Fang Geng

Simply and effectively preparing high‐purity phosvitin using polyethylene glycol and anion‐exchange chromatography

An attempt was made to develop a new protocol for preparing phosvitin that could be easy scaled up using polyethylene glycol (PEG6000). Influence of PEG6000 concentration and pH of sample solution on phosvitin isolation was investigated. Phosvitin of high purity (99%) was obtained in good yield (47%) with the optimal condition of pH 4.0 and 3% PEG6000 precipitation. In addition, through evaluating different anion-exchange chromatography methods, the DEAE procedure at pH 7.5 was finally selected as the best procedure to obtain metal-free phosvitin that lost the least protein. Furthermore, it is observed that the purity and characterizations of prepared phosvitin were similar to those of the standard phosvitin from Sigma, and the random coil of phosvitin converted into more compact structure after removing metal ion. In conclusion, the developed method was simple and suitable for scaled up preparation of phosvitin.

Phosvitin phosphorus is involved in chicken embryo bone formation through dephosphorylation

The aim of this study was to investigate the role of phosvitin in bone formation in chicken embryos. The yolk P content, P/N ratio and secondary structure of phosvitin, alkaline phosphatase (ALP) activity of the tibia, and body length were determined during incubation. A high correlation was found between the phosphate group content of phosvitin and both secondary structure and bone metabolism (ALP activity in the tibia, body length). The ALP activity and body length growth slightly lagged behind changes in the P/N ratio and the secondary structure of phosvitin. The phosphate content of phosvitin decreased, the γ-random coil and β-turn gradually transformed into α-helixes, and the secondary structure of protein tended to become more orderly; these changes mainly occurred on d 13 to 16. Bone formation of the chicken embryos occurred primarily on d 14 to 18, whereas ALP activity and body length growth increased substantially. The results indicate that phosvitin P is involved in chicken embryo bone formation through dephosphorylation.

Mass Spectrometry and Two-Dimensional Electrophoresis To Characterize the Glycosylation of Hen Egg White Ovomacroglobulin.

Glycosylation of proteins plays an important role in their biological functions, such as allergenicity. Ovomacroglobulin (OVMG) is a glycoprotein from hen egg white, but few studies have been done so far to delineate the glycosylated sites of OVMG. The present study characterized the glycosylation of OVMG using mass spectrometry and two-dimensional electrophoresis. MALDI-TOF-MS showed that the OVMG subunit [M + H](+) ion has a peak at m/z 183297; therefore, the carbohydrate moiety is calculated as 11.5% of the whole OVMG molecule. HPLC-ESI-MS/MS confirmed that of 13 potential N-glycosylation sites of OVMG, 11 sites were glycosylated; 1 site (N(1221)) was found in both glycosylated and nonglycosylated forms. On the two-dimensional electrophoresis gel, a series of OVMG spots horizontally distributed at 170 kDa, with an isoelectric point range of 5.03-6.03, indicating the heterogeneity of glycosylation of OVMG. These results provided important information for understanding of structure, function, and potential allergenic sites of OVMG.

A simple method for isolating chicken egg yolk immunoglobulin using effective delipidation solution and ammonium sulfate

Chicken egg yolk immunoglobulin (IgY) is a superior alternative to mammalian immunoglobulin. However, the practical application of IgY in research, diagnostics, and functional food is limited due to complex or time-consuming purification procedures. The objective of this study was to develop a simple, safe, large-scale separation method for IgY from egg yolk. Egg yolk was diluted with 6-fold delipidation solutions made of different types (pectin, λ-carrageenan, carboxymethylcellulose, methylcellulose, and dextran sulfate) and concentrations (0.01, 0.05, 0.1, 0.15, and 0.2%) of polysaccharides, respectively. The yolk solution was adjusted to pH 5.0, and then kept overnight at 4°C before being centrifuged at 4°C. The resulting supernatant was added to 35% (w/v) (NH4)2SO4 and then centrifuged. The precipitant, which contained IgY, was dissolved in distilled water and then dialyzed. SDS-PAGE and Western blotting were utilized to conduct qualitative analysis of IgY; high-performance liquid chromatography (HPLC) was used for quantitative analysis. The immunoreactivity of IgY was measured by ELISA. The results showed that yield, purity, and immunoreactivity varied with types and concentrations of polysaccharides. The optimal isolation of IgY for pectin, λ-carrageenan, dextran sulfate, and carboxymethylcellulose was at the concentration of 0.1%; for methylcellulose, optimal isolation was at 0.15%. The best results were obtained in the presence of 0.1% pectin. In this condition, yield and purity can reach 8.36 mg/mL egg yolk and 83.3%, respectively, and the negative effect of IgY on immunoreactivity can be minimized. The procedure of isolation was simplified to 2 steps with a higher yield of IgY, avoiding energy- and time-consuming methods. Therefore, the isolation condition under study has a great potential for food industry production of IgY on a large scale.

