Synthesis, Kinetic Study, and Spectroscopic Analysis of Peroxidase-like Pinch-Porphyrin Fe(III) Complexes.

In the present manuscript, we report the kinetic and spectroscopic analysis of six new pinch-porphyrins: protoporphyrin-picpenta 1, mesoporphyrin-picpenta 2, deuteroporphyrin-picpenta 3, protoporphyrin-picocta 4, mesoporphyrin-picocta 5, and deuteroporphyrin-picocta 6. The Michaelis-Menten enzymatic pathway and the guaiacol test confirmed the ability of the compounds to function like new peroxidase models. UV-vis, 1H NMR, and electron spin resonance studies are in accordance with porphyrin-Fe(III) molecules with the quantum phenomena called quantum mixed spin (qms, s = 3/2, s = 5/2). Importantly, the influence of the presence of the s = 3/2 spin state in the compounds and its critical role for the catalytic capacity is proven here, which was the original hypothesis in our research group. The compounds with higher populations of the s = 3/2 spin state have increased peroxidase activity.

Spectroscopic and Kinetic Characterization of Peroxidase-Like π-Cation Radical Pinch-Porphyrin-Iron(III) Reaction Intermediate Models of Peroxidase Enzymes.

The spectroscopic and kinetic characterization of two intermediates from the H2O2 oxidation of three dimethyl ester [(proto), (meso), (deuteroporphyrinato) (picdien)]Fe(III) complexes ([FePPPic], [FeMPPic] and [FeDPPic], respectively) pinch-porphyrin peroxidase enzyme models, with s = 5/2 and 3/2 Fe(III) quantum mixed spin (qms) ground states is described herein. The kinetic study by UV/Vis at λmax = 465 nm showed two different types of kinetics during the oxidation process in the guaiacol test for peroxidases (1-3 + guaiacol + H2O2 → oxidation guaiacol products). The first intermediate was observed during the first 24 s of the reaction. When the reaction conditions were changed to higher concentration of pinch-porphyrins and hydrogen peroxide only one type of kinetics was observed. Next, the reaction was performed only between pinch-porphyrins-Fe(III) and H2O2, resulting in only two types of kinetics that were developed during the first 0-4 s. After this time a self-oxidation process was observed. Our hypotheses state that the formation of the π-cation radicals, reaction intermediates of the pinch-porphyrin-Fe(III) family with the ligand picdien [N,N'-bis-pyridin-2-ylmethyl-propane-1,3-diamine], occurred with unique kinetics that are different from the overall process and was involved in the oxidation pathway. UV-Vis, ¹H-NMR and ESR spectra confirmed the formation of such intermediates. The results in this paper highlight the link between different spectroscopic techniques that positively depict the kinetic traits of artificial compounds with enzyme-like activity.

Determination of moderately polar arsenolipids and mercury speciation in freshwater fish of the River Elbe (Saxony, Germany).

Arsenic and mercury are frequent contaminants in the environment and care must be taken to limit their entrance into the food chain. The toxicity of both elements strongly depends upon their speciation. Total amounts of As and Hg as well as their species were analyzed in muscle and liver of 26 fishes of seven freshwater fish species caught in the River Elbe. The median concentrations of As were 162 µg kg(-1) w.w. in liver and 92 µg kg(-1) w.w. in muscle. The median concentrations of total Hg were 241 µg kg(-1) w.w. in liver and 256 µg kg(-1) w.w. in muscle. While this level of Hg contamination of the freshwater fish in the River Elbe is significantly lower than 20 years ago, it exceeds the recommended environmental quality standard of 20 µg Hg kg(-1) w.w. by a factor of 5-50. However, the European maximum level of 500 µg Hg kg(-1) for fish for human consumption is rarely exceeded. Arsenic-containing fatty acids and hydrocarbons were determined and partially identified in methanolic extracts of the fish by HPLC coupled in parallel to ICP-MS (element specific detection) and ESI-Q-TOF-MS (molecular structure detection). While arsenobetaine was the dominant As species in the fish, six arsenolipids were detected and identified in the extracts of liver tissue in common bream (Abramis brama), ide (Leuciscus idus), asp (Aspius aspius) and northern pike (Esox lucius). Four arsenic-containing fatty acids (AsFA) and two arsenic-containing hydrocarbons (AsHC) are reported in freshwater fish for the first time. With respect to mercury the more toxic MeHg(+) was the major species in muscle tissue (>90% of total Hg) while in liver Hg(2+) and MeHg(+) were of equal importance. The results show the high relevance of element speciation in addition to the determination of total element concentrations to correctly assess the burden of these two elements in fish.

