Anne-Christine Schmidt

Evaluation of extraction procedures for the ion chromatographic determination of arsenic species in plant materials

The determination of arsenic species in plants grown on contaminated sediments and soils is important in order to understand the uptake, transfer and accumulation processes of arsenic. For the separation and detection of arsenic species, hyphenated techniques can be applied successfully in many cases. A lack of investigations exists in the handling (e.g., sampling, pre-treatment and extraction) of redox- and chemically labile arsenic species prior to analysis. This paper presents an application of pressurized liquid extraction (PLE) using water as the solvent for the effective extraction of arsenic species from freshly harvested plants. The method was optimized with respect to extraction time, number of extraction steps and temperature. The thermal stability of the inorganic and organic arsenic species under PLE conditions (60-180 degrees C) was tested. The adaptation of the proposed extraction method to freeze-dried, fine-grained material was limited because of the insufficient reproducibility in some cases.

New clusters of arsenite oxidase and unusual bacterial groups in enrichments from arsenic-contaminated soil

In the present study cultivation-dependent and molecular methods were applied in combination to investigate the arsenite-oxidizing communities in enrichment cultures from arsenic and lead smelter-impacted soils with respect to both 16S rRNA and arsenite oxidase gene diversity. Enrichments with arsenite as the only electron donor resulted in completely different communities than enrichments with yeast extract and the simultaneous presence of arsenite. The lithoautotrophic community appeared to be dominated by Ferrimicrobium-related Actinobacteria, unusual Acidobacteria, Myxobacteria, and α-Proteobacteria but the heterotrophic community comprised many Dokdonella-related γ-Proteobacteria. Gene sequences of clones encoding arsenite oxidase from the enrichment for lithoautotrophs belonged to three major clusters with sequences from non-cultivated microorganisms. So, primers used to detect arsenite oxidase genes could amplify the genes from many α-, β- and γ-Proteobacteria, but not from various strains of the other phyla present in the enrichment for lithotrophs. This was also observed for the isolates where arsenite oxidase genes from new proteobacterial isolates of the genera Burkholderia, Bosea, Alcaligenes, Bradyrhizobium and Methylobacterium could be amplified but the genes of the new Rhodococcus isolate S43 could not. The results indicate that the ability to oxidize arsenite is widespread in various unusual taxa, and molecular methods for their detection require further improvement.

Analysis of accumulation, extractability, and metabolization of five different phenylarsenic compounds in plants by ion chromatography with mass spectrometric detection and by atomic emission spectroscopy

Phenylated arsenic compounds occur as highly toxic contaminants in former military areas where they were formed as degradation products of chemical warfare agents. Some phenylarsenic compounds such as roxarsone and aminophenylarsonic acids were applied as food additive and veterinary drugs in stock-breeding and therefore pose an environmental risk in agricultural used sites. Very few data exist in the literature concerning uptake and effects of phenylarsenic compounds in plants growing on contaminated soils. In this study, the accumulation, extractability, and metabolization of five different phenylarsenic compounds, phenylarsonic acid, p- and o-aminophenylarsonic acid, phenylarsine oxide, and 3-nitro-4-hydroxyphenylarsonic acid called roxarsone, by the terrestrial plant Tropaeolum majus were investigated. Ion chromatography coupled to inductively coupled plasma mass spectrometry was used to differentiate these arsenic compounds, and inductively coupled plasma atomic emission spectroscopy was used for total arsenic quantification. All compounds considered were taken up by the roots and transferred to stalks, leaves, and flowers. The strongest accumulation was observed for unsubstituted phenylarsonic acid followed by its trivalent analogue phenylarsine oxide that was mostly oxidized in soil whereas the amino- or nitro- and hydroxy-substituted phenylarsonic acids were accumulated to a smaller degree. The highest extraction yield of 90% for ground leaf material was achieved by 0.1M phosphate buffer, pH 7.7, in a two-step extraction with a total extraction time of 24h. The extraction of higher amounts of arsenic (50-70% of total arsenic present in leaves depending on arsenic species application) from non-ground intact leaves with deionized water in comparison with the buffer (20-40% of total arsenic) is ascribed to osmotic effects. The arsenic species analysis revealed a cleavage of the amino groups from the phenyl ring for plants treated with aminophenylarsonic acids. A further important metabolic effect consisted in the production of inorganic arsenate and arsenite from the phenylated arsonic acid groups.

