Impact of glutathione on the allergenicity of the peach lipid transfer protein Pru p 3.

BACKGROUND AND OBJECTIVE: The allergenic potential of proteins can be altered under various physicochemical conditions. Glutathione (GSH) is a reducing agent that is used as an antioxidant in food products. We aimed to characterize the natural folding of peach proteins and test the allergenicity of reduced and natural Pru p 3, the major peach allergen. METHODS: Pru p 3 was purified from peach, and its conformation was analyzed by means of circular dichroism. Using a thiol fluorescent probe, reduced proteins were detected in fresh peach. GSH-reduced Pru p 3 was tested in vitro for T-cell proliferation and in vivo using skin prick testing. RESULTS: GSH-reduced Pru p 3 produced variable skin prick reactions in peach-allergic patients. The proliferative response of peripheral blood mononuclear cells from allergic patients to reduced Pru p 3 tended to be less intense, whereas secretion of the cytokines IFN-γ, IL-5, and IL-10 was comparable. In a pool of sera from peach-allergic patients, reduction hardly impaired IgE-binding. Moreover, the stability of reduced Pru p 3 to gastrointestinal digestion was similar to that of the natural form. CONCLUSIONS: GSH can at least transiently reduce Pru p 3. We found that the effect of reduction on the allergenicity of Pru p 3 varied. Therefore, as an additive, GSH does not seem to eliminate the risk of reactions for peach-allergic patients.

Fractionation of membrane proteins on immobilized lectins by high-performance liquid affinity chromatography.

Detergent-solubilized glycoproteins were fractionated on high-performance affinity columns employing A concanavalin and Pisum sativum agglutinin as ligands immobilized on microparticulate silica via a propyl spacer. The separations were characterized by high recovery (90-95%) and high specificity (enrichment factor 6- and 58-fold for the bound fractions on A concanavalin and P. sativum agglutinin columns, respectively, compared with the crude extract), as estimated by enzyme-linked lectin assay and chromatographic criteria. In addition, the short running times (30-40 min) make this method highly useful for a first characterization of complex glycoprotein samples and of individual molecules.

Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography. IV. Mobile phase and surface-mediated effects on recovery of native proteins in gradient elution on non-porous, monodisperse 1.5-microns reversed-phase silicas.

The reversed-phase chromatography of proteins by gradient elution with acidic, low-ionic-strength aqueous-organic eluents is often associated with losses of the biological activity of the protein. In this study, the enzymatic activities of catalase, horseradish peroxidase and pepsin were examined under static and dynamic column conditions on non-porous, monodisperse 1.5-microns reversed-phase silicas with various n-alkyl ligands. Catalase readily lost its enzymatic activity under the influence of the acidic aqueous-organic eluents in the absence of the reversed-phase packing, whereas peroxidase was partially deactivated as a result of combined mobile phase and stationary phase effects but regained its activity on storage after elution. The enzymatic activity of pepsin was found to be very dependent on the column residence time and on the type of bonded n-alkyl ligand employed.

Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography. V. Performance of non-porous monodisperse 1.5-microns bonded silicas in the separation of proteins by hydrophobic-interaction chromatography.

Non-porous monodisperse 1.5-microns silicas were allowed to react with (A) and (B) N-acetylaminopropyltriethoxysilane to generate bonded phases useful in high-performance hydrophobic-interaction chromatography (HIC). Differences in the selectivity were observed between the amide and the ether phase. Peak capacities between 10 and 30 were achieved for several proteins with the amide and ether phase packed into columns of 36 X 8 mm I.D. and elution of the proteins under chromatographic conditions in which the gradient volume, VG, was held constant by varying the gradient time between 20 and 2.5 min and the flow-rate between 0.5 and 4.0 ml/min. The S values derived from the dependences of log k' on the volume fraction of the low ionic strength buffer, phi b, were of the same magnitude as reported for porous HIC silicas and showed a dependence on the molecular weight of the protein. Using these HIC stationary phases based on non-porous 1.5-microns supports, fast separations (less than 5 min) could be carried out with high biological recoveries.

Separation of plasma membrane proteins of cultured human fibroblasts by affinity chromatography on bonded microparticulate silicas.

Adsorbents for high-performance affinity chromatography were prepared by bonding proteins and reactive Procion triazine dyes to 3-isothiocyanatopropyl- and 3-aminopropylsilicas. The materials prepared were used successfully in the separation of hydrophobic plasma membrane proteins of cultured human fibroblasts. The data obtained show that the reaction of 3-isothiocyanatopropyltriethoxysilane (ITCPS) with the surface hydroxyl groups of silica yields a new and convenient route to preparing an "activated carrier" that is capable of coupling with potential affinity ligands containing amino functional groups. The reaction and bonding procedures of 3-isothiocyanatopropyltriethoxysilane and 3-aminopropyltriethoxysilane with silica were optimized with regard to a controlled and reproducible ligand density by adjusting the type of solvent and organic base as reaction catalyst. The ligand content of reactive triazine dyes directly coupled to 3-aminopropylsilica was significantly higher than that of the 6-aminohexyl derivatives coupled to 3-isothiocyanatopropylsilica. The stability of Procion Blue MX-R bonded to 3-aminopropylsilica and 3-isothiocyanatopropylsilica in phosphate-buffered aqueous solution at pH 5.0 and 8.0 was examined.

Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography. III. Retention and selectivity of proteins and peptides in gradient elution on non-porous monodisperse 1.5-microns reversed-phase silicas.

Following previous studies of the use of non-porous monodisperse 1.5-microns n-octyl- and n-octadecyl-bonded silicas in gradient elution of proteins, this work was aimed at elucidating further the properties of this novel column material for peptide and protein separations in comparison with wide-pore silicas. First, it is demonstrated that with short columns (e.g., 35 X 8 mm I.D.) packed with these non-porous reversed-phase materials, mixtures of small peptides and mixtures of proteins can be very efficiently resolved. When the chain length of the bonded ligand was varied, the retention of a test set of proteins in gradient elution followed the ligand sequence C18 greater than C8 approximately C4 approximately phenyl greater than C2 under constant elution conditions, and the selectivity remained unchanged. Comparison of the S values of these proteins, as determined from evaluation of the log k' vs. phi dependences with non-porous silicas and with a LiChrospher Si 1000 C8 with identical accessible ligand surface areas per unit column volume, indicated lower values for the non-porous materials (k' = capacity factor; phi = molar fraction of organic solvent; S = slope of the plot of log k' vs. phi). The origin of this behaviour is discussed.

Polymer support synthesis. XV. Behaviour of non-porous surface-coated silica gel microbeads in oligonucleotide synthesis.

Non-porous silica gel microbeads of diameter 1.5 microns have been investigated as supports for oligonucleotide synthesis. In the preparation of oligothymidylates of chain length up to 150 bases, with 5'-di-p-anisylphenylmethyl-3'-phosphoramidite as an intermediate, the average yields per chain elongation were up to 99%. Lower overall yields were observed in the case of a support which developed a strong tendency towards aggregation after the build up of an oligonucleotide coating.