Mass spectrometric detection, identification, and fragmentation of arseno-phytochelatins.

Phytochelatins (PC) are cystein-rich oligopeptides in plants for coordination with toxic metals and metalloids via their thiol groups. The composition, structure, and mass spectrometric fragmentation of arseno-PC (As-PC) with PC of different degree of oligomerization (PC2-PC5) in solution were studied using liquid chromatography coupled in parallel to inductively coupled plasma mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry. As-PC were detected from As(PC2) to As(PC5) with an increasing number of isomers that differ in the position of thiol groups bound to As. Thermodynamic modeling supported the identification process in case of these isomers. Mass spectrometric fragmentation of the As-PC does not follow the established pattern of peptides but is governed by the formation of series of As-containing annular cations, which coordinate to As via S, N, or O. Structure proposals for 30 As-PC fragment ions in the range m/z 147.92 to m/z 1290.18 are elaborated. Many of these fragment ions are characteristic to several As-PC and may be suited for a screening for As-PC in plant extracts. The mass spectrometric data offer the perspective for a future more sensitive determination of As-PC by means of liquid chromatography tandem mass spectrometry with multiple reaction monitoring.

Species determination of Culicoides biting midges via peptide profiling using matrix-assisted laser desorption ionization mass spectrometry.

BACKGROUND: Culicoides biting midges are vectors of bluetongue and Schmallenberg viruses that inflict large-scale disease epidemics in ruminant livestock in Europe. Methods based on morphological characteristics and sequencing of genetic markers are most commonly employed to differentiate Culicoides to species level. Proteomic methods, however, are also increasingly being used as an alternative method of identification. These techniques have the potential to be rapid and may also offer advantages over DNA-based techniques. The aim of this proof-of-principle study was to develop a simple MALDI-MS based method to differentiate Culicoides from different species by peptide patterns with the additional option of identifying discriminating peptides. METHODS: Proteins extracted from 7 Culicoides species were digested and resulting peptides purified. Peptide mass fingerprint (PMF) spectra were recorded using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and peak patterns analysed in R using the MALDIquant R package. Additionally, offline liquid chromatography (LC) MALDI-TOF tandem mass spectrometry (MS/MS) was applied to determine the identity of peptide peaks in one exemplary MALDI spectrum obtained using an unfractionated extract. RESULTS: We showed that the majority of Culicoides species yielded reproducible mass spectra with peak patterns that were suitable for classification. The dendrogram obtained by MS showed tentative similarities to a dendrogram generated from cytochrome oxidase I (COX1) sequences. Using offline LC-MALDI-TOF-MS/MS we determined the identity of 28 peptide peaks observed in one MALDI spectrum in a mass range from 1.1 to 3.1 kDa. All identified peptides were identical to other dipteran species and derived from one of five highly abundant proteins due to an absence of available Culicoides data. CONCLUSION: Shotgun mass mapping by MALDI-TOF-MS has been shown to be compatible with morphological and genetic identification of specimens. Furthermore, the method performs at least as well as an alternative approach based on MS spectra of intact proteins, thus establishing the procedure as a method in its own right, with the additional option of concurrently using the same samples in other MS-based applications for protein identifications. The future availability of genomic information for different Culicoides species may enable a more stringent peptide detection based on Culicoides-specific sequence information.

Different strategies of cadmium detoxification in the submerged macrophyte Ceratophyllum demersum L.

