Simultaneous purification of biotin-binding proteins-I and -II from chicken egg yolk and their characterization.
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ABSTRACT:
SUBMITTER: Subramanian N
PROVIDER: S-EPMC1136964 | biostudies-other | 1995 Jun
REPOSITORIES: biostudies-other
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Project description:The plasma of laying hens contains a specific biotin-binding protein that appears to be identical with an egg-yolk biotin-binding protein. Both proteins are saturated with biotin and require elevated temperatures to effect the exchange of [14C]biotin for the protein-bound vitamin. The heat-exchange curve in each case is the same and differs sharply from that of avidin, the egg-white biotin-binding protein. On Sephadex G-100 gel filtration, plasma and yolk biotin-binding proteins were each eluted slightly ahead of avidin (mol.wt. 68,000), suggesting that they are of similar molecular weight. Plasma and yolk biotin-binding proteins required the same ionic strength to be eluted from a phosphocellulose ion-exchange column. Both the plasma and yolk biotin-binding proteins had a pI of 5; avidin has a pI of 10. Plasma biotin-binding protein cross-reacted with antiserum to yolk biotin-binding protein and showed a precipitin line of identity with purified yolk biotin-binding protein. It is suggested that biotin-binding plays an important role in mediating the transport of the vitamin from the bloodstream to the developing oocyte.
| S-EPMC1186112 | biostudies-other
Project description:1. Biotin in chicken egg yolk is non-covalently bound to a specific protein that comprises 0.03% of the total yolk protein (0.8 mg/yolk). This biotin-binding protein is not detectable by the normal avidin assay owing to the biotin being tightly bound. Exchange of [14C]biotin for bound biotin at 65 degrees C is the basis of an assay for this protein. 2. Biotin-binding protein from egg yolk is distinguishable from egg-white avidin on Sephadex G-100 gel filtration, although the sizes of the two proteins appear quite similar. 3. Biotin-binding protein is denatured at a lower temperature and freely exchanges biotin at lower temperatures than does avidin. 4. The biotin-binding protein in egg yolk is postulated to be responsible for the deposition of biotin in egg yolk. D-[carboxyl-14C]Biotin injected into laying hens rapidly appears in the egg bound to yolk biotin-binding protein and avidin. Over 60% of the radioactivity is eventually deposited in eggs. The kinetics of biotin deposition in the egg suggests a 25 day half-life for an intracellular biotinyl-coenzyme pool in the laying hen.
| S-EPMC1163865 | biostudies-other
Project description:BBP-II, the major biotin-binding protein from chicken oocytes, was purified 12,000-fold with a 22% yield. The purification procedure includes butan-1-ol extraction of yolk lipids, phosphocellulose chromatography of the water-soluble proteins, DEAE-cellulose chromatography at pH 7.4 and hydroxyapatite column chromatography. Final purification was obtained by using a second DEAE-cellulose column chromatography at pH 6.0. BBP-I activity separated from BBP-II activity during elution from the first DEAE-cellulose column. Purified BBP-II was homogeneous on both polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis under conditions that would detect a 1% impurity. The subunit Mr determined from SDS/polyacrylamide-gel electrophoresis was 18,200 (72,600 for tetramer), which compares favourably with an Mr value of 17,300 (69,100) calculated from the amino acid analysis. A single precipitin line formed when rabbit antiserum to the protein was directed against a crude chicken egg-yolk sample. BBP-II purified by this procedure lacked carbohydrate and phosphate, was stable indefinitely when frozen, and was quite stable at room temperature. The N-terminal amino acid sequence showed polymorphism at three positions in the first 23 residues and was about 45% identical with the N-terminal 22 residues of avidin. Antiserum to BBP-II cross-reacted with BBP-I and similar proteins in the yolk of eggs from various birds and alligator as judged by immunodiffusion and enzyme-linked immunosorbent assays. No cross-reaction was observed with chicken egg-white by either of these methods.
