Bioactivities of hen's egg yolk phosvitin and its functional phosphopeptides in food industry and health.

Phosvitin, one of the most noteworthy bioactive components of hen egg yolk, is an amphiphilic protein that stands out with its unique composition and functionality in the food industry and health. Phosvitin consists of 4% of egg yolk dry matter and 11% of egg yolk proteins. It is considered as the most phosphorylated protein with 10% phosphorus. Besides, some potential novel phosphopeptides containing clusters of phosphoserines can be derived from hen's egg yolk phosvitin. Phosvitin, which has many functional features thanks to its unique structure, is known primarily for its metal bonds binding (iron, calcium, etc.) feature. On the other hand, its phosphopeptides may increase the bioavailability of metals compared to phosvitin. Although this feature of phosvitin may partially decrease the bioavailability of especially iron in the egg, it allows the phosvitin to have many bioactivities in the food industry and health. Lipid oxidation, which is a serious problem in the food industry, can be inhibited by adding phosvitin and its derived phosphopeptides to the food production chain via inhibiting bivalent iron. Because phosvitin is an amphiphilic protein capable of chelating, it also shows potential antibacterial effects against the Gram-negative bacteria. Moreover, the literature has recently been attempting to define the promising relationship between phosvitin and its phosphopeptides and plenty of health-promoting activities such as immune-enhancing, melanogenesis inhibitor, anti-ageing, and anticancer. In this review, current information on the hen's egg yolk phosvitin and its phosphopeptides and their bioactivities in the food industry and health are discussed and some future directions are given.

In-Tip Lanthanum Oxide Monolith for the Enrichment of Phosphorylated Biomolecules.

Polymeric monoliths fabricated in tips with embedded materials of choice are important in separation science. Polymeric backbone however interferes in the enrichment and thus affects efficiency. This work focuses on the in-tip fabrication of lanthanum oxide porous monolith and its application in the enrichment of phosphorylated peptides and lipids. Polycondensation reaction uses an aqueous solution of LaCl3·7H2O with N-methyl formamide as porogen and propylene oxide as initiator. The aging time of monolith and temperature condition for the reaction are optimized to attain porous monolithic tip. A comparison of (i) solid phase batch extraction using La2O3, (ii) La2O3 embedded in poly(glycidyl methacrylate (GMA)/divinylbenzene (DVB)) tip, and (iii) pure La2O3 monolithic tip shows improved enrichment efficiency in the case of pure La2O3 monolithic tip. The monolithic tip achieves selectivity of 1:4500 as compared to solid phase extraction (SPE)(1:3500) and limit of detection down to 0.25 fmol. The in-tip La2O3 monolith strategy has better batch to batch reproducibility, reduced time of enrichment, and ease of operation in comparison to solid phase batch extraction. The developed strategy enriches phospho- content from biological samples like phosvitin and lipovitellin from egg yolk and phospholipids/phosphopeptides from human serum. The enriched phospho- moieties are analyzed by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) except the phospholipids where laser desorption ionization (LDI)-MS is employed.

Comparison of a novel TiO2/diatomite composite and pure TiO2 for the purification of phosvitin phosphopeptides.

A novel TiO2/diatomite composite (TD) was prepared and then characterized by scanning electron microscope (SEM) and Fourier Transform Infrared (FTIR). The results of SEM showed that after modification, the porous surface of diatomite was covered with TiO2. Both diatomite and TD had clear disc-shaped structures with average grain diameters of around 25 µm. Then TD and pure TiO2 were applied in the purification of phosvitin phosphopeptides (PPPs) from the digest of egg yolk protein, and a comparative study of adsorption properties of PPPs on TD and TiO2 was performed. In the study of adsorption kinetics, the adsorption equilibrium of PPPs on TD and TiO2 fitted well with the Langmuir model, and the time needed to reach adsorption equilibrium were both around 10 min. The maximum dynamic adsorption capacity of TD (8.15 mg/g) was higher than that of TiO2 (4.96 mg/g). The results of repeated use showed that TD and TiO2 were very stable after being subjected to ten repeated adsorption-elution cycles, and TD could easily be separated from aqueous solution by filtration. On the other hand, the present synthetic technology of TD was very simple, cost-effective, organic solvent-free and available for large-scale preparation. Thus, this separation method not only brings great advantages in the purification of PPPs from egg yolk protein but also provides a promising purification material for the enrichment of phosphopeptides in proteomic researches.

Highly efficient enrichment of phosvitin phosphopeptides by novel magnetic carboxymethyl chitosan nanoparticles decorated with Fe (III) ions.

