A Pitfall in Heavy Metal Separation with Amino-modified Silica Adsorbents from Aqueous Solution: The Occurring pH Shift.

Selective separation of heavy metal ions from acidic aqueous solutions is of strong interest for certain industrial processes, such as electroplating, as well as environmental protection, for example battery recycling. Amino-functionalized adsorbents are often discussed as suitable material for this purpose. Herein, two silica-based adsorbents functionalized with 3-aminopropyl- and 3-[2-[2-aminoethylamino]-ethylamino]-propyl-ligands resulting in adsorbents MonoA and TriA, respectively, were investigated regarding their separation behavior with focus on nickel(II) and cobalt(II) in batch as well as continuous flow experiments in acidic aqueous solutions. For both adsorbents, pH shifts into the alkaline range were observed in the process solutions, causing precipitation of metal hydroxides mainly in the particle pores in case of adsorbent MonoA and a combination of precipitation and adsorption regarding adsorbent TriA. Contrary to prior studies, our findings evidence that amino-functionalized adsorbents are not applicable for nickel(II) and cobalt(II) in selective adsorption processes and additionally demonstrate that, besides batch investigations, continuous flow experiments are essential for well-founded adsorbent selections in process development.

Investigation on the proteome response of transplanted blue mussel (Mytilus sp.) during a long term exposure experiment at differently impacted field stations in the German Bight (North Sea).

In a pilot field study the proteome response of Mytilus sp. was analyzed in relation to the concentration of different trace metal contaminants. Over a period of eight month test organisms have been exposed at a near-shore station in the anthropogenic impacted estuary of the river Elbe and at an off-shore station in the vicinity of the Island of Helgoland in the German Bight (North Sea). The stations differ in their hydrological as well as chemical characteristics. The physiological biomarkers, such as condition index which have been continuously monitored during the experiment clearly indicate the effects of the different environmental conditions. Multiple protein abundance changes were detected utilizing the techniques of two dimensional gel electrophoresis (2dGE) and consequently proteins arising as potential candidates for ecotoxicological monitoring have been identified by MALDI-ToF and ToF/ToF mass spectrometry. Different cytoskeletal proteins, enzymes of energy metabolism, stress proteins and one protein relevant for metal detoxification have been pointed out.

Isolation of a Nav channel blocking polypeptide from Cyanea capillata medusae - a neurotoxin contained in fishing tentacle isorhizas.

Jellyfish are efficient predators which prey on crabs, fish larvae, and small fish. Their venoms consist of various toxins including neurotoxins that paralyse prey organisms immediately. One possible mode of action of neurotoxins is the blockage of voltage-gated sodium (Na(v)) channels. A novel polypeptide with Na(v) channel blocking activity was isolated from the northern Scyphozoa Cyanea capillata (L., 1758). For that purpose, a bioactivity-guided multidimensional liquid chromatographic purification method has been developed. A neurotoxic activity of resulting chromatographic fractions was demonstrated by a bioassay, which based on the mouse neuroblastoma cell line Neuro2A. The purification process yielded one fraction containing a single polypeptide with proven activity. The molecular weight of 8.22 kDa was determined by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-ToF MS). Utilising Laser Microdissection and Pressure Catapulting (LMPC) for the separation of different nematocyst types in combination with direct MALDI-ToF MS analysis of the intact capsules, the neurotoxin was found to be present in all types of fishing tentacle isorhizas (A-isorhizas, a-isorhizas, O-isorhizas) of C. capillata medusae.

A novel proteinaceous cytotoxin from the northern Scyphozoa Cyanea capillata (L.) with structural homology to cubozoan haemolysins.

