Immunoaffinity LC-MS/MS Quantification of the Sepsis Biomarker Procalcitonin Using Magnetic- and Polystyrene-Bead Immobilized Polyclonal Antibodies.

Procalcitonin (PCT) is a biomarker for bacterial sepsis, and accurate quantification of PCT is critical for sepsis diagnosis and treatment. Immunological PCT quantification methods are routinely used in clinical laboratories, yet there is a need for harmonization of PCT quantification protocols. An orthogonal method to clinical immunological assays, such as LC-MS/MS, is required. In this study, a highly sensitive and robust immunoaffinity LC-MRM quantitative method for detecting procalcitonin in human serum has been developed. An initial comparison of immunocapture of PCT with a polyclonal anti-PCT antibody immobilized on polystyrene nanoparticles (Latex) and magnetic beads demonstrated superior performance with magnetic beads. Three tryptic PCT peptides from the N- and C-terminal regions of PCT were selected for LC-MS/MS quantification. For PCT quantification, an LLOQ of 0.25 ng/mL of PCT in human serum was achieved using a sample volume of 1 mL. The method's trueness and precision consistently lie within the 15% margin. The parallel measurement of three PCT peptides may allow future differentiation of intact PCT vs other PCT forms originating from potential degradation, processing, or polymorphisms. An established and validated LC-MRM-based quantification of PCT will be relevant as an orthogonal method for harmonization and standardization of clinical assays for PCT.

Endoscopic Submucosal Dissection of a Giant Rectal Adenoma Manifesting as McKittrick-Wheelock Syndrome.

McKittrick-Wheelock syndrome (MKWS) is an uncommon clinical manifestation of large, villous, epithelial lesions of the distal colon and rectum. Excessive secretion of electrolyte-rich mucus from these lesions leads to secretory diarrhea, electrolyte disorders and acute renal failure. Several cases of MKWS have been reported since its initial description in 1954. The definitive treatment for the great majority of MKWS cases has consisted of surgical resection of the affected part of the colorectum, usually in the form of a low anterior resection or an abdominoperineal resection with the formation of an ostomy. Recent developments in endoscopic resection techniques now offer new, minimally invasive treatment alternatives for MKWS patients. We present the first reported case in the Western world of MKWS caused by a rectal adenoma with a size of 19 × 10 cm, treated through endoscopic submucosal dissection. Through the lessons learned by this case, as well as by a thorough review of the literature, we discuss this uncommon syndrome, focusing on treatment alternatives.

Comprehensive Comparison of the Capacity of Functionalized Sepharose, Magnetic Core, and Polystyrene Nanoparticles to Immuno-Precipitate Procalcitonin from Human Material for the Subsequent Quantification by LC-MS/MS.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The fast and accurate diagnosis of sepsis by procalcitonin (PCT) has emerged as an essential tool in clinical medicine. Although in use in the clinical laboratory for a long time, PCT quantification has not yet been standardized. The International Federation of Clinical Chemistry working group on the standardization of PCT (IFCC-WG PCT) aims to provide an LC-MS/MS-based reference method as well as the highest metrological order reference material to address this diagnostic need. Here, we present the systematic evaluation of the efficiency of an immuno-enrichment method, based on functionalized Sepharose, magnetic-core, or polystyrene (latex) nano-particles, to quantitatively precipitate PCT from different human sample materials. This method may be utilized for both mass spectrometric and proteomic purposes. In summary, only magnetic-core nano-particles functionalized by polyclonal PCT antibodies can fulfil the necessary requirements of the international standardization of PCT. An optimized method proved significant benefits in quantitative and specific precipitation as well as in the subsequent LC-MS/MS detection of PCT in human serum samples or HeLa cell extract. Based on this finding, further attempts of the PCT standardization process will utilize a magnetic core-derived immuno-enrichment step, combined with subsequent quantitative LC-MS/MS detection.

Distributions for time, interspecies and intraspecies extrapolation for deriving occupational exposure limits.