Functional immobilized metal affinity magnetic carboxymethyl chitosan nanoparticles (abbreviated as Fe(3)O(4) (PEG+CM-CTS) @ Fe (III)) were conveniently applied for phosvitin phosphopeptides (PPPs) enrichment for the first time. The morphology of magnetic nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of Fe(3)O(4) (PEG+CM-CTS) @ Fe (III) was about 20 nm, and could easily aggregate by a magnet when suspending in the aqueous solution. In the PPPs enrichment study, the results obtained emphasized the role of pH, temperature and the initial concentration of the peptides solution in governing the efficiency and mechanism of affinity interactions. Due to the large specific surface area, the enrichment of PPPs onto the Fe(3)O(4) (PEG+CM-CTS) @ Fe (III) nanoparticles was promising. The adsorption equilibrium of PPPs onto the obtained magnetic nanoparticles fitted well with the Langmuir model, and the nitrogen/phosphorus molar ratio (N/P) which at the maximum enrichment capacity for PPPs was 4.83. Due to the small diameter, the decrease of the N/P is particularly rapid in the early enrichment stages (0-30 min) to reach a plateau after 60 min. Compared with traditional methods, the need for preparation of phosvitin before purification is obviated and PPPs of higher purity were obtained. Since the preparation, surface modification and affinity separation processes of the magnetic nanoparticles are cost-effective, convenient and efficient, this type of Fe(3)O(4) (PEG+CM-CTS) @ Fe (III) nanoparticles would bring advantages compared to conventional separation techniques of PPPs from chicken egg yolk, as well as for phosphopeptides enrichment in proteomics research.

Voltammetric sensing of phosphoproteins using a gallium(III) acetylacetonate-modified carbon paste electrode.

The voltammetric detection of phosphoproteins was developed using a gallium(III) acetylacetonate-modified carbon paste electrode. Because phosphate groups of the protein interacted with the gallium(III) ion, the protein was accumulated on the electrode surface. A hexaammine ruthenium(III) ion, which combined with the functional groups, was used to monitor the interaction. When phosvitin and hexaammine ruthenium(III) ions were incubated in 0.1 M acetate buffer (pH 3.2), a reduction peak of hexaammine ruthenium(III) ion at the electrode decreased as the concentration of the protein increased. In contrast, an increase in the peak current was observed with a plain carbon paste electrode. These results were caused by a competitive reaction of the phosphate groups with the hexaammine ruthenium(III) and gallium(III) ions. In the presence of α-, ß- and κ-caseins, the electrode response decreased due to the order of the numbers of phosphate groups. This method could be applied to the sensing of phosphoproteins at the 10(-10) M level.

Carp (Cyprinus carpio) vitellogenin: characterization of yolk proteins, development of immunoassays and use as biomarker of exposure to environmental estrogens.

The precursor protein of egg yolk, vitellogenin (Vg), is cleaved into three major components (lipovitellin, phosvitin and beta'-component) at the time of incorporation by growing oocytes. We purified three yolk proteins (YP1, YP2 and YP3) from ovaries of the common carp (Cyprinus carpio) by a combined method of ammonium sulfate precipitation and column chromatography. Biochemical analyses of the purified proteins of this species suggest that YP1, YP2 and YP3 are lipovitellin, beta'-component and phosvitin, respectively. A specific antiserum against purified carp YP1 (lipovitellin) was used to develop a single radial immunodiffusion (SRID) technique and an enzyme-linked immunosorbent assay (ELISA) for carp Vg. By SRID and ELISA, we measured the circulating carp Vg level to be in the ranges of 12.5-400 microg/ml and 2.0-1000 ng/ml, respectively, which cover a wide range of Vg levels. From 1997-1998, male and female carp were captured at points of effluent discharge from a sewage treatment plant connected to the Tama River, where estrogenic compounds were later detected, and the presence of Vg in their circulation was examined. Vg was detected in both male and female carp at the mg/ml level, suggesting that estrogens such as estrone and estradiol were sufficiently high to induce Vg in male carp inhabiting this area. The result of this study supports the use of carp Vg as a biomarker of fish exposure to environmental estrogens.

Relationship between vitellogenin and its related egg yolk proteins in Sakhalin taimen (Hucho perryi).