It is well known that jellyfish are producers of complex mixtures of proteinaceous toxins for prey capture and defence. Nevertheless, studies on boreal scyphozoans concerning venom composition and toxic effects are rare. Here the isolation of a novel cytotoxic protein from the fishing tentacle venom of Cyanea capillata (L. 1758) using bioactivity-guided, multidimensional liquid chromatography is described. The crude venom was purified utilising preparative size-exclusion, ion-exchange, and reversed-phase chromatography. The cytotoxicity of resulting chromatographic fractions has been proven by a dye-uptake assay with the human hepatocyte cell line HepG2. The final purification step yielded, among other fractions, a fraction containing a single protein (named CcTX-1) with a molecular weight of its main isoform of 31.17 kDa The purification process leads to an increased cytotoxic activity per protein equivalents and the finally isolated CcTX-1 caused a nearly total loss of cell viability at a protein concentration of 1.3 µg mL⁻¹ corresponding to 0.4 µg/105 cells. De novo sequencing of CcTX-1 was conducted after enzymatic digestion and subsequent matrix-assisted laser desorption ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-ToF/ToF MS/MS). The obtained sequence data provide an approximate 85% description of the amino acid sequence. This sequence information partially matched that of two known haemolytic proteins of two cubozoan species: CaTX-1 from Carybdea alata Reynaud, 1830 and CrTX-1 from Carybdea rastonii Haacke, 1886.

A new method for the separation of different types of nematocysts from scyphozoa and investigation of proteinaceous toxins utilizing laser catapulting and subsequent mass spectrometry.

Jellyfish have an increasing impact on marine ecology. Cnidocysts bearing stinging cells afford, amongst others, prey capture and defence. Several different types of stinging capsules are found in one species and they are supposed to have specific functions, e.g. paralysing prey or adhering to it. Due to these assumed different roles of the capsules, it is suggested that toxins, which are contained in the capsules, differ in composition. Analysis of distinct types of nematocysts requires an appropriate method for the separation of the different types. Mixtures of types of nematocysts were obtained of two species of jellyfish, Aurelia aurita and Cyanea lamarckii, by maceration of the tissue. These mixtures were treated with a method called laser microdissection and pressure catapulting (LMPC). Optimized maceration methods, which were firstly introduced as a method for this purpose, in conjunction with optimized LMPC parameters lead to sufficient amounts of separated capsules of individual types for subsequent mass-spectrometric analyses. In case of A. aurita, the resulting mass spectra had some constituents in common, whereas in the overall pattern, the two distinct nematocyst types differed.

Characterization of haptoglobin in the blood plasma of harbor seals (Phoca vitulina).

Haptoglobin (Hp) is one of the acute phase proteins (APP) in the blood of vertebrates that is involved in immune responses. Hp concentrations are found to vary under conditions of, for example, infection, trauma or cancer. These variations and the changes in its constitution are frequently used to assess the health status of mammals. In this work, Hp from the blood plasma of diseased and healthy harbor seals was isolated and structurally characterized. The process developed for the isolation of Hp is based on glycoprotein enrichment from crude plasma samples by means of ConA lectin affinity separation. Structural features of the protein backbone and the N-glycans were determined using MALDI-TOF/TOF-MS. De novo sequencing of seal Hp revealed an alpha-chain composed of 84 amino acids and a beta-chain comprising 245 amino acids. Comparison with Hp of the phylogenically related dog and human Hp 1-1 reveals the conserved and variable regions. All cysteine residues responsible for disulfide bonds and one glycosylation site have identical positions in the primary structures. Altogether, four possible glycosylation sites were identified. The glycoprofile is dominated by disialylated biantennary complex-type glycans.

Isolation of a cytotoxic glycoprotein from the Scyphozoa Cyanea lamarckii by lectin-affinity chromatography and characterization of molecule interactions by surface plasmon resonance.

