Ultrasound-based protein determination in maize seeds.

The need for a simple and accurate method for protein estimation in alcoholic extracts led to the reexamination of the optimum conditions of a colorimetric assay based on the biuret reaction. Sonication time and the other experimental parameters were optimized after kinetics study on the extraction of either zein or total proteins. Zein extraction and purity were investigated by (1)H and (13)C NMR spectroscopy, SDS-PAGE electrophoresis, and UV-visible spectrophotometry (UV-vis). A zein assay was proposed, which involves the reaction of copper ions in copper phosphate powder with zein extracted in ethanolic solutions under strong alkaline environment. Furthermore, we extended this procedure to determine total proteins in maize samples simultaneously with their ultrasonic-assisted (US) extraction with an alkaline-alcoholic solution. Proteins in both types of extracts were well characterized by UV-vis spectroscopy. However, the 545 nm absorbance of the violet-colored supernatants which is proportional to the protein content was found to be the key parameter of the improved biuret-based protein assay. Comparison of values obtained by this procedure and by Micro-Kjeldahl method was in excellent agreement. A scaled-down procedure agreed well with the standard procedure. Enhanced accuracy and repeatability was found in protein determination in maize using the modified biuret method. The optimization of reagent concentrations and incubation times were studied as well.

NOSH aspirin may have a protective role in Alzheimer's disease.

Evidence indicates that inflammation, oxidative stress, and the disruption of normal conformation of proteins might be directly linked to Alzheimer's disease (AD). The present study was undertaken using literature data to find a possible drug to address the multiple disorders involved in AD-associated Aß accumulation and plaque formation. We consider NOSH-aspirin a drug of choice for reducing the inflammatory areas in the brain (aspirin moiety), removing the noxious heavy metals from plaques (hydrogen sulfide), and increasing the oxygen supply to neurons since nitrogen oxide is a potent vasodilator and an anti-inflammatory agent. Several confirmatory data in literature and possible mechanisms for cellular defence as well as novel therapeutical pathways are discussed.

Biostructural theory of the living systems.

Eugen Macovschi is among the few scientists who tried, and partly succeeded, to explain the differences between "dead" and "living" in biological sciences. He discovered and characterized the so-called biostructure of the living bodies and worked out a biostructural theory, which is the first supramolecular conception in biology. Nevertheless, complex biological systems are currently considered only from the molecular point of view, although they may be regarded as specific phenomena on highly structured bodies within the four-dimensional Universe. According to Macovschi, the biostructure provides organisms with life properties and controls their life processes and chemical changes. Nevertheless, plant cells or bacterial ones differ much from the animal or human cells. In fact, there are various biostructures which are related with cell properties. Hence, this theory creates confusions and cannot be easily used to explain all the properties of the biosystems. Consequently, it is our goal to highlight the principles, advantages, limitations, and applications of the biostructural theory, which might support new ideas and theories in modern life sciences.

Silver-induced conformational changes of polypeptides: a CD study.

The role of silver ions in various pathologies, as well as their effect on peptide conformation and properties are less understood. Consequently, we synthesized several peptides with various residues in their sequence to investigate silver-induced conformational changes at various pH values by Circular Dichroism spectroscopy. Uniquely, the glycine-based, histidine-containing peptide showed a severe change from a random coil and ß-turn conformation to large α-helices during silver binding. When comparing the effect of silver ions on the conformation of bradykinin a similar tendency was found. Besides, silver ions reduced the amyloid-ß peptide tendency to aggregation. Our results suggest a specific and protective role for silver ions in brain pathologies, which is related to their high affinity toward physiologically and pharmacologically active peptides. Fourier transform infrared spectroscopy studies as well as the mass spectrometric ones support our conclusions.

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 H(2)N-AAAAHAAAAHAAAAHAAAA-COOH (P19-H5) and H(2)N-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.

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.

Interaction of beta-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.

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 Beer's 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.

Synthesis and mass spectrometric characterization of a metal-affinity decapeptide: copper-induced conformational changes.

A decapeptide with high affinity toward heavy metal ions (RCHQYHHNRE) has been prepared by Fmoc strategy using TGR resin as solid support. The model peptide provides a simple system that can be used for a systematic study of the impact of different metal ions on peptide secondary structure on a molecular level; histidine residues were incorporated into the peptide in a sequence similar to beta-amyloid peptide (Abeta1-40) to generate possible complexation sites for Cu (2+) ions. The peptide secondary structure, as investigated by circular dichroism, and self-assembled nanostructures were observed to depend strongly on the presence of copper and sodium dodecyl sulfate (SDS). Atomic force microscopy (AFM) revealed also that copper and SDS affected slightly the Abeta1-40 nanostructures. An explanation for the effect of metal ions and SDS on the self-assembly of peptides was proposed. The extensive beta-sheet formation may further promote peptide self-assembly into longer fibers.

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 ((203)Hg), 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 oligomer-ization 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.

Highly selective assay of proteins in dilute solutions.

A very simple, highly selective assay of proteins has been developed. Solutions of 6 M hydrochloric acid containing 0-20 mug ml(-1) protein are subjected to acid hydrolysis, in the presence of air, during their evaporation to dryness on a steam bath. Then, the resulted amino acids are determined with a 2,2-dihydroxy-1,3-indanedione reagent. To eliminate any interference and to increase in selectivity of the assay, a blank with the unhydrolyzed protein is carried out. The protein being analyzed or alanine is used to plot calibration curves. The absorbance of the colored solution is read at 516 nm. The color system obeys Beer's law in the range 0.1-20 mug ml(-1) protein. The molar absorptivity for alanine was found to be 1.69x10(3) (+/-9.7) l mol(-1) cm(-1). Also, the relative molar absorptivity for peptone was found to be 1.23x103 (+/-6.5) l mol(-1) cm(-1). The lowest limits for the estimation of serum albumin, gelatin, and peptone are around 0.25,0.19 and 0.31 mug ml(-1), respectively. The method is highly selective and the determination is little affected by the presence of other substances. All other important analytical parameters were studied and practical applicability of the method has been verified by the analysis of some biological materials.