The heavy metal cadmium (Cd) is highly toxic to plants. To understand the mechanisms of tolerance and resistance to Cd, we treated the rootless, submerged macrophyte Ceratophyllum demersum L. with sub-micromolar concentrations of Cd under environmentally relevant conditions. X-ray fluorescence measurements revealed changing distribution patterns of Cd and Zn at non-toxic (0.2 nM, 2 nM), moderately toxic (20 nM) and highly toxic (200 nM) levels of Cd. Increasing Cd concentrations led to enhanced sequestration of Cd into non-photosynthetic tissues like epidermis and vein. At toxic Cd concentrations, Zn was redistributed and mainly found in the vein. Cd treatment induced the synthesis of phytochelatins (PCs) in the plants, with a threshold of induction already at 20 nM Cd for PC3. In comparison, in plants treated with Cu, elevated PC levels were detected only at the highest concentrations (100-200 nM Cu). Our results show that also non-accumulators like C. demersum store toxic metals in tissues where the heavy metal interferes least with metabolic pathways, but remaining toxicity interferes with micronutrient distribution. Furthermore, we found that the induction of phytochelatins is not proportional to metal concentration, but has a distinct threshold, specific for each PC species. Finally we could show that 20 nM Cd, which was previously regarded as non-toxic to most plants, already induces detoxifying mechanisms.

Determination and identification of hydrophilic and hydrophobic arsenic species in methanol extract of fresh cod liver by RP-HPLC with simultaneous ICP-MS and ESI-Q-TOF-MS detection.

The present study was focused on the determination and identification of arsenic species in methanolic extracts of cod liver. Arsenic species were fractionated and the fractions analysed by RP-HPLC-ICP-MS coupled with ESI-Q-TOF-MS. The total concentration of arsenic in the fresh cod liver was analysed by ICP-MS to be 1.53±0.02 mg As kg(-1)w.w. and the extraction recovery was ca. 100% and the column recovery >93%. Besides polar inorganic and methylated arsenic species (>70%) more hydrophobic arsenic-containing fatty acids and hydrocarbons occurred. Based on the mass spectrometric data proposals for molecular structures were elaborated for 20 of the organic As species included 10 arsenic-containing fatty acids (AsFA) and an arsenic-containing hydrocarbon (AsHC) mentioned for the first time in fresh cod liver. Arsenobetaine was found as main water-soluble arsenic compound in cod liver followed by higher molecular mass arsenic-containing fatty acids and hydrocarbons.

Identification of degradation products of phenylarsonic acid and o-arsanilic acid in contact with suspensions of soils of volcanic origin.

A set of organoarsenicals were identified in aqueous phenylarsonic acid (PA) and o-arsanilic acid (AA) solutions treated with soil of volcanic origin in batch systems. The transformation products were separated by liquid chromatography (RP-LC) and identified with element selective inductively coupled plasma-mass spectrometry (ICP-MS) as well as molecular selective electrospray ionization-mass spectrometry (ESI-MS) detection after their HPLC separation. The identification of the main degradation products by means of ESI-MS, ESI-MS/MS and ESI-TOF-MS showed the occurrence of nitrophenylarsonic acid and methylphenylarsinic acid in the solutions containing AA and PA in contact with soils, respectively. Using irradiation of PA solution with visible light, new compounds related from PA appeared with increasing irradiation times which were identified as 4-hydroxyphenylarsonic acid, 3-hydroxyphenylarsonic acid and 2-hydroxyphenylarsonic acid. Additionally, a dihydroxyphenylarsonic compound was identified as impurity of PA.

Fast separations by capillary electrophoresis hyphenated to electrospray ionization time-of-flight mass spectrometry as a tool for arsenic speciation analysis.

Fast capillary electrophoresis (CE) hyphenated to time-of-flight mass spectrometry (TOF-MS) of four organoarsenic species (glycerol oxoarsenosugar, sulfate oxoarsenosugar, arsenobetaine, arsenocholine) are presented using short length CE capillaries under high electric field strengths of up to 1.3 kV cm(-1) with small inner diameter (ID). The separation of arsenosugars by CE is demonstrated for the first time. An aqueous formic acid solution was employed as the background electrolyte (BGE) for the separation. Various acid concentrations were evaluated for their influence on migration times, separation efficiency as well as with regard to controlling the charge of the arsenic species. A 0.1 M formic acid/ammonium formate buffer (pH 2.8) proved to be suitable for the separation of the four species. A non-aqueous BGE was tested as an alternative buffer system for fast speciation analysis. Separation of arsenobetaine and arsenocholine could even be achieved within 10 s by pressure-assisted CE. Application of the optimized method for the analysis of extracts of a seagrass and a Wakame algae sample as well as the brown algae homogenate reference material IAEA-140/TM revealed a clear signal for the glycerol arsenosugar.