This work aimed at improving the empirical database of time (i.e., exposure duration), interspecies and intraspecies extrapolation when deriving occupational exposure limits (OELs). For each extrapolation step, a distribution was derived, which can be used to model the associated uncertainties. For time and interspecies extrapolation, distributions of ratios of dose descriptors were derived from studies of different length or species. National Toxicology Program (NTP) study data were manually assessed, and data from REACH (Registration, Evaluation and Authorisation of Chemicals) registration dossiers were evaluated semi-automatically. Intraspecies extrapolation was investigated by compiling published studies on human toxicokinetic and toxicodynamic variability. A new database was established for toxicokinetic differences in interindividual susceptibility, including many inhalation studies. Using NTP data produced more reliable results than using REACH data. The geometric mean (GM) for time extrapolation subacute/chronic agreed with previous evaluations (GM = 4.11), whereas the GM for subchronic/chronic extrapolation was slightly higher (GM = 2.93) than the GMs found by others. No significant differences were observed between systemically and locally acting substances. Observed interspecies differences confirmed the suitability of allometric scaling, with the derived distribution describing remaining uncertainty. Distributions of intraspecies variability at the 1% and 5% incidence level had medians of 7.25 and 3.56, respectively. When compared with assessment factors (AFs) currently used in the EU, probabilities that these AFs are protective enough span a wide range from 10% to 95%, depending on the extrapolation step. These results help to select AFs in a transparent and informed way and, by allowing to compare protection levels achieved, to harmonise methods for deriving OELs.

Derivation of occupational exposure limits: Differences in methods and protection levels.

Frameworks for deriving occupational exposure limits (OELs) and OEL-analogue values (such as derived-no-effect levels [DNELs]) in various regulatory areas in the EU and at national level in Germany were analysed. Reasons for differences between frameworks and possible means of improving transparency and harmonisation were identified. Differences between assessment factors used for deriving exposure limits proved to be one important reason for diverging numerical values. Distributions for exposure time, interspecies and intraspecies extrapolation were combined by probabilistic methods and compared with default values of assessment factors used in the various OEL frameworks in order to investigate protection levels. In a subchronic inhalation study showing local effects in the respiratory tract, the probability that assessment factors were sufficiently high to protect 99% and 95% of the target population (workers) from adverse effects varied considerably from 9% to 71% and 17% to 87%, respectively, between the frameworks. All steps of the derivation process, including the uncertainty associated with the point of departure (POD), were further analysed with two examples of full probabilistic assessments. It is proposed that benchmark modelling should be the method of choice for deriving PODs and that all OEL frameworks should provide detailed guidance documents and clearly define their protection goals by stating the proportion of the exposed population the OEL aims to cover and the probability with which they intend to provide protection from adverse effects. Harmonisation can be achieved by agreeing on the way to perform the methodological steps for deriving OELs and on common protection goals.

A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease.

There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway-regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1(SER727) is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.

Evidence that formation of vimentin mitogen-activated protein kinase (MAPK) complex mediates mast cell activation following FcεRI/CC chemokine receptor 1 cross-talk.

Accumulating evidence points to cross-talk between FcεRI and CC chemokine receptor (CCR)-mediated signaling pathways in mast cells. Here, we propose that vimentin, a protein comprising type III intermediate filament, participates in such cross-talk for CCL2/monocyte chemotactic protein 1 (MCP-1) production in mast cells, which is a mechanism for allergic inflammation. Co-stimulation via FcεRI, using IgE/antigen, and CCR1, using recombinant CCL3/macrophage inflammatory protein-1α (MIP-1α), increased expression of phosphorylated, disassembled, and soluble vimentin in rat basophilic leukemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells) and bone marrow-derived murine mast cells, both models of mucosal type mast cells. Furthermore, co-stimulation enhanced production of CCL2 as well as phosphorylation of MAPK. Treating the cells with p38 MAPK inhibitor SB203580, but not with MEK inhibitor PD98058, reduced CCL2 production, suggesting that p38 MAPK, but not ERK1/2, plays a critical role in the chemokine production. Immunoprecipitation analysis showed that vimentin interacts with phosphorylated ERK1/2 and p38 MAPKs in the co-simulated cells. Preventing disassembly of the vimentin by aggregating vimentin filaments using ß,ß'-iminodipropionitrile reduced the interaction of vimentin with phosphorylated MAPKs as well as CCL2 production in the cells. Taken together, disassembled vimentin interacting with phosphorylated p38 MAPK could mediate CCL2 production in mast cells upon FcεRI and CCR1 activation.

Biomechanics and injury risk of a headbutt.

