Gabi Drochioiu

Interaction of β-amyloid(1-40) peptide with pairs of metal ions: An electrospray ion trap mass spectrometric model study

The stoichiometries and the affinity toward simple and paired metal ions of synthetic amyloid-beta(1-40) peptide (Abeta1-40) were investigated by electrospray ion trap mass spectrometry (ESI-MS), circular dichroism (CD), and atomic force microscopy (AFM). The results lead to the working hypothesis that pH-dependent metal binding to Abeta1-40 may induce conformational changes, which affect the affinity toward other metals. A significant copper and zinc binding to Abeta1-40 peptide at pH 5.5 was found, whereas nickel ions commonly bind to each molecule of beta-amyloid peptide. Some complexes of Abeta1-40 with more than one nickel ion were identified by ESI-MS. In addition, nickel ions proved to enhance Abeta oligomerization. On increasing pH, up to 12 ions of zinc may bind to a single Abeta molecule. Under the same pH and concentration conditions, the binding pattern of the independent copper and silver ions to Abeta1-40 was different from that of the equimolecular mixture of the two metal ions. One might assume that some conformational changes due to water loss altered the capacity of Abeta peptide to bind certain heavy metal ions. As a consequence, copper-silver interaction with the binding process to Abeta1-40 became highly complex. A competition between silver and nickel ions for Abeta1-40 binding sites at high pH was also observed. New strategies were proposed to identify the characteristic signals for some important metal ion-peptide complexes in the spectra recorded at high pH or high concentrations of metal ions. To explain the formation of such a large number of high metal ion-Abeta complexes, we took into consideration the participation of both histidine residues and free amino groups as well as carboxylate ones in the binding process. Finally, CD and AFM studies supported the mass spectrometric data.

Model peptide-based system used for the investigation of metal ions binding to histidine-containing polypeptides

The reaction of histidine-containing polypeptides with toxic and essential metals and the molecular mechanism of complexation has yet to be determined, particularly with respect to the conformational changes of the interacting macromolecules. Therefore, a system of oligopeptides containing histidine residues in various positions of Ala or Gly sequences has been designed and used in heavy metal comparatively binding experiments. The role of spacing residues (Gly and Ala repeats) in selecting the various conformations was investigated. The newly synthesized peptides and metal ion adducts have been characterized by Fourier transform infrared spectroscopy (FTIR) as well as electrospray ion trap mass spectrometry (ESI-MS) and circular dichroism (CD). The analysis of CD-spectra of the four peptides in water revealed that the secondary structure depends much on the position of each amino acid in the peptide backbone. Our peptides system reveals various binding mechanisms of metal ions to peptides depending on the position of histidine residue and the corresponding conformations of Ala or Gly sequences. Biological and medical consequences of conformational changes of metal-bound peptides are further discussed. Thus, the binding of heavy metals to four peptides may serve as a model system with respect to the conformational consequences of the metal addition on the amino acid repeats situated in prion protein.

Effect of radioactive and non-radioactive mercury on wheat germination and the anti-toxic role of glutathione

Studies to test the noxious effect of mercury ions on wheat germination and seedling growth showed that germination rate, shoot length, and fresh weights varied as a function of their concentration in the treatment solutions. At the same concentration, the radioactive mercury proved to be more harmful to the living seeds and seedlings. The detoxification action of glutathione for both radioactive and non-radioactive mercury was also followed. After a seven-day period of germination in the presence of the investigated compounds, the wheat plantlets were cut from the seeds, and their height, weight, and residual radioactivity were measured. The shoot length decreased from 8.1 (blank) to 4.6 cm (non-radioactive mercury) or even to 2.5 cm (203Hg), while glutathione had both an anti-toxic and an anti-radiotoxic effect (6.4 and 6.0 cm, respectively). The root weight of the lot decreased from 1.7 to 0.7 g and 0.4 g, respectively, while glutathione showed a healing action (1.5 and 1.7 g). The radioactive ions accumulated especially in roots (35.5 %), and less in shoots (11.2 %). Results were statistically validated.

Copper-induced oligomerization of peptides: a model study

In this work, copper-binding of the tetraglycine peptide (Gly–Gly–Gly–Gly) was studied by electrospray ionization mass spectrometry. Experiments were performed under alkaline conditions, in the presence of ethanolamine (pH 10.95). We observed that the presence of copper(II) ions induces the aggregation of the peptide and the formation of copper-bound complexes with higher molecular mass is favored, such as the oligomer complexes [3M + 2Cu – 3H]+ and [4M + 3Cu – 5H]+. At 1:1 peptide–copper(II) ion ratio, the singly charged [3M + 2Cu – 3H]+ oligomer complex is the base peak in the mass spectrum. Metal ion-induced oligomerization of neurotoxic peptides is well known in the literature; however, there are very few examples in which such oligomerization was directly observed by mass spectrometry. Our results show that application of short peptides can be useful to study the mechanism of metal ion binding and metal ion-induced oligomerization of peptides.