Formation of sulfonyl aromatic alcohols by electrolysis of a bisazo reactive dye.

Five sulfonyl aromatic alcohols, namely 4-((2-hydroxyethyl)sulfonyl)phenol, 4-((2-(2-((4-hydroxyphenyl)sulfonyl)ethoxy)vinyl)sulfonyl)phenol, 4-(ethylsulfonyl)phenol, 4-(vinylsulfonyl)phenol and 5-((4-aminophenyl)sulfonyl)-2-penten-1-ol were identified by LC-ESI-Qq-TOF-MS as products formed by electrolysis of the bisazo reactive dye Reactive Black 5 (RB5). Since electrolyses were performed in an undivided cell equipped with Ni electrodes in alkaline medium, amines like 4-(2-methoxyethylsulfonyl)benzene-amine (MEBA) with m/z 216 were also suspected to be formed due to the plausible chemical reaction in the bulk or the cathodic reduction of RB5 and its oxidation by-products. Aiming to check this hypothesis, a method was used for the preparation of MEBA with 98% purity, via chemical reduction also of the dye RB5. The logP of the synthesized sulfonyl aromatic compounds was calculated and their logkw values were determined chromatographically. These data were discussed in regard to the relationship between hydrophobicity/lipophilicity and toxicity.

Retention of phenylarsenicals in soils derived from volcanic materials.

Sorption of phenylarsenicals including 4-hydroxy-3-nitrophenylarsonic acid (roxarsone), an animal feed additive widely used for growth stimulation, on soils was investigated in batch systems. Phenylarsonic acid, o-arsanilic acid and roxarsone were retained differently by unpolluted, non-sterilized soils. Sorption isotherms were analyzed by the Henry, Tóth and Langmuir-Freundlich equations. The saturation capacity of the Acrisol soil was 3.4 for o-arsanilic acid, 10.9 for phenylarsonic acid and 1.9 g(As) kg(soil)(-1) (dry mass) for roxarsone. The iron content in the soil was not the only factor determining retention of the studied phenylarsenicals. The order of retention on the three soils after 24 h was: roxarsone>o-arsanilic acid>phenylarsonic acid. Besides arsenite and arsenate, new arsenic-containing compounds were detected.

Detection of arsenic-containing hydrocarbons in canned cod liver tissue.

Arsenic is a metalloid well known to be potentially toxic depending of its species. Lipid-soluble arsenicals (arsenolipids) are present in a wide range of biological samples in which they could play a role in the biosynthesis of organoarsenic compounds from inorganic arsenic compounds. Arsenolipids have recently attracted considerable interest. In order to gain deeper insights into the impact of arsenolipids new analytical approaches for reliable determination of this class of arsenic-containing hydrocarbons in various matrices are needed. High concentrations of arsenolipids were found in seafood which served as sample material in this study. We report the investigation of three arsenolipids found in canned cod liver from which they were extracted and purified by solid phase extraction (SPE) using a silica gel column and ethyl acetate/methanol as eluent. Analytical studies were conducted by means of gas chromatography coupled with ICP-MS, MIP-AES and EI-qMS and by TOF-MS. The results obtained by GC-ICP-MS and GC-MIP-AES showed the existence of numerous arsenic compounds in the SPE fractions collected. Three major peaks were found within a retention time window between 10 and 25 min. The presence of arsenic compounds in the fish tissue could be confirmed using GC-EI-qMS analysis. Corresponding information of the molecular weights of the major arsenic species were provided by TOF-MS which allows highly accurate mass determinations. The results showed the presence of the arsenic-containing hydrocarbons with the following molecular formulas: C(17)H(37)AsO (calculated for [M+H](+) 333.2133; found 333.2136; Deltam=0.90 ppm); C(19)H(41)AsO (calculated for [M+H](+) 361.2446; found 361.2446; Deltam=0.00 ppm); C(23)H(37)AsO (calculated for [M+H](+) 405.2133; found 405.2145; Deltam=2.96 ppm). Suggestions for the corresponding structures are discussed.