Headbutt is a relevant type of a criminal assault that can result in injuries. The aim of this study was to collect basic biomechanical data and assess the injury risk associated with a headbutt. Series of measurements were carried out with volunteers with and without relevant soccer heading experience, and the impact velocity of the striking head was measured. A soccer ball was used as a surrogate of the stationary victim's head. Two scenarios were considered: one corresponding to the typical headbutt situation, i.e. short movement of the assailant's head without backswing, and one representing the worst case, i.e. the most severe head impact without time or space constraints for the assailant. The results as well as epidemiological data from court cases and a large Munich's university clinic show that a typical headbutt is not likely to lead to life-threatening injuries, but bony injuries of the face can easily occur. Under certain circumstances (support of the victim's head, secondary impact on the ground etc.), severe injuries with potentially lethal outcomes are possible. A thorough analysis of each case is thus an imperative. The (soccer) heading experience does not influence the velocity of the headbutt.

Nature's polyoxometalate chemistry: X-ray structure of the Mo storage protein loaded with discrete polynuclear Mo-O clusters.

Some N(2)-fixing bacteria prolong the functionality of nitrogenase in molybdenum starvation by a special Mo storage protein (MoSto) that can store more than 100 Mo atoms. The presented 1.6 Å X-ray structure of MoSto from Azotobacter vinelandii reveals various discrete polyoxomolybdate clusters, three covalently and three noncovalently bound Mo(8), three Mo(5-7), and one Mo(3) clusters, and several low occupied, so far undefinable clusters, which are embedded in specific pockets inside a locked cage-shaped (αß)(3) protein complex. The structurally identical Mo(8) clusters (three layers of two, four, and two MoO(n) octahedra) are distinguishable from the [Mo(8)O(26)](4-) cluster formed in acidic solutions by two displaced MoO(n) octahedra implicating three kinetically labile terminal ligands. Stabilization in the covalent Mo(8) cluster is achieved by Mo bonding to Hisα156-N(ε2) and Gluα129-O(ε1). The absence of covalent protein interactions in the noncovalent Mo(8) cluster is compensated by a more extended hydrogen-bond network involving three pronounced histidines. One displaced MoO(n) octahedron might serve as nucleation site for an inhomogeneous Mo(5-7) cluster largely surrounded by bulk solvent. In the Mo(3) cluster located on the 3-fold axis, the three accurately positioned His140-N(ε2) atoms of the α subunits coordinate to the Mo atoms. The formed polyoxomolybdate clusters of MoSto, not detectable in bulk solvent, are the result of an interplay between self- and protein-driven assembly processes that unite inorganic supramolecular and protein chemistry in a host-guest system. Template, nucleation/protection, and catalyst functions of the polypeptide as well as perspectives for designing new clusters are discussed.

Estimation of effective mass of longish rigid instruments in head impacts.

Impacts to the head are a common form of body violence and thus a relevant legal medical issue. Biomechanical assessment of injury potential has been traditionally based on qualitative analysis and experience. The aim of this study was to collect benchmark data that would facilitate the assessment of the maximum force in head impacts with longish rigid instruments. Series of measurements were performed with a specially designed modifiable impactor, and the relationship between its inertial properties and its effective mass during the impact was studied. The effective mass was defined as the amount of point mass that would, if exposed to the same velocity change as the striking end of the instrument, produce the same area under the force-time curve as the impactor. The results show that the effective mass decreases from approximately 100% of the total body mass for very short impactors to about 50% for longer (approximately 70 cm) impactors. No influence of the hand/grip force on the effective mass of the impactor was found if it was used in a hammer-like manner; other striking techniques can lead to substantial increase of the effective mass attributable to the hand/grip force.

Tracheoinnominate fistula: acute bleeding and hypovolemic shock due to a trachea-innominate artery fistula after long-term tracheostomy, treated with a stent-graft.

BACKGROUND: A tracheo-innominate fistula is a rare but life-threatening complication of tracheostomy and has a mortality rate of 100% without therapy. The underlying cause is an acquired fistula between the brachiocephalic trunk and the trachea, induced by a tracheostomy cannula's mechanical impact. CASE PRESENTATION: A 25-year-old female was admitted with pulsatile bleeding from a tracheostomy. The cause of the bleeding was a tracheo-innominate artery fistula, which was difficult to recognize. Said fistula was treated with implantation of a self-expanding stent-graft. The bleeding stopped immediately after the implantation of the stent-graft. Dual antiplatelet medication with aspirin IV and ticagrelor PO, bridged with a bolus of eptifibatide IV, was started right after the stent deployment. CONCLUSIONS: Endovascular self-expanding stent-graft implantation is a viable treatment option for tracheo-innominate artery fistulae, especially in hemorrhagic emergencies.

Consumer exposure to biocides--identification of relevant sources and evaluation of possible health effects.