Analysis of arsenic species accumulation by plants and the influence on their nitrogen uptake

Terrestrial plants are able to accumulate arsenic to a substantial extent but survive the stress to differing degrees of vitality. The influence of arsenic on important energy and metabolic cycles does not yet have sufficient explanation. Parallel to the uptake and processing of arsenic species such as As(III) and As(V) by Silene vulgaris, the nitrogen uptake using a 15N tracer method was investigated. The results showed that the nitrogen uptake decreases with increasing arsenate concentrations applied to the plants. The reaction of the plants treated with arsenite changed from a depression at low arsenite concentrations to a strong increase with the largest quantity applied, exceeding the 15N-incorporation of the control plants. This behaviour underlines the divergent behaviour of the N-metabolism caused by both arsenic species. As(III) can be detoxified by complexation with peptides rich in SH-groups. As(V) acts as a phosphate analogue and interrupts diverse phosphorylation reactions.

Evaluation of the arsenic binding capacity of plant proteins under conditions of protein extraction for gel electrophoretic analysis.

As prerequisite for the investigation of arsenic-binding proteins in plants, the general influence of different extraction parameters on the binding behaviour of arsenic to the plant protein pool was investigated. The concentration of the extraction buffer affected the extraction yield both for proteins and for arsenic revealing an optimal buffer concentration of 5mM Tris/HCl, pH 8. The addition of 1 or 2% (w/v) SDS to the extraction buffer produced a two- to threefold enhancement of the total protein extraction yield but strongly suppressed the simultaneous extraction of arsenic from 80+/-8% extraction yield obtained without SDS to 48+/-2% in presence of 2% (w/v) SDS. The arsenic binding capacity of the protein fraction obtained after extraction with Tris buffer and protein precipitation by trichloroacetic acid in acetone was estimated to be 1.4+/-0.6% independently on the original spiking concentration of arsenic provided in the form of monomethylarsonate to the extracts. Due to the low total protein concentrations of the plant extracts that varied in the range from 75 to 412 microgmL(-1) depending on the extraction parameters, high arsenic concentrations of 263-1001 mg (kgproteinmass)(-1) resulted for spiking concentrations of 10 mgAsL(-1). The optimized protein isolation procedure was applied to plants grown under arsenic exposure and revealed a similar arsenic binding capacity as for the spiked protein extracts.

Size exclusion chromatography coupled to electrospray ionization mass spectrometry for analysis and quantitative characterization of arsenic interactions with peptides and proteins

Arsenic-binding proteins are of toxicological importance since enzymatic activities can be blocked by arsenic interactions. In the present work, a novel methodology based on size exclusion chromatography coupled to electrospray ionization mass spectrometry (SEC-ESI-MS) was developed with special emphasis to preserve the intact proteins and their arsenic bindings. The eluent composition of 25 mMTris/HCl, pH 7.5, with the addition of 100-mM NaCl optimized for SEC with UV detection provided the highest SEC separation efficiency, but was not compatible with the ESI-MS because of the non-volatility of the buffer substance and of the salt additive. In order to find the best compromise between chromatographic separation and ionization of the arsenic-binding proteins, buffer type and concentration, pH value, portion of organic solvent in the SEC eluent as well as the flow rate were varied. In the optimized procedure five different arsenic-binding peptides and proteins (glutathione, oxytocin, aprotinin, alpha-lactalbumin, thioredoxin) covering a molar mass range of 0.3-14 kDa could be analyzed using 75% 10-mM ammonium formate, pH 5.0/25% acetonitrile (v : v) as eluent and a turbo ion spray source operated at 300 degrees C and 5.5 kV. A complete differentiation of all peptides and proteins involved in the arsenic-binding studies as well as of their arsenic-bound forms has become feasible by means of the extracted ion chromatograms (XIC) of the mass spectrometric detection. The new method offered the possibility to estimate equilibrium constants for the reaction of phenylarsine oxide with different thiol-containing biomolecules by means of the XIC peak areas of reactants and products. Limits of detection in the range of 2-10 microM were obtained by SEC-ESI-MS for the individual proteins.

