Binding mechanism and bioavailability of a novel phosvitin phosphopeptide (Glu-Asp-Asp-pSer-pSer) calcium complex.

Phosvitin has excellent calcium binding capacity, related to its phosphopeptides. The phosphopeptides may be used as functional ingredients for improving calcium bioavailability, but the calcium-binding mechanism is unclear. In this study, a novel phosvitin phosphorylated pentapeptide (Glu-Asp-Asp-pSer-pSer, EDDpSpS) was selected to prepare an EDDpSpS calcium complex (EDDpSpS-Ca), and the calcium-binding mechanism and bioavailability investigated. The calcium-binding capacity of EDDpSpS was up to 468 ± 152.80 mg/g. Calcium ions prompted the folding of the EDDpSpS structure to form spherical nanoparticles. The calcium binding sites of EDDpSpS involved peptide bonds, carboxyl, amino, and phosphate groups. Molecular forces involved in these interactions were electrostatic in nature. Moreover, EDDpSpS-Ca had excellent bioavailability when compared to CaCO3, calcium lactate, and d-calcium gluconate. This study revealed the calcium-binding mechanism of phosvitin phosphopeptide, and suggested that EDDpSpS-Ca has the potential to be a novel, efficient, and promising calcium supplement.

How Do Actinyls Interact with Hyperphosphorylated Yolk Protein Phosvitin?

The development of the nuclear industry has raised multiple questions about its impact on the biotope and humans. Proteins are key biomolecules in cell machinery and essential in deciphering toxicological processes. Phosvitin was chosen as a relevant model for phosphorylated proteins because of its important role as an iron, calcium, and magnesium storage protein in egg yolk. A multitechnique spectroscopic investigation was performed to reveal the coordination geometry of two oxocations of the actinide family (actinyl UVI , NpV ) in speciation with phosvitin. IR spectroscopy revealed phosphoryl groups as the main functional groups interacting with UVI . This was confirmed through laser luminescence spectroscopy (U) and UV/Vis absorption spectroscopy (Np). For UVI , X-ray absorption spectroscopy at the LIII edge revealed a small contribution of bidentate binding present, along with predominantly monodentate binding of phosphoryl groups; for NpV , uniquely bidentate binding was revealed. As a perspective to this work, X-ray absorption spectroscopy speciation of UVI and NpV in the extracted yolk of living eggs of the dogfish Scyliorhinus canicula was determined; this corroborated the binding of phosphorous together with a reduction of the actinyl moiety. Such data are essential to pinpoint the mechanisms of heavy metals (actinyls) accumulation and toxicity in oviparous organisms, and therefore, contribute to a shift from descriptive approaches to predictive toxicology.

Role of polysaccharide conjugation in physicochemical and emulsifying properties of egg phosvitin and the calcium binding capacity of its phosphopeptides.

Phosvitin phosphopeptides (PPP) effectively enhanced calcium bioavailability via inhibiting calcium-phosphate deposition. It is difficult to hydrolyze native phosvitin (PSV) to release PPP due to its compact structure. Polysaccharide conjugation could improve the biofunctional properties of proteins via altering their structures. In this research, PSV was subjected to conjugation with pectin, and changes in physicochemical characteristics and functionalities were determined. The results showed that PSV underwent an unfolding process when conjugated with pectin at a mass ratio of 1 : 2, exposing more hydrophobic groups. Excessive glycosylation induced a refolded structure with a lower surface hydrophobicity and a higher thermal stability. These secondary and tertiary structural changes improved the emulsifying properties of PSV and allowed the production of emulsions with smaller oil droplets. Simultaneously, due to redistribution of phosphate groups, the PPP derived from copolymers exhibited a stronger calcium binding capacity, especially at a mass ratio of 1 : 6, possessing a potential to be utilized in functional foods.

Egg yolk phosvitin and functional phosphopeptides--review.

Phosphopeptides are among the most interesting biomolecules with characteristic molecular structure and functions. They usually contain clusters of phosphoserines, which can effectively bind calcium and iron, and inhibit formation of insoluble calcium phosphates or iron complexes. Therefore, phosphopeptides can increase calcium or iron bioavailability and prevent lipid oxidation in foods. Milk protein casein has been currently used by industry to produce phosphopeptides. Egg yolk phosvitin is considered as the most phosphorylated protein found in the nature. Phosvitin from egg yolk can be much better source for producing phosphopeptides with varying sizes and functions than casein because it contains much greater number of phosphates in the molecule than casein. However, still phosvitin has not been subjected to considerable attention with regard to bioactive peptides production.

Complexation of U(VI) with highly phosphorylated protein, phosvitin A vibrational spectroscopic approach.

The complexation of uranium(VI) to variant functional groups of the highly phosphorylated protein phosvitin in aqueous solution was investigated by attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy. For the verification of the affinity of the actinyl ions to carboxyl and phosphate groups of the amino acid side chains, samples with different phosphate to uranium(VI) (P/U) ratios were investigated under denaturing conditions as well as in aqueous medium. From a comparative study with other heavy metal ions, i.e. Ba(2+) and Pb(2+), a strong coordination of U(VI) to carboxyl and phosphoryl groups can be derived. Furthermore, with increasing P/U ratios, a preferential binding of U(VI) to phosphoryl groups is indicated by the spectra of the batch samples. These findings are confirmed by spectra of aqueous U(VI)-phosvitin complexes reflecting an explicit coordination of the uranyl ions to phosphate groups at a high P/U ratio. Our study provides a deeper insight into the molecular interactions between actinyl ions and protein, and can be conferred to other basic biomolecules such as polysaccharides and nucleic acids.

