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

Poster P3: Toxicity and Detoxification of Cadmium in the Aquaticmacrophyte Ceratophyllum Demersum; session "Metal homeostasis and detoxification"

The heavy metal cadmium (Cd) is an important pollutant and poisonousto many organisms. We studied the effects of Cd on C.demersum under environmentally relevant conditions. High, moderateand low concentrations of Cd had different effects. Lethally toxicconcentrations (100–200 nM) led to growth stop and the plants’ability to perform photosynthesis (measured as Fv/Fm) decreased morethan twofold, consistent with decreased pigment content. Moderatelytoxic concentrations (10–50 nM) led to reduced growth, slightlyreduced pigment content, but hardly affected photosynthesis (measuredas O2 exchange and as Fv/Fm). Lower concentrations(0.2–5 nM) even had beneficial effects, like enhanced growth rate.When applied in low concentrations, Cd was homogeneously distributedin the whole cross-section of the leaves like a nutrient.Moderate and high Cd concentrations led to sequestration of Cd in thevascular bundle and the epidermis cells, where Cd does not affectphotosynthetic molecules. At toxic Cd concentrations, Zn was redistributedand mainly found in the vein along with Cd, indicatinginhibition of Zn transporters. Consistently, by metalloproteomics(HPLC-ICP-MS) we found that during Cd toxicity concentrations of Cdincreased in fractions of the major photosynthetic complexes while Mgdecreased, suggesting the replacement of Mg by Cd in chlorophylls.Furthermore, the induction of phytochelatins was not proportionalto metal concentration, but had distinct thresholds, specific for each PCspecies. PC3 especially was switch-like induced already at 20 nM Cd,which was previously regarded as non-toxic to most plants. Phytochelatinlevels at the lowest Cd concentrations were not detectable orbelow 0.1 % of the level at sublethally toxic concentrations, suggestingthat they do not have another function than metal detoxification

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

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1756-3305-7-392) contains supplementary material, which is available to authorized users

Identification of roxarsone metabolites produced in the system: soil – chlorinated water – light by using HPLC-ICP-MS/ESI-MS, HPLC-ESI-MS/MS and high resolution mass spectrometry (ESI-TOF-MS)

Roxarsone (4-hydroxy-3-nitrophenylarsonic acid) in contact with soil of volcanic origin and chlorine containing water generated a set of organoarsenicals. The transformation products were identified with element-specific (ICP-MS) as well as molecular-specific (ESI-MS) detection after their HPLC separation. The identification of the main transformation products by means of ESI-MS, ESI-MS/MS and ESI-TOF-MS adduce evidence of chlorinated phenylarsonic acids and a phenylarsine oxide derivative which contains arsenic in the trivalent state. Traces of chlorine in water used for sorption experiments are suggested to be responsible for the formation of chlorinated products. After irradiation of a roxarsone solution with visible light, different transformation product so far not identified were detected

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