BACKGROUND: Products containing biocides are used for a variety of purposes in the home environment. To assess potential health risks, data on products containing biocides were gathered by means of a market survey, exposures were estimated using a worst case scenario approach (screening), the hazard of the active components were evaluated, and a preliminary risk assessment was conducted. METHODS: Information on biocide-containing products was collected by on-site research, by an internet inquiry as well as research into databases and lists of active substances. Twenty active substances were selected for detailed investigation. The products containing these substances were subsequently classified by range of application; typical concentrations were derived. Potential exposures were then estimated using a worst case scenario approach according to the European Commission's Technical Guidance Document on Risk Assessment. Relevant combinations of scenarios and active substances were identified. The toxicological data for these substances were compiled in substance dossiers. For estimating risks, the margins of exposure (MOEs) were determined. RESULTS: Numerous consumer products were found to contain biocides. However, it appeared that only a limited number of biocidal active substances or groups of biocidal active substances were being used. The lowest MOEs for dermal exposure or exposure by inhalation were obtained for the following scenarios and biocides: indoor pest control using sprays, stickers or evaporators (chlorpyrifos, dichlorvos) and spraying of disinfectants as well as cleaning of surfaces with concentrates (hydrogen peroxide, formaldehyde, glutardialdehyde). The risk from aggregate exposure to individual biocides via different exposure scenarios was higher than the highest single exposure on average by a factor of three. From the 20 biocides assessed 10 had skin-sensitizing properties. The biocides isothiazolinone (mixture of 5-chloro-2-methyl-2H-isothiazolin-3-one and 2-methyl-2H-isothiazolin-3-one, CMI/MI), glutardialdehyde, formaldehyde and chloroacetamide may be present in household products in concentrations which have induced sensitization in experimental studies. CONCLUSIONS: Exposure to biocides from household products may contribute to induction of sensitization in the population. The use of biocides in consumer products should be carefully evaluated. Detailed risk assessments will become available within the framework of the EU Biocides Directive.

ERASSTRI - European risk assessment study on synthetic turf rubber infill - Part 3: Exposure and risk characterisation.

As the final part of a Europe-wide study on the risk from synthetic turf infill consisting of rubber granules derived from end-of-life tyres (ELT), exposure of sportspeople was assessed and compared with health-based reference values for various chemical substances. Based on information from previous project phases, exposure scenarios were established and exposure was calculated for oral, dermal and inhalation routes. Calculated cancer risks for exposure to polycyclic aromatic hydrocarbons were below 1:1 million. Risk characterisation ratios (RCRs) for non-carcinogenic substances were below 1, indicating no health concerns. For 2-hydroxybenzothiazole no toxicological data were found from which to derive a substance-specific reference value. A threshold-of-toxicological concern approach revealed maximum RCRs slightly above 1, which are acceptable, given the conservativism of the approach. ERASSTRI substantially improved the data available for assessing human health risks from using ELT-derived infill material. Overall, no health concerns could be identified for the use of synthetic turfs with ELT-derived infill material.

ERASSTRI - European Risk Assessment Study on Synthetic Turf Rubber Infill - Part 2: Migration and monitoring studies.

End-of-life tyre (ELT)-derived rubber granules are used as synthetic turf infill on sports fields. They contain various chemical substances and there are concerns that exposure to these substances might be harmful for human health. In this second part of a Europe-wide study to address these concerns migration of substances from rubber granules to artificial body fluids (sweat, saliva, gastric juice) was tested and exposure measurements at sports fields were performed to improve the database for exposure assessment. Some PAHs, aluminium, cobalt, benzothiazole, tert-butylamine, MIBK, 4-tert-octylphenol, bisphenol A, and the phthalates DINP and DEHP were found in at least some samples of sweat simulant. The migration rates calculated with these data were used to inform the dermal exposure assessment. In artificial saliva and gastric juice, only aluminium, cobalt, 4-tert-octylphenol and MIBK were detected and migrated fractions were calculated. Bioaccessibility from rubber granules in the gastrointestinal tract was estimated conservatively, assuming complete availability for most substances. In addition, air samples from 17 sports fields in six European countries were analysed. There were no increased concentrations of metals (aluminium, cobalt), PAHs, or other semivolatile substances in air samples, but some volatiles (MIBK: 95th percentile: 18 µg/m3, benzothiazole: 95th perc.: 7 µg/m3, tert-butylamine: 95th perc.: 31 µg/m3, 2-heptanone: 95th perc.: 0.4 µg/m3, cyclohexanone: 95th perc.: 1.5 µg/m3, and saturated aliphatic hydrocarbons >C9: 95th perc.: 26 µg/m3) were slightly increased in a few samples. In addition, skin wipe samples were obtained from 43 sportspeople after playing on synthetic turfs. Only aluminium was detected above the limit of quantification in these samples (95th perc.: 0.84 mg/sample). These data are important input for risk characterisation as performed in the final study phase. Bioaccessibility data are used for estimating oral and dermal exposure of sportspeople, and air measurements are essential for inhalation exposure assessment.