Laboratory analyses of 60Co2+, 65Zn2+ and 55+59Fe3+ radiocations uptake by Lemna minor

The living Lemna minor vascular plant and two different sorbents obtained by chemical treatment of this plant were tested to study the removal process of 60Co2+, 65Zn2+ and 55+59Fe3+ from low radioactive wastewaters. The most effective sorbent was the protonated biomass, indicating the decisive contribution of the complexation process in the assembly of the uptake mechanisms. The uptake performance of the biosorbent obtained from the L. minor can be increased with ∼20% by treatment with 0.1 N HNO3. Concerning the metabolically active mechanism, it can be notice the slow elimination of 65Zn2+ and the continuously increase of 55+59Fe3+ uptake degree. The Na2CO3 generated in situ in systems participates to a double exchange reaction with the metallic cations during the uptake. 60Co2+, 65Zn2+ and 55+59Fe3+ radiocations prefer for coordination N-donor ligands at the expense O-donors ones.

Analysis of cyanogens with resorcinol and picrate

The total cyanogenic potential of various substrates (flax seed, stones of peach, plum, nectarine and apricot as well as apple seeds, and various model compounds) was investigated by using the acid hydrolysis method, picrate method, and a novel method based on the reaction of cyanide liberated from plants with resorcinol and picrate. The hydrocyanic acid liberated from cyanogens was trapped by using a 1% sodium bicarbonate. Then, 1 ml of extract was mixed with 1 ml of working reagent containing 160 microg of resorcinol, 320 microg of picric acid, and 30 mg of sodium carbonate, and heated on a boiling water bath for 10 min. The absorbance was measured at 488 nm in 1cm glass cuvettes at room temperature. The color system obeys Beers law in the range of 0-5 microg ml(-1) total HCN. Using model compounds and real samples including replicate analyses on prunasin, the resorcinol method proved to be more accurate, reproducible, and especially more sensitive than the known spectrophotometric methods such as the acid hydrolysis method and the picrate method.

A modified ninhydrin micro-assay for determination of total cyanogens in plants.

Indirect quantification of total cyanogenic compounds (cyanogens) in plants was studied using a novel ninhydrin-based spectrophotometric micromethod. The ninhydrin-cyanide system obeys Beers law in the range from 20 μg L(-1) to 800 μg L(-1) CN(-) with molar absorptivity ε=1.4×10(5) L mol(-1)cm(-1). Recovery at 100 μg L(-1) CN(-) was 100.5±0.1% and LOD and LOQ were 8 and 22 μg L(-1), respectively. The conditions for ninhydrin reagent preparation were discussed. The extraction and separation solutions, extraction and incubation time, and solvent volume were also investigated for maximum recovery of total cyanogens. 0.1% NaHCO3 was used as an extraction solvent for cyanide formed after enzymatic hydrolysis of cyanogens. The procedure was suitable for samples containing more than 90 mg CN(-)/100 g sample. When cyanogen concentrations were lower, the resulted cyanide was separated by microdiffusion in a Conway cell. Water was used as a hydrolysis medium and a donor solvent, while 2% Na2CO3 as an absorbing solution. Total cyanogen content in plum and almond kernels, as well as apple and flax seeds was determined.

Electrospray ionization mass spectrometric approach of conformationally-induced metal binding to oligopeptides

Electrospray ionization mass spectrometry was used to measure the binding of copper and nickel ions to the newly synthesized model peptides H2N–AAAAHAAAAHAAAAHAAAA–COOH (P19–H5) and H2N–AAAHAAAHAAAHAAAAAAA–COOH (P19–H4). The affinity of histidine-containing peptides toward heavy metal ions proved to be related to the position of each histidine residue in the peptide sequence. In contrast to P19–H5, P19–H4 peptide bound no nickel or copper ions in the gas phase, whereas its spectra showed an intense fragmentation. The role of spacing residues (Ala repeats) in selecting the various conformations was also investigated. Finally, the circular dichroism and Fourier transform infrared spectra indicated that these isomer peptides have quite different conformations. A close relationship between the conformation of alanine-based peptides and their affinity toward metal ions may result in different patterns of metal ion-peptide systems.

4-Methylpyrimidinium ylides. Part 7: 3+2 Dipolar cycloadditions to non-symmetrical substituted alkenes and alkynes

The ability of 4-methylpyrimidinium ylides (as 1,3-dipoles) to react with activated non-symmetrical substituted dipolarophiles (alkenes and alkynes) is presented. 4-Methylpyrimidinium ylides did not react with alkenes. With alkynes the reactions are regiospecific, a single regioisomer being obtained. A possible mechanism for the reaction pathway is proposed. For the first time in the pyrimidinium ylides series both isomers resulting from bonding to the 2-and 6-positions of the heterocycle ring were obtained. The appropriate conditions in order to increase the selectivity of one of the isomers were determined.

IR, MS and CD Investigations on Several Conformationally-Different Histidine Peptides

Solid phase synthetic methodology has been used to prepare four peptides which form a system able to monitor metal ion binding to conformationally different peptides. The 19-residues oligopeptides containing histidine residues in various positions of Ala or Gly sequences, namely GGGGHGGGGHGGGGHGGGG, GGGHGGGHGGGHGGGGGGG, AAAAHAAAAHAAAA-HAAAA, and AAAHAAAHAAAHAAAAAAA have been synthesized by Fmoc strategy and characterized by Fourier transform infrared spectroscopy (FT-IR) as well as electrospray ion trap mass spectrometry (ESI-MS) and circular dichroism (CD). The analysis of CD-spectra of the four peptides revealed that the secondary structure depends much on the amino acid sequence. Biological and medical consequences of conformational changes of metal-bound peptides are further discussed.