| S-EPMC1135486 | biostudies-other
Project description:Besides being a common food component broadly consumed worldwide, egg yolk immunoglobulin Y (IgY) has essential therapeutic potentials. In fact, in a time of ever-increasing risk of antibiotic resistance, it is crucial to find new ways to battle infection, and oral administration of preformed specific antibodies represents one of the most attractive approaches against infection. Infectious diseases of bacterial and viral origin in humans and animals can be controlled and passively cured by orally applied IgYs isolated from chicken egg yolks. Despite multiple obvious advantages of oral administration of IgY, harvesting IgY from egg yolk in a pure form is a challenging task. In this study, we developed a fast, simple, cost-effective, and efficient protocol for IgY isolation from chicken egg yolks. First, egg yolk was collected and diluted with 5 volumes of cold distilled water, homogenized, pH adjusted, and centrifuged. Next, the supernatant was collected, to which caprylic acid at concentration of 2% v/v was added, followed by pH adjustment to pH 5.0, centrifugation at 4°C, and collection of the resulting supernatant. This step was repeated twice, with adding 2% v/v of caprylic acid each time. The final supernatant was concentrated using ultrafiltration, and the IgY purity and activities were checked by SDS-PAGE, western blotting, and ELISA. The sequential (2, 2, 2%) addition of caprylic acid yielded IgY with a purity of 63.5, 90.6, and 95.8%, respectively, and reached 97.9% after ultrafiltration at pH 9.0. The IgY activity increased exponentially to reach 99% after the ultrafiltration step. The proposed caprylic-acid-based protocol of IgY purification from the yolk of chicken eggs seems to be simple, fast, direct, and very cheap. This indicates that this protocol has great potential for scale-up processing.
| S-EPMC7936219 | biostudies-literature
Project description:Aminopeptidase Ey (EC 3.4.11.20) from chicken (Gallus gallus domesticus) egg yolk is a homodimeric exopeptidase with a broad specificity for N-terminal amino acid residues at P1 position of the substrate. Aminopeptidase Ey is a 300-k metalloexopeptidase, containing 1.0 g atom of zinc per mole of a subunit with a relative molecular mass of 150 k. A full-length cDNA was cloned from chicken (female) liver cDNA library. Analysis of the 3196-base pairs (bp) nucleotide sequence of the cDNA revealed a single open reading frame coding for 967 amino acid residues. The coding region of aminopeptidase Ey gene, apdE, occupies 2901 bp of the cDNA. The predicted amino acid sequence of the enzyme is 66, 65, 64 and 63% identical with those of aminopeptidases N (EC 3.4.11.2) from human, pig, rabbit and rat, respectively. Aminopeptidase Ey contains the metallo-binding sequence motif, His-Glu-Xaa-His, found in zinc metallopeptidases. Zinc binding sites, His-386, His-390 and Glu-409, and catalytic site, Glu-387, were conserved in the homologous aminopeptidases N.
| S-EPMC7172579 | biostudies-literature
Project description:BackgroundToxoplasma gondii may cause abortions, ocular and neurological disorders in warm-blood hosts. Immunized mammals are a wide source of hyperimmune sera used in different approaches, including diagnosis and the study of host-parasite interactions. Unfortunately, mammalian antibodies present limitations for its production, such as the necessity for animal bleeding, low yield, interference with rheumatoid factor, complement activation and affinity to Fc mammalian receptors. IgY antibodies avoid those limitations; therefore they could be an alternative to be applied in T. gondii model.Methodology/principal findingsIn this study we immunized hens with soluble tachyzoite antigens of T. gondii (STAg) and purified egg yolk antibodies (IgY) by an inexpensive and simple method, with high yield and purity degree. IgY anti-STAg antibodies presented high avidity and were able to recognize a broad range of parasite antigens, although some marked differences were observed in reactivity profile between antibodies produced in immunized hens and mice. Interestingly, IgY antibodies against Neospora caninum and Eimeria spp. did not react to STAg. We also show that IgY antibodies were suitable to detect T. gondii forms in paraffin-embedded sections and culture cell monolayers.Conclusions/significanceDue to its cost-effectiveness, high production yield and varied range of possible applications, polyclonal IgY antibodies are useful tools for studies involving T. gondii.
| S-EPMC3395712 | biostudies-literature
Project description:A simple, rapid and efficient procedure for the purification of thiamin-binding protein from chicken egg yolk was developed. The method involved removal, by exclusion, of lipoproteins from DEAE-cellulose and subsequent elution of water-soluble proteins held on the ion-exchanger with 1 M-NaCl, followed by treatment of the eluted protein fraction with an aqueous suspension of dextran/charcoal to generate apoprotein from the holoprotein. The resultant protein fraction was subjected to bioaffinity chromatography on thiamin pyrophosphate--AE (aminoethyl)-Sepharose. The protein eluted specifically with 10 microM-thiamin at pH 7.0, was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, had a mol.wt. of 38 000 +/- 2000 and was not a glycoprotein. The purified thiamin-binding protein specifically interacted with riboflavin-binding protein with no detectable deleterious affect on its (14C)thiamin-binding capacity. The protein bound [14C]thiamin with a molar ratio of 1.0, with dissociation constant (Kd) 0.41 microM. This protein-ligand interaction was inhibited by thiamin analogues and antagonists. The absorption spectrum of the protein in the presence of thiamin exhibited significant hypochromism at the 278 nm band, indicating the involvement of aromatic amino acid residues of the protein, during its binding to the ligand. The protein cross-reacted with the monospecific antiserum to egg-white thiamin-binding protein, showing thereby that thiamin-binding proteins present in chicken egg yolk and white are the products of the same structural gene.