ERASSTRI - European Risk Assessment Study on Synthetic Turf Rubber Infill - Part 1: Analysis of infill samples.

End-of-life tyre (ELT)-derived rubber granules are used as synthetic turf infill on sports fields. They contain various chemical substances and there are concerns that exposure to these substances might be harmful for human health. This Europe-wide risk assessment study addresses these concerns. As the first part, chemical substances in samples from recycling companies and from sports fields were analysed. 86 coated and non-coated ELT granule samples from sites in 14 European countries were investigated, together with ten non-ELT materials. An extensive list of potentially relevant substances was compiled, and the infill materials were analysed for these substances, using GC and HPLC methods. Volatilisation of substances was studied in emission chambers. Polycyclic aromatic hydrocarbons (sum of 8 REACH PAHs) were identified at average concentrations below 10 mg/kg. Substances found at higher concentrations in rubber granules were aluminium (arithmetic mean in uncoated samples from sports fields 5383 mg/kg) and cobalt (168 mg/kg), benzothiazole (48 mg/kg) and 2-hydroxybenzothiazole (34 mg/kg), 6PPD (571 mg/kg) and DPG (51 mg/kg), and 4-tert-octylphenol (14 mg/kg). In addition, the following volatiles were found to evaporate from crumb rubber in emission chambers: benzothiazole, tert-butylamine, cyclohexanone, methyl isobutyl ketone, 2-heptanone and saturated aliphatic hydrocarbons higher than C9. With this comprehensive survey we created a profound database on concentrations of chemical substances in ELT-derived infill material, which is essential for a reliable risk assessment. The results were used to inform subsequent investigations (migration studies, exposure monitoring survey).

Oral exposure to inorganic arsenic: evaluation of its carcinogenic and non-carcinogenic effects.

Inorganic arsenic, which is extensively metabolised in humans into even more toxic methylated arsenicals, is a potent carcinogen, causing tumours of the skin, lung, urinary bladder, and other organs. It also induces a number of non-cancer effects. Consumption of drinking water highly contaminated by arsenic causes serious health problems in some countries in southeastern Asia, and arsenic poses problems for drinking-water safety world-wide. Existing risk assessments are based on epidemiological studies from regions with high exposure concentrations (in the mg/L range). It is a matter of debate whether these findings are useful at predicting arsenic-induced effects at low concentrations. In recent years numerous epidemiological studies on cancer and non-cancer effects of inorganic arsenic have been published. This work aims at reviewing recent toxicological and epidemiological data on inorganic arsenic with emphasis on effects at low exposure concentrations. Information obtained from epidemiological studies is supplemented with mechanistic data from in vitro and in vivo studies. Various modes of action for arsenic carcinogenicity are discussed. The information gathered was used to evaluate the reliability of existing cancer-risk assessments and to improve current assessments of non-cancer health effects. A tolerable daily dose, based on epidemiological studies on arsenic-induced skin disorders, is presented.

Consumer behaviour survey for assessing exposure from consumer products: a feasibility study.

Evaluating chemical exposures from consumer products is an essential part of chemical safety assessments under REACH and may also be important to demonstrate compliance with consumer product legislation. Modelling of consumer exposure needs input information on the substance (e.g. vapour pressure), the product(s) containing the substance (e.g. concentration) and on consumer behaviour (e.g. use frequency and amount of product used). This feasibility study in Germany investigated methods for conducting a consumer survey in order to identify and retrieve information on frequency, duration, use amounts and use conditions for six example product types (four mixtures, two articles): hand dishwashing liquid, cockpit spray, fillers, paints and lacquers, shoes made of rubber or plastic, and ball-pens/pencils. Retrospective questionnaire methods (Consumer Product Questionnaire (CPQ), and Recall-Foresight Questionnaire (RFQ)) as well as protocol methods (written reporting by participants and video documentation) were used. A combination of retrospective questionnaire and written protocol methods was identified to provide valid information in a resource-efficient way. Relevant information, which can readily be used in exposure modelling, was obtained for all parameters and product types investigated. Based on the observations in this feasibility study, recommendations are given for designing a large consumer survey.

