Analizie bioinformatycznej poddano 89 peptydów immunoaktywnych dostępnych w bazie BIOPEP. Obecność fragmentów potencjalnie immunoaktywnych stwierdzono w 90 ze 150 sekwencji analizowanych białek. Sekwencje aminokwasowe badanych peptydów analizowano z uwzględnieniem: długości łańcucha, udziału procentowego poszczególnych aminokwasów, pI, molowego współczynnika ekstynkcji, indeksu hydropatii i ładunku wypadkowego. Ponadto określono możliwości ich uwalniania in silico przez enzymy proteolityczne. Na podstawie komputerowej analizy z zastosowaniem programu ProtParam stwierdzono, że immunoaktywne peptydy to głównie fragmenty hydrofilowe, w sekwencji których przeważają takie aminokwasy, jak: Lys, Arg i Pro, obdarzone ładunkiem dodatnim w neutralnym pH. W komputerowej symulacji proteolizy in silico wybranych 11 białek żywności o największej częstości występowania peptydów immunoaktywnych (parametr A > 0,02) wykazano, że tylko trzy enzymy: chymaza (EC 3.4.21.39), elastaza trzustkowa (EC 3.4.21.36) i endopeptydaza glicylowa (EC 3.4.22.25), spośród dostępnych w bazie BIOPEP, wykazywały specyficzność pozwalającą na uwalnianie peptydów immunoaktywnych w układzie jednego enzymu. Produktami hydrolizy analizowanych białek, przy użyciu wybranych enzymów proteolitycznych, były głównie 2 - 3 aminokwasowe fragmenty peptydowe.
Studies were carried out to identify and detect potentially toxic proteins of wheat. The gliadin fractions were subjected to chromatographic and spectroscopic analyses to develop the relevant discriminants. The spectral analysis showed that these proteins differ considerably in their tryptophan-to-tyrosine molar ratios. A standard curve was used. The gliadin fractions were identified by comparing the calculated values of the tryptophan-to-tyrosine molar ratio with the values obtained based on the amino acid sequence of wheat gliadins in the Swiss-Prot/TrEMBL database. Based on the retention times and second derivatives of UV spectra, the particular wheat gliadin fractions were classified as α/β or γ-gliadins.
Products of proteolysis of food proteins may play a role of agents regulating different functions in the human organism. The peptides derived from plant-, meat- and egg proteins may decrease the blood pressure, act as opioid agonists and antagonists, cause contraction of smooth muscles, inhibit platelet aggregation and oxidation. The coeliac-toxic peptides are an example of peptides with a negative influence on the human organism. The activity of food-originated peptides has mainly been investigated in vitro. Information published so far refers to biologically active peptides obtained in laboratory scale. Their application as components of so called "functional foods" (specially designed foods with desired biological activity) can however be expected possible.
W pracy przeprowadzono analizę in silico w celu oceny reaktywności krzyżowej białek orzecha ziemnego (Arachis hypogaea L.), ze szczególnym uwzględnieniem białka Ara h 9 – markera reaktywności krzyżowej w odniesieniu do białek owoców. Określono możliwość występowania reakcji krzyżowych między różnymi białkami orzecha ziemnego, jak również między białkami orzecha ziemnego a białkami innych alergenów pokarmowych, w tym powszechnie znanych alergenów soi, soczewicy, pomidora, orzecha laskowego, jabłoni zwyczajnej i brzoskwini oraz m.in. chińskiego liczi i daktylowca. Podobieństwo sekwencji epitopów białek orzecha ziemnego na poziomie > 80 %, oceniane na podstawie komputerowych baz danych: UniProtKB i BIOPEP oraz programu EVALLER, stwierdzono głównie w stosunku do epitopów soi, pomidora, orzecha laskowego, jabłoni i brzoskwini.