Vitellogenin (Vg) and its three egg yolk protein products, lipovitellin (Lv), phosvitin (Pv) and beta'-component, were isolated from mature female Sakhalin taimen (Hucho perryi). Vg had an apparent molecular weight of 540 kDa and appeared as a major 240 kDa band in SDS-PAGE, which resolved into two major bands (165 and 125 kDa) after reduction. The estimated molecular weights of purified Lv, Pv, and beta'-component were 330, 23, and 30 kDa, respectively. Lv appeared as a main band of 150 kDa in SDS-PAGE which resolved into two smaller bands (92 and 29 kDa) after reduction. beta'-component appeared as a 34 kDa band before and as a 17kDa band after reduction. Except for Pv, the purified proteins all reacted with an antiserum to Vg. In SDS-PAGE, Pv appeared as a 23 kDa band and a second < 6.5 kDa diffuse band. An antiserum to Pv dephosphorylated by alkaline phosphatase (Ap) was prepared. In Western blots, the antiserum reacted with dephosphorylated Pv and Vg, but not with Lv and beta'-component. This is the first immunological proof that three egg yolk proteins (Lv, Pv, and beta'-component) are derived from Vg in fish.

Preparation and rapid resolution of Xenopus phosvitins and phosvettes by high-performance liquid chromatography.

The presence of acidic phosivitins phosvettes in Xenopus laevis yolk platelets and their purification by (NH4)2SO4 precipitation of associated lipovitellin were documented by polyacrylamide gel electrophoresis followed by staining with Stains-all. Procedures were further developed to resolve the various entities present in the crude phosvitin/phosvette fraction by size-exclusion, anion-exchange, and hydrophobic interaction chromatography, using a Pharmacia FPLC system, and their resolution was documented by both electrophoresis and two-dimensional chromatography. Four major entities (phosvitins 1 and 2; phosvettes 1 and 2) were observed, but microheterogeneity was also apparent, particularly by hydrophobic interaction chromatography. The new separation procedures require min/h rather than h days.

Coadsorption onto hydroxyapatite of human salivary mucins and phosvitin, a phosphoprotein from egg yolk.

The adsorption of human salivary mucins (HWSM, 0.4 mg/ml) onto hydroxyapatite (HAP) was studied in the presence of varying amounts of the phosphoprotein phosvitin by three different procedures. a. Preadsorption of HWSM onto HAP for 20 h, followed by 4 h coadsorption with phosvitin, resulted in a decrease of 50% in HWSM binding to HAP with 0.3 mg/ml phosvitin and a complete desorption with 1.0 mg/ml phosvitin. b. Preincubation of HAP with phosvitin for 20 h, followed by 4 h coadsorption with HWSM, resulted in decrease of 50% in HWSM binding to HAP with 0.15 mg/ml phosvitin and the adsorption of HWSM was prevented completely with 1.0 mg/ml phosvitin. c. Simultaneous incubation of HWSM and phosvitin gave the least adsorption of HWSM to HAP: a decrease of 50% with as little as 0.025 mg/ml phosvitin and a nearly complete desorption with 0.3 mg/ml phosvitin. Similarly, the adsorption of phosvitin was strongly inhibited by HWSM after either simultaneous adsorption or preadsorption with HWSM. However, after preincubation of HAP with phosvitin, desorption of phosvitin by HWSM was not achieved. Release of phosphate increased by preadsorption with HWSM followed by incubation with phosvitin, but was lowered by about 50% after preadsorption with phosvitin. After simultaneous incubation of HAP with both species, the adsorption did not result in release of phosphate ions. Calcium release was only substantial when phosvitin was in excess in solution. The smallest release of calcium ions was observed when HAP was preincubated with phosvitin, followed by coadsorption with HWSM.

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.

Genetic variants of phosvitin in egg yolk of the Japanese quail, Coturnix coturnix japonica.

Phosvitin polymorphism in egg yolk of the Japanese quail was found by horizontal polyacrylamide gradient gel electrophoresis. Six phenotypes of yolk phosvitin designated A, B, C, AB, AC, and BC were observed in a population of 281 birds. Analysis of family data revealed that the phenotypic variation of quail yolk phosvitins was controlled by an autosomal Pv locus with three codominant alleles, Pva, Pvb and Pvc. The gene frequencies of Pva, Pvb and Pvc were 0.064, 0.824 and 0.112, respectively.

Calcium transport in nonmammalian vertebrates.