A systematic study on extraction of total arsenic from down-scaled sample sizes of plant tissues and implications for arsenic species analysis.

An easily feasible, species-conserving and inexpensive protocol for the extraction of total arsenic and arsenic species from terrestrial plants was designed and applied to the investigation of accumulation and metabolization of arsenite (As(III)), arsenate (As(V)), monomethylarsonate (MMA(V)), and dimethylarsinate (DMA(V)) by the model plant Tropaeolum majus. In contrast to existing extraction methods hazardous additives and elaborate procedures to enhance the extraction yields were omitted. The proposed protocol is suited to down-scale the sample sizes used for the extractions and to promote a compartmentally resolved analysis of the arsenic distribution within individual leaves, leaf stalks, and stems instead of the conventional extraction of pooled samples. In a two-step extraction, the high extraction efficiencies (85-92%) for arsenic achieved by phosphate buffer from larger amounts (200mg) of homogenized leaf material in a one-step extraction, could be enhanced to 94-100% in a second extraction step. A strong dependence of the arsenic extractability on the type of arsenic species accumulated in the tissue as well as on the type of the tissue (leaf, leaf stalk, stem) was found. For the extraction of 5mm long segments cut from individual leaves without previous homogenization of the plant parts yields between 75 and 93% depending on arsenic species prevailing in the cells were obtained using 1 or 10mM phosphate buffer. The total extraction and analysis protocol was validated using a standard reference material as well as by spiking experiments. The arsenic species analysis by IC/ICPMS revealed a number of nine unidentified metabolites in the plant extracts in addition to the species MMA(V), DMA(V), As(III), and As(V) that were provided to the plants during their growth phase.

Some critical aspects in the determination of binding constants by electrospray ionisation mass spectrometry at the example of arsenic bindings to sulphur-containing biomolecules.

The influences of reactant concentrations, solvent type, acid strength, pH conditions and ionic strength on the determination of apparent gas-phase equilibrium constants K using electrospray ionisation mass spectrometry (ESI-MS) were elucidated. As example serves the interaction of the tripeptide glutathione (GSH) with phenylarsine oxide (PAO). It was shown that rising initial concentrations of both reactants were not adequately compensated by increasing signal intensities of the reaction products in the mass spectra. The equilibrium constant for the formation of the phenylarsenic-substituted peptide species decreased from 1.42 x 10(5) +/- 1.81 x 10(4) l micromol(-1) to 1.54 x 10(4) +/- 1.5 x 10(3) l micromol(-1) with rising initial GSH concentrations from 1 to 10 microM at fixed PAO molarity of 50 microM. K values resulting from a series with a fixed GSH molarity of 5 microM and a PAO molarity varied from 10 to 100 microM remained in a narrower range between 4.59 x 10(4) +/- 2.15 x 10(4) l micromol(-1) and 1.07 x 10(4) +/- 4.0 x 10(3) l micromol(-1). In contrast, consumption numbers calculated from the ion intensity ratios of reaction products to the unreacted peptide were not influenced by the initial reactant concentrations. In a water-acetonitrile-acetic acid mixture (48:50:2, v:v), the consumption of 5 micro M GSH increased from 8.3 +/- 1.4% to 39.6 +/- 1.6% with increased molar excess of PAO from 2 to 20, respectively. The GSH consumption was considerably enhanced in a changed solvent system consisting of 25% acetonitrile and 75% 10 mM ammonium formate, pH 5.0 (v:v) up to 80% of the original peptide amount at an only threefold molar arsenic excess.