Investigation of Reduced ELISA Recovery of Almond and Hazelnut Traces from Roasted Nut Samples by SDS-PAGE and Mass Spectrometry.

Western society is facing an increase in the number of food-allergic individuals, with rising incidence in the past years. Therefore, allergen-free food and accurate and reliable analysis of allergen contamination are essential for the protection of consumers. Yet, there is limited understanding on the effect of food processing on allergenicity and on the ability of available methods to detect trace contamination in processed food. Available studies addressing this have relied on sample processing on a laboratory scale. In this study, industry-like processing under precisely defined conditions (ranging from 110 to 150°C roasting temperatures) was employed to better understand the limitations of state-of-the-art methods for detecting traces of hazelnut and almond in processed food. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated an overall reduction in extracted proteins from roasted nut samples, and with matrix-assisted laser desorption ionization time-of-flight Cor a 9 and Prunin, were identified as majorly expressed proteins for hazelnut and almond, respectively. A commercial ELISA kit detected nut traces only up to a 130°C roasting temperature. Untargeted MS (Orbitrap) analysis was able to detect traces of nuts roasted up to 150°C while also confirming Cor a 9 and Prunin as the major expressed proteins for hazelnut and almond, respectively. Preparing cookie dough spiked with roasted nut samples, a complex food matrix was simulated. Analysis by ELISA showed the same limitations encountered for pure nuts samples, hardly detecting traces of nuts roasted above 130°C. Targeted MS (linear ion trap) using multiple reaction monitoring methods for one proteotypic peptide for Cor a 9 and Prunin, respectively, enabled a detection of nut traces up to 150°C. The results indicated that a reduced extractability because of temperature-related effects (e.g., protein denaturation, cross-linking, poor solubility) caused the significant differences between the ELISA and MS analysis. Overall, the results of this study may form the basis to improve allergen detection after roasting through improved extraction methods and refined ELISA formats.

Azotobacter vinelandii metal storage protein: "classical" inorganic chemistry involved in Mo/W uptake and release processes.

The release of Mo (as molybdate) from the Mo storage protein (MoSto), which is unique among all existing metalloproteins, is strongly influenced by temperature and pH value; other factors (incubation time, protein concentration, degree of purity) have minor, though significant effects. A detailed pH titration at 12 degrees C revealed that three different steps can be distinguished for the Mo-release process. A proportion of approximately 15% at pH 6.8-7.0, an additional 25% at pH 7.2-7.5 and ca. 50% (up to 90% in total) at pH 7.6-7.8. This triphasic process supports the assumption of the presence of different types of molybdenum-oxide-based clusters that exhibit different pH lability. The complete release of Mo was achieved by increasing the temperature to 30 degrees C and the pH value to >7.5. The Mo-release process does not require ATP; on the contrary, ATP prevents, or at least reduces the degree of metal release, depending on the concentration of the nucleotide. From this point of view, the intracellular ATP concentration is suggested to play-in addition to the pH value-an indirect but crucial role in controlling the extent of Mo release in the cell. The binding of molybdenum to the apoprotein (reconstitution process) was confirmed to be directly dependent on the presence of a nucleotide (preferably ATP) and MgCl2. Maximal reincorporation of Mo required 1 mM ATP, which could partly be replaced by GTP. When the storage protein was purified in the presence of ATP and MgCl2 (1 mM each), the final preparation contained 80 Mo atoms per protein molecule. Maximal metal loading (110-115 atoms/MoSto molecule) was only achieved, if Mo was first completely released from the native protein and subsequently (re-) bound under optimal reconstitution conditions: 1 h incubation at pH 6.5 and 12 degrees C in the presence of ATP, MgCl2 and excess molybdate. A corresponding tungsten-containing storage protein ("WSto") could not only be synthesized in vivo by growing cells, but could also be constructed in vitro by a metalate-ion exchange procedure by using the isolated MoSto protein. The high W content of the isolated cell-made WSto (approximately 110 atoms/protein molecule) and the relatively low amount of tungstate that was released from the protein under optimal "release conditions", demonstrates that the W-oxide-based clusters are more stable inside the protein cavity than the Mo-oxide analogues, as expected from the corresponding findings in polyoxometalate chemistry. The optimized isolation of the W-loaded protein form allowed us to get single crystals, and to determine the crystal X-ray structure. This proved that the protein contains remarkably different types of polyoxotungstates, the formation of which is templated in an unprecedented process by the different protein pockets. (Angew. Chem. Int. Ed. 2007, 46, 2408-2413).