| S-EPMC1162655 | biostudies-other
Project description:Boiling has important effects on the texture of chicken eggs, but its effects on egg nutrients have not been systematically investigated. In this study, changes in the metabolites of egg yolks boiled under different heating intensities were quantified and compared by metabolomic analysis. A total of 797 metabolites were identified, and the abundance of 162 metabolites changed significantly after boiling. The significant reduction of L-lysine and D-fructose suggested that Maillard reactions occurred in over-boiled egg yolks. Egg yolk endogenous enzymes might induce a partial hydrolysis of proteins and phospholipids during the warm-up period of boiling, as the abundance of dipeptides, lysophospholipids, and free fatty acids was significantly increased in boiled egg yolks. Boiling increased the detectable abundance of fat-soluble vitamins, riboflavin, and biotin, possibly by altering the complex structure of protein-lipid-lipophilic compounds or denaturing vitamin-binding proteins. The results of metabolomic analyses provide important information for understanding the nutritional changes of egg yolk boiled under different heating intensities. Graphical abstract Image 1 Highlights • Metabolites of boiled egg yolks at different heating intensities were compared.• 797 metabolites were quantified, 162 metabolites changed significantly after boiling.• Maillard reactions occur in over-boiled egg yolk as the reduce of L-lysine and D-fructose.• Endogenous enzymes may induce partial hydrolysis of proteins and phospholipids.• Boiling increased the detectable fat-soluble vitamins, riboflavin and biotin.
| S-EPMC9792406 | biostudies-literature
Project description:Egg yolk constitutes about a third of the structure of the chicken egg however, the molecular structure and physiological effects of egg yolk-derived lipid membranous vesicles are not clearly understood. In this study, for the first record, the egg yolk nanovesicles (vitellovesicles, VVs) were isolated, characterized, and used as a supplement for porcine embryo culture. Yolks of ten freshly oviposited eggs were filtered and ultracentrifuged at 100,000 × g for 3 h to obtain a pellet. Cryogenic transmission electron microscopy and nanoparticle tracking analysis of the pellet revealed bilipid membranous vesicles. Protein contents of the pellet were analyzed using tandem mass spectrometry and the miRNA content was also profiled through BGISEQ-500 sequencer. VVs were supplemented with the in vitro culture medium of day-7 hatched parthenogenetic blastocysts. After 2 days of blastocyst culture, the embryonic cell count was increased in VVs supplemented embryos in comparison to the non-supplemented embryos. TUNEL assay showed that apoptotic cells were increased in control groups when compared with the VVs supplemented group. Reduced glutathione was increased by 2.5 folds in the VVs supplemented group while reactive oxygen species were increased by 5.3 folds in control groups. Quantitative PCR analysis showed that VVs significantly increased the expression of lipid metabolism-associated genes (monoglyceride lipase and lipase E), anti-apoptotic gene (BCL2), and superoxide dismutase, while significantly reducing apoptotic gene (BAX). Culturing embryos on Matrigel basement membrane matrix indicated that VVs significantly enhanced embryo attachment and embryonic stem cell outgrowths compared to the non-supplemented group. This considers the first report to characterize the molecular bioactive cargo contents of egg yolk nanovesicles to show their embryotrophic effect on mammalian embryos. This effect might be attributed to the protein and miRNA cargo contents of VVs. VVs can be used for the formulation of in vitro culture medium for mammalian embryos including humans.
| S-EPMC10014936 | biostudies-literature
Project description:Chicken egg yolk was found to contain a unique glycosphingolipid pattern not seen in other types of tissue or cell. These glycosphingolipids were isolated in pure form and their structures established by sequential enzymic hydrolysis and permethylation analysis. The major gangliosides in chicken egg yolk are N-acetylneuraminosylgalactosylceramide, N-acetylneuraminosyl-lactosylceramide and di-N-acetylneuraminosyl-lactosylceramide. The only neutral glycosphingolipid found in chicken egg yolk is galactosylceramide.
| S-EPMC1185825 | biostudies-other