard deviation adjustment factor. This approach was implemented in a user-friendly MS-Excel file and was successfully applied to the analysis of As, Cd, Ni and Pb in marine sediment extracts by Absorption Spectroscopy. Evaluations were tested by the metrological compatibility of estimated and reference values of control standards for confidence levels of 95% and 99%. The success rates of the compatibility tests were statistically equivalent to the confidence level (p-value>0.01).

Assessing electron transfer reactions and catalysis in multicopper oxidases with operando X-ray absorption spectroscopy.

Here we propose an experimental setup based on operando X-ray absorption spectroscopy (XAS) to understand why copper-containing oxidoreductase enzymes show exceptional performance as catalysts for the oxygen reduction reaction (ORR). An electrode based on carbon nanoparticles organized in mesoporous structures with bilirubin oxidase (BOD) was developed to be used in a home-made operando XAS electrochemical cell, and we probed the electron transfer under ORR regime. In the presence of molecular oxygen, the BOD cofactor containing 4 copper ions require an overpotential about 150 mV to be reduced as compared to that in the absence of oxygen. A second electron transfer step, which occurs faster than the cofactor reduction, suggests that the cooper ions act as a tridimensional redox active electronic bridges for the electron transfer reaction.

A general covalent binding model between cytotoxic selenocompounds and albumin revealed by mass spectrometry and X-ray absorption spectroscopy.

Selenocompounds (SeCs) are promising therapeutic agents for a wide range of diseases including cancer. The treatment results are heterogeneous and dependent on both the chemical species and the concentration of SeCs. Moreover, the mechanisms of action are poorly revealed, which most probably is due to the detection methods where the quantification is based on the total selenium as an element. To understand the mechanisms underlying the heterogeneous cytotoxicity of SeCs and to determine their pharmacokinetics, we investigated selenium speciation of six SeCs representing different categories using liquid chromatography-mass spectrometry (LC-MS) and X-ray absorption spectroscopy (XAS) and the cytotoxicity using leukemic cells. SeCs cytotoxicity was correlated with albumin binding degree as revealed by LC-MS and XAS. Further analysis corroborated the covalent binding between selenol intermediates of SeCs and albumin thiols. On basis of the Se-S model, pharmacokinetic properties of four SeCs were for the first time profiled. In summary, we have shown that cytotoxic SeCs could spontaneously transform into selenol intermediates that immediately react with albumin thiols through Se-S bond. The heterogeneous albumin binding degree may predict the variability in cytotoxicity. The present knowledge will also guide further kinetic and mechanistic investigations in both experimental and clinical settings.