Influence of one- and two-dimensional gel electrophoresis procedure on metal–protein bindings examined by electrospray ionization mass spectrometry, inductively coupled plasma mass spectrometry, and ultrafiltration

Three independent methods, (i) electrospray ionization mass spectrometry (ESI-MS), (ii) carrying out the complete protein preparation procedure required for protein gel electrophoresis (GE) including extraction, precipitation, washing, and desalting with subsequent microwave digestion of the produced protein fractions for metal content quantification, and (iii) ultrafiltration for separating protein-bound and unbound metal fractions, were employed to elucidate the influences of protein sample preparation and GE running conditions on metal-protein bindings. A treatment of the protein solution with acetone instead of trichloroacetic acid or ammonium sulfate for precipitate formation led to a strongly enhanced metal binding capacity. The desalting step of the resolubilized protein sample caused a metal loss between 10 and 35%. The omission of some extraction buffer additives led to a diminished metal binding capacity of protein fractions obtained from the sample preparation procedure for GE, whereas a tenside addition to the protein solution inhibited metal-protein bindings. The binding stoichiometry of Cu and Zn-protein complexes determined by ESI-MS was influenced by the type of the metal salt which was applied to the protein solution. A higher pH value of the sample solution promoted the metal ion complexation by the proteins. Ultrafiltration experiments revealed a higher Cu- and Zn-binding capacity of the model protein lysozyme in both resolubilization buffers for 1D- and 2D-GE compared to the protein extraction buffer. Strongly diminished metal binding capacities of lysozyme were recorded in the running buffer of 1D-GE and in the gel staining solutions.

Optimization of peptide and protein separation with a monolithic reversed-phase column and application to arsenic-binding studies

A separation method for a mixture of eight sulfur-containing peptides and proteins characterized by a wide molar mass (1-18.4 kDa) and pI range (4.5-10.7) was developed onto a monolithic phenyl phase. Based on the first optimization steps that revealed an increase of the acetonitrile content to 45 vol.% as sufficient for the elution of all biomolecules and the addition of the ion pairing reagent trichloroacetic acid (TCA) as preferable over the eluent additives formic acid or ammonium acetate buffer, the critical variables TCA concentration, gradient time, and eluent flow rate were optimized using a Box-Behnken experimental design. To achieve optimum values for separation factors of all peak pairs, a TCA content of 0.025% (m/v), a gradient time of 10 min, and a flow rate of 3.5 mL min(-1) were selected. Arsenic binding studies were undertaken under conditions optimized with respect to the crucial separation factor of the nonapeptides vasotocin (Vtc) and vasopressin (Vpr) in a shortened gradient time of 7.5 min. A complete separation of phenylarsenic-substituted and unmodified forms of these peptides allowed the calculation of both consumptions and apparent equilibrium constants K from HPLC-UV peak areas. The nonapeptide consumptions by the reaction with phenylarsine oxide (PAO) increased from 7% up to 100% in dependence on the molar ratio of the reaction components. Due to an enhanced UV absorption of the phenylarsenic-substituted biomolecules, the calculation of apparent equilibrium constants led to increasing K values with rising PAO molarities from 9.6×10(5) to 1.2×10(8) in case of Vtc and from 2.2×10(6) to 1.4×10(9) in case of Vpr. For α-lactalbumin, a consumption of 59.2±6.1% by the reaction with molar excesses of PAO varying from 1.4 to 21 can be derived from the chromatograms. The quantitative evaluation of the reaction of the small protein aprotinin with PAO was hindered by a pronounced peak broadening that occurred after reduction of the disulfide bridges.