A biospecific lectin-affinity-based isolation process for a novel glycoprotein (ClGp1) from the venom of the pelagic jellyfish Cyanea lamarckii, is described and the isolated glycoprotein is chemically and biologically characterized according to size, molecular interaction and toxicity. The molecular mass of the isolated protein is 25.7 kDa as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). The carbohydrate content was calculated after enzymatic deglycosylation as 6.85 kDa. The glycoprotein is cytotoxic and could be isolated from cnidocysts of mesenteric and fishing tentacles. The binding behaviour of the glycoprotein to the lectins Concanavalin A (ConA) and Wheat Germ Agglutinin (WGA) was analyzed by surface plasmon resonance (SPR) and affinity constants in the range of K(D)=3.0 x 10(-7) M for ConA and 2.1 x 10(-6) M (pH 5.0) and 2.6 x 10(-6) M (pH 7.4) for WGA were obtained.

Nickel species analysis of human colonic tissue using liquid chromatography, gel electrophoresis and mass spectrometry.

Studies to specify metal-binding species, such as metalloproteins that are present in trace amounts in colonic cell cytosol, using chromatographic separation methods in combination with inductively coupled plasma mass spectrometry (ICP-MS) as element-specific detection require an optimised sample preparation regarding the solubilisation of the proteins. Focus should be taken to avoid metal contamination, enzymatic digestion by different proteases and oxidation. In this article different sample preparation methods are studied to find a suitable method for the isolation and characterisation of Ni species previously found in cytosols from normal and malignant tissues of the human colon. The total Ni concentrations of the cytosols were determined as well as the total protein content. Thus, a Ni-containing protein could be isolated from cytosols of malignant human colonic tissues using size-exclusion chromatography with ICP-MS for element-specific detection. Ni-containing species in the molecular mass range from 10,000 to 20,000 Da were found and pre-concentrated. The determination of the molecular mass of the species was performed through online coupling of reversed-phase chromatography with electrospray ionisation quadrupole time-of-flight MS. Using identical chromatographic conditions and ICP-MS the detected protein was shown to contain Ni.

Distribution of elements binding to molecules with different molecular weights in aqueous extract of Antarctic krill by size-exclusion chromatography coupled with inductively coupled plasma mass spectrometry.

The distribution of silver, arsenic, cadmium, cobalt, chromium, copper, iron, manganese, nickel, lead, selenium and zinc binding to species with different molecular weight in aqueous extract of krill was studied by on-line size-exclusion chromatography (SEC)/inductively coupled plasma mass spectrometry (ICP-MS). The extract was fractionated in three fractions with different molecular weight (MW) ranges (>20,000 relative molecular mass (rel. mol. mass), 2000-20,000 rel. mol. mass and <2000 rel. mol. mass), which were further analyzed by SEC with columns having different optimum fractionation ranges in order to obtain more detailed information about the MW distribution of the elements. Various distribution profiles for the target elements among different MW ranges were observed. The results obtained indicated that manganese, zinc, silver, cadmium and lead species were mostly distributed in the higher MW range (>20,000 rel. mol. mass). In the case of chromium, iron, cobalt, arsenic and selenium, most of them bind to species with lower MW (<2000 rel. mol. mass). Only copper and nickel species was predominantly present in middle MW range (2000-20,000 rel. mol. mass). Further speciation of arsenic compounds in the small MW fraction was carried out with anion exchange chromatography (AEC) coupled with ICP-MS. The results showed that the dominant arsenic species in this fraction is As(III) (63% of extractable arsenic), while As(V) (13%) and two unknown arsenic species (19% and 5%, respectively) are present in lower amounts.

Determination of the absolute configuration of selenomethionine from antarctic krill by RP-HPLC/ICP-MS using chiral derivatization agents.

A fast and sensitive method was developed for the determination of the absolute configuration of selenomethionine. The enantiomers of selenomethionine were converted into diastereomeric isoindole derivatives by reaction with o-phthaldialdehyde and N-isobutyryl-L-cysteine. This easy-to-handle reaction proceeds quantitatively in a few minutes at room temperature. Separation and detection of the diastereomers was achieved by reversed-phase high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (RP-HPLC/ICP-MS) using a conventional C18 reversed-phase column. Detection limits of about 4 microg L(-1) were obtained. The method was applied to the determination of the configuration of selenomethionine extracted from antarctic krill, which turned out to possess the L-configuration.