A colorimetric protein phosphatase inhibition assay for the determination of cyanobacterial peptide hepatotoxins based on the dephosphorylation of phosvitin by recombinant protein phosphatase 1.

A colorimetric protein phosphatase inhibition assay based on the dephosphorylation of phosvitin by recombinant protein phosphatase 1 was developed for analysis of waters for cyanobacterial hepatotoxins. The phosphate released in the assay was determined using a malachite green reagent. Good agreement with toxin concentrations determined by HPLC was obtained. The assay was capable of determining these toxins at concentrations around 1 microgram/L with high precision and without sample concentration. This is of considerable benefit as the World Health Organisation specifies a provisional guideline of 1 microgram/L for microcystin-LR. There was evidence, however, that the sample matrix might affect quantification, leading to false positive results. Thus the assay should be viewed as a screening procedure, and confirmatory analyses by an alternative procedure should be carried out for positive results. Further work is required to resolve the question of matrix interferences if phosphatase inhibition assays are used directly for measuring toxin levels in water, especially if this information is used to check compliance with water quality guidelines.

A new method for in vitro calcification using acrylamide gel and bovine serum.

To investigate the mechanism of biological calcification in vitro, a model system consisting of an acrylamide gel block (1 x 3 x 3 mm) and fetal bovine serum was developed. Mineral deposition was induced in gel blocks which were immersed in 300 microliters of fetal bovine serum at 37 degrees C for 7 days in a CO2 incubator. X-ray diffraction indicated that the mineral was hydroxyapatite with low crystallinity. Effects of the concentration of acrylamide gel, the partial pressure of CO2 and matrix proteins within the gel on the mineral formation were investigated. In the gel concentration range of 10-60%, the largest amount of crystal grew in 40% acrylamide gel, where the serum protein did not penetrate. With an increase in the partial pressure of CO2 the Ca content in the gel block increased, reached the highest level at about 3.5% CO2 and then began to decrease. In 40% gel and at 5% CO2, the mineral formation was enhanced by phosvitin, phosphophoryn, demineralized dentin powder and alkaline phosphatase. Mineral deposition occurred around the collagen fibers immobilized in 40% acrylamide gel. These results indicate that 1) a putatively serum-derived inhibitor of calcification with high-molecular weight was prevented from penetrating into the 40% acrylamide gels, 2) immobilized polyanionic proteins and alkaline phosphatase were able to increase mineral deposition and 3) the partial pressure of CO2 greatly influenced the mineral deposition. It was concluded that this gel system is useful to investigate the mechanism of biological calcification in vitro.

Analysis of mosquito vitellogenin cDNA. Similarity with vertebrate phosvitins and arthropod serum proteins.

The cDNA coding for vitellogenin of the mosquito Aedes aegypti was cloned and sequenced. An immunological analysis of expressed deletions from the 5'-end of the vitellogenin cDNA clones using vitellogenin subunit-specific antibodies showed that the small vitellogenin subunit is located at the N terminus and the large one at the carboxy-portion of the pre-provitellogenin. The position of the cleavage between the vitellogenin subunits in the pre-provitellogenin was identified by locating the N terminus of the large subunit. The cleavage site has a consensus RXRR for the subtilisin-processing endoprotease. Mosquito vitellogenin is highly hydrophilic with 17 putative N-linked glycosylation sites and 13 potential tyrosine sulfation sites. In contrast to known invertebrate vitellogenins, mosquito vitellogenin contains three polyserine domains that are similar to those of phosvitins in vertebrate vitellogenins. These polyserine domains, originally presumed to be vertebrate-specific, have several phosphorylation consensus sites in their sequences. Unlike other known vitellogenins, mosquito vitellogenin is rich in aromatic amino acid residues, tyrosine and phenylalanine, and in this respect is similar to insect serum proteins, arylphorins. This similarity suggests that mosquito vitellogenin may supply aromatic amino acids to the cuticle of rapidly developing embryos.

Comparative studies of phosvitin from chicken and salmon egg yolk.

1. A method developed for the isolation of phosvitin from chicken egg yolk was successfully applied to the isolation of phosvitin from salmon eggs. 2. Salmon roe phosvitin is smaller in molecular size than chicken egg phosvitin. 3. Circular dichroism spectra of all phosvitins investigated displayed good similarities with spectra showing characteristics of unordered and beta-sheet secondary structure. 4. The main component in the Fourier transform infrared spectra of chicken egg phosvitin is indicative of unordered conformation, whereas the Fourier infrared data of the salmon egg phosvitin are consistent with more of beta-sheet structure compared to the chicken egg phosvitin.

Phosphorus nuclear magnetic resonance of diverse phosvitin species.

1. High resolution 31P nuclear magnetic resonance (NMR) spectra, with and without proton decoupling, of the principal egg phosphoproteins--phosvitins--of a bird (Gallus gallus), an amphibian (Xenopus laevis) and a fish (Salmo gairdneri) were obtained. 2. The spectra were evaluated with special reference to available amino acid sequences and the major NMR resonance in all three spectra was assigned to phosphoserine clusters. 3. The resolution of numerous additional phosphorus resonances provides the basis for further investigation of the particular molecular environments of phosvitin-bound phosphoryl groups and their involvement in the diverse binding modes for metal complex formation by phosvitins.