The title of this paper commemorating the contributions made by Professor Urist has an interesting bearing upon basic skeletal tissue biology. This is because the calcium-binding proteins (vitellogenins), upon which Professor Urist and Schjeide have focused much of their attention in non-mammalian vertebrates, although produced by the liver and present in the blood plasmas of non-mammalian vertebrates during vitellogenesis, are, in effect, substitutes for the protein casein present in the milk of mammalian vertebrates. Vitellogenins (180,000-250,000 daltons) appear to be produced solely for deposition in the yolk of the egg so that the calcium they carry (considerably more than is associated with casein of milk) and the amino acids of which they are comprised can be utilized during embryonic development. In many instances the progeny of non-mammalian vertebrates emerge from the shell as miniatures of the adult, capable of rapid movement and thus requiring a well developed skeletal as well as muscular system. Vitellogenins are not found in any other cells (phagocytes excepted) other than hepatocytes wherein they are made, nor are they present in the intercellular matrix of developing or remodeling bone. In non-vitellogenic females and in males of non-mammalian vertebrates, they are absent from the blood plasma altogether, so that nonionized calcium therein is solely bound to such proteins as albumin and the lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorus-31 NMR as a probe for phosphoproteins.

Nuclear magnetic resonance methodology continues to advance such that phosphorus-31 NMR experiments can be profitably applied to elucidate some aspects of proteins which are covalently phosphorylated. This review introduces NMR spectral parameters pertinent to using phosphorus-31 NMR for investigation of structure and dynamics. The techniques of two-dimensional NMR, solid state NMR, and isotopic substitution are also introduced. Characteristics of phosphorylated amino acids and peptides, as revealed by phosphorus-31 NMR, are described. Studies of phosphorylated containing phosphomonoesters, phosphoramidates, acyl phosphates, and disubstituted phosphorus bridges are discussed. Among these phosphoproteins are several examples where phosphorus residues evidently play a role as polyelectrolytes, in enzyme catalysis, and in regulation of protein function.

Amino acid sequence of phosvitin derived from the nucleotide sequence of part of the chicken vitellogenin gene.

The amino acid sequence of the egg yolk storage protein phosvitin has been deduced from the nucleotide sequence of part of the chicken vitellogenin gene. Of the phosvitin sequence, 210 amino acids including the N-terminal residue are contained on one large exon, whereas the remaining six amino acids are encoded on the next exon. Phosvitin contains a core region of 99 amino acids, consisting of 80 serines, grouped in runs of maximally 14 residues interspersed by arginines, lysines, and asparagines. The serines of the core region are encoded by AGC and AGT codons exclusively and the arginines by AGA and AGG, which results in a continuous stretch of 99 codons with adenine in the first position. The N-terminal quarter of the phosvitin sequence contains 16 serines grouped in a cluster with alanines and threonines and coded mainly by TCX triplets. The C-terminal part includes 27 serines, preferentially coded by AGC and AGT, 13 histidine residues, and the sequence ...Asn-Gly-Ser... at which the carbohydrate moiety of phosvitin is attached. Heteroduplex formation between cloned DNAs from chicken and Xenopus vitellogenin genes shows that the phosvitin sequence contains a stretch of highly conserved sequence.

Lipovitellin-phosvitin crystals with orthorhombic features: thin-section electron microscopy, gel electrophoresis, and microanalysis in teleost and amphibian yolk platelets and a comparison with other vertebrates.

Yolk-platelet crystals in the teleosts Pelvicachromis pulcher and Noemacheilus barbatulus and the amphibians Xenopus laevis, Rana temporaria, R. esculenta, and Triturus sp. have been studied by electron diffraction and imaging using a standardized processing (glutaraldehyde-osmium tetroxide fixation, glutaraldehyde-urea embedding, thin-section staining), by X-ray microanalysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of their constituents. The crystal lattice is orthorhombic having--following standardized processing--in three amphibians a = 9.0 nm, b = 17.6 nm, c = 19.2 nm, and in the two teleosts a = 8.9 nm, b = 17.6 nm, c = 20.0 nm (averages). These values are very close to X-ray data from wet crystals (Xenopus laevis). Crystal images in the three axial projections point to the presence of space group P212121 (or an approximation of it since the lipovitellin dimers cannot be fully equivalent in some cases), to differences between the phosvitins of the two teleosts, and to a highly unusual stain exclusion from large crystal constituents interpreted as representing lipovitellin dimers. Microanalysis in ultrathin cryosections and other preparations revealed K and Cl to be the prominent ions in the crystals (and in the superficial layer of the platelet). Gel electrophoresis (including data of cyclostomes) showed considerable molecular variations despite a closely similar crystal architecture, emphasizing a physiological significance of the architecture, which may have remained conserved for nearly 400 million years according to paleontologic views.

Chemical and physical modification of proteins by the hydroxide ion.