Background. Proteomic analysis is emerging as a highly useful tool in food research, including studies of food allergies. Two-dimensional gel electrophoresis involving isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis is the most effective method of separating hundreds or even thousands of proteins. In this study, albumin and globulin fractions of pea seeds cv. Ramrod were subjected to proteomic analysis. Selected potentially alergenie proteins were identified based on their molecular weights and isoelectric points. Material and methods. Pea seeds (Pisum sativum L.) cv. Ramrod harvested over a period of two years (Plant Breeding Station in Piaski-Szelejewo) were used in the experiment. The isolated albumins, globulins and legumin and vicilin fractions of globulins were separated by two-dimensional gel electrophoresis. Proteomic images were analysed in the ImageMaster 2D Platinum program with the use of algorithms from the Melanie application. The relative content, isoelectric points and molecular weights were computed for all identified proteins. Electrophoregrams were analysed by matching spot positions from three independent replications. Results. The proteomes of albumins, globulins and legumin and vicilin fractions of globulins produced up to several hundred spots (proteins). Spots most characteristic of a given fraction were identified by Computer analysis and spot matching. The albumin proteome accumulated spots of relatively high intensity over a broad range of pi values of -4.2-8.1 in 3 molecular weight (MW) ranges: I - high molecular-weight albumins with MW of -50-110 kDa, II - average molecular-weight albumins with MW of -20-35 kDa, and III - Iow molecular-weight albumins with MW of-13-17 kDa. 2D gel electrophoregrams revealed the presence of81 characteristic spots, including 24 characteristic of legumin and 14-of vicilin. Conclusions. Two-dimensional gel electrophoresis proved to be a useful tool for identifying pea proteins. Pattems of spots with similar isoelectric points and different molecular weights or spots with different isoelectric points and similar molecular weights play an important role in proteome analysis. The regions characteristic of albumin, globulin and legumin and vicilin fractions of globulin with typical MW and pl values were identified as the results of performed 2D electrophoretic separations of pea proteins. 2D gel electrophoresis of albumins and the vicilin fraction of globulins revealed the presence of 4 and 2 spots, respectively, representing potentially allergenic proteins. They probably corresponded to vicilin fragments synthesized during post-translational modification of the analysed protein.
Przedstawiono możliwości aktualnie stosowanych metod rozdziału peptydów i białek oraz metod stosowanych do analizy struktury tych związków. Omówiono zastosowania metod rozdziału takich, jak elektroforeza na żelu poliakryloamidowym w obecności dodecylosiarczanu sodu (SDS-PAGE), izoelektryczne ogniskowanie (IEF), elektroforeza kapilarna (CE) i wysokosprawna chromatografia cieczowa (HPLC), a także metody badania struktury białek, takie jak spektrometria masowa (MS), spektroskopia w nadfiolecie (UV), spektroskopia w podczerwieni (FTIR) wraz ze spektroskopią ramanowską, magnetyczny rezonans jądrowy (NMR), krystalografia rentgenowska oraz dichroizm kołowy (CD).
The aim of the present review was to describe the application of diode-array detectors for the identification of HPLC-separated chemical compounds on the basis of their UV/Vis spectra. Diode-array detectors serve to identify compounds containing conjugated double bonds, especially aromatic compounds, and to evaluate their purity. This group of compounds includes: peptides and proteins (in that case aromatic amino acid residues are chromophores), anthocyanins and other flavonoids, other phenolic compounds, carotenoids and other dyes. The most simple strategy of interpreting UV/Vis spectra is to determine the location of absorbance maximum in zero order spectra (such a strategy is usually applied in dye analysis). The absorbance ratio at different wavelengths was used to identify and investigate structural changes in proteins and peptides. Chemometrical analysis of zero order spectra provides a basis for evaluating peak purity and choosing the background spectrum. Derivatives of spectra may be characterised via determination of convexity intervals, location of minima of second derivative, calculation of amplitudes of second and third derivatives or similarity indices between derivatives of individual spectra. The possibilities offered by UV/Vis spectroscopy cover, among others, discrimination between spectra of proteins with different contents of aromatic amino acids (mainly tyrosine and tryptophan), as well as discrimination between spectra of anthocyanins containing the same chromophore but different glycosidic moieties.
The aim of this study was to evaluate plots showing a linear correlation between the parameters characterising protein content in the spots obtained using two-dimensional electrophoresis (scatter plots). The correlation coefficient of normalized spot volume may be used to determine regions with increased reproducibility. In our experiment, conducted using pea seed proteins, the correlation coefficient reached 0.980 in such regions, compared with 0.917 noted for the entire gel surface. The correlation coefficient calculated based on scatter plots may serve as a tool for the preliminary selection of regions characteristic of a given proteome.
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