Proteins are exposed to alkaline conditions during solubilization and/or purification, during food storage and processing, in removal of toxic constituents, and for characterization. During alkali treatment, there are changes in solubility and aggregation, hydrolysis, elimination reactions involving the side chains of certain amino acids, racemization of amino acid residues, addition of compounds to proteins, fragmentation of the peptide chain, as well as modification or elimination of nonprotein constituents. The rates of these reactions are affected by pH, temperature, cations (in some cases), ionic strength (in some cases), protein concentration, and to some extent by the specific nature of the protein. The general mechanisms and stoichiometry of these reactions are described. Other constituents of high protein foods also undergo reactions in alkaline solutions and the products of these reactions may in turn react with proteins. We have described the effect of alkali on enediol formation and fragmentation of carbohydrates, the hydrolysis of lipids in alkaline solution and effect on rate of peroxidation of the polyunsaturated fatty acids, the oxidation of amino acid residues, especially methionine, the oxidation of phenols to benzoquinones, and the catalytic effect of metal ions in alkaline solutions. Alkali treatment is also used in the specific modification of proteins to distinguish between O-glycosyl and amide-linked glycosyl groups, to effect specific cleavage of peptide bonds via beta elimination, in the formation of anhydrotrypsin, anhydrochymotrypsin, anhydrosubtilisin and thiol-subtilisin, and in formation of intrachain crosslinking in proteins.

Standards for measuring spatial resolution in biological X-ray microanalysis.

Lateral resolution in biological X-ray microanalysis is usually severely limited by the necessity to obtain an adequate number of X-ray counts, which requires the use of a relatively large electron beam spot size. Resolution is further degraded by spreading of the beam within the specimen. Although it is possible to make theoretical calculations of resolution, no straightforward and general method of measuring lateral resolution in biological X-ray microanalysis appears to have been proposed hitherto. In this paper, standards are proposed consisting of distinctive protein-bound elements embedded in araldite, and their use is described.

Phosphotyrosine in proteins. Stability and quantification.

The acid and base stability of the phosphoryl bond of phosphotyrosine (Tyr-P) was studied using conditions for rapid and complete hydrolysis of protein peptide bonds. A method was developed for the quantification of Tyr-P in proteins using rapid base hydrolysis and an amino acid analyzer equipped with a fluorometric detection system. The recovery of [32P]Tyr-P from base digests of radiolabeled samples of phosphotyrosyl glutamine synthetase, transforming protein of Rous sarcoma virus, casein, and rabbit anti-sarcoma IgG was 80 +/- 2%. Phosphotyrosine could not be detected in several commercial histone samples, but Tyr-P was detected in phosvitin samples. The putative Tyr-P from the phosvitin hydrolysate was separated from normal amino acids by Dowex 50-H+ chromatography. Treatment of the partially purified Tyr-P with bacterial alkaline phosphatase produced tyrosine in near equivalent quantities to the measured level of Tyr-P. These results show that basic hydrolysis of phosphotyrosyl proteins yields Tyr-P in constant and good yields which can be quantified in amounts greater than or equal to 100 pmol or radiochemically detected in smaller amounts with an amino acid analyzer.

Localization of phosphoproteins and of protein kinases in chromatin from hepatoma tissue-cultured cells.

An important role in the control of gene expression has been attributed to phosphoproteins present among chromatin non-histone proteins. In a previous work we have shown that at least part of these phosphoproteins are associated with nucleosomes. In this work we wanted to establish whether this association occurs with all nucleosomes or with the nucleosomes present in fragments preferentially released by a mild micrococcal nuclease digestion, which originated essentially from active parts of chromatin. Phosphoproteins were labelled in vivo by incubating hepatoma tissue-cultured cells with [32P]phosphate and chromatin was submitted to a limited micrococcal nuclease digestion. The released fragments were fractionated by preparative gel electrophoresis. [32P]Phosphoproteins were essentialy found in the smallest released fragments: monomers and dimers of nucleosomes. The same result was obtained when the phosphoproteins were labelled in vitro by incubating each fragment obtained by the preparative electrophoresis in the presence of [gamma-32P]ATP. It indicates that part of the protein kinase activity was strongly bound to the particles. The bound phosphoproteins were analysed by sodium dodecylsulfate/polyacrylamide gel electrophoresis. Two main polypeptides were characterized: phosphopeptide a, Mr 41000, present in all small fragments; phosphopeptide b, Mr 31000, present in all small fragments, except in the fastest moving nucleosomes. Phosvitin kinase was found associated with the small released fragments, its specific activity was by far the highest in the fraction which includes the dimers of nucleosomes. It is concluded that phosphoproteins and protein kinases are associated with the nucleosomes of the active parts of chromatin, which suggests a role of these proteins in the control of gene expression.