Calcium binding characteristics and structural changes of phosvitin.

Phosvitin is a unique highly phosphorylated protein that plays a role in the regulation of calcification. We conducted a comprehensive study of the chemical, thermodynamic and structural aspects of the interaction of phosvitin with calcium ions using a calcium ion selective electrode (ISE), isothermal titration calorimetry (ITC), circular dichroism spectrum (CD) and fluorescence spectroscopy, respectively. The results showed that under neutral and alkaline conditions, distinct high affinity and low affinity binding modes existed in the interaction between phosvitin and calcium. The high affinity association constant was approximately 10(4)mol(-1), while the binding sites contained nearly 30mol of calcium per mole of phosvitin. This reaction was driven by enthalpy. The unordered and β-turn conformations of phosvitin increased, while the β-sheet conformation decreased. The main interaction forces were electrostatic force, hydrogen bonds or van der Waals force. The low affinity association constant and binding sites were not constant, as many calcium ions were sequestered by phosvitin. The binding reaction was driven by entropy, and the β-sheet conformation of phosvitin increased while the unordered conformation decreased. The main interaction force was hydrophobic force. However, under acidic conditions, the interaction between phosvitin and calcium was an entropy-driven endothermic reaction, and the main interaction force was weak hydrophobic force. This calcium-binding characteristic of phosvitin may play a specific role in its biological function.

Hen egg yolk phosvitin stimulates osteoblast differentiation in the absence of ascorbic acid

BACKGROUND Egg yolk phosvitin, one of the most highly phosphorylated extracellular matrix proteins known in nature, has a strong calcium binding and reducing capacity. Here, we investigated the effects of phosvitin on osteoblast differentiation and osteogenic gene expression in cultured mouse osteoblastic MC3T3-E1 cells by using alkaline phosphatase activity analysis, alizarin red S staining and real-time PCR assay. RESULTS Alkaline phosphatase activity and alizarin red S staining analyses demonstrated no significant difference between differentiating MC3T3-E1 cells cultured in the presence of phosvitin and those cultured in the presence of ascorbic acid after 21 days of differentiation. Our real-time PCR assay also indicated the two groups were similar in the expression of the osteogenic gene markers, collagen type I, osteocalcin, runt-related transcription factor 2, and bone morphogenetic protein-2. CONCLUSION Our findings indicate that phosvitin plays a similar role to that of ascorbic acid in osteoblast differentiation and mineralisation.

Hen Egg White Ovomacroglobulin Promotes Fibroblast Migration via Mediating Cell Adhesion and Cytoskeleton

BACKGROUND Hen egg white ovomacroglobulin (OVM) possesses a variety of bioactivities and could potentially be used as a pharmaceutical agent. It has been reported that OVM is involved in wound healing and cancer pathological processes, and previous results suggest that OVM plays a potential role in cell proliferation and migration; however, this has not yet been proven. In the present study, the effects of OVM on fibroblast proliferation and migration were evaluated. RESULTS Results of cell counting, cell viability, and cell cycle indicated that proliferation of fibroblasts was not altered by OVM treatment. However, scratch assays showed that OVM could promote the migration of 3 T6 mouse embryonic fibroblasts and human skin fibroblasts (HSF). Also, the adhesion of HSF to the collagen matrix was also enhanced by OVM treatment. RT-qPCR and western blot analysis showed that β1 -integrin, β-tubulin, and β-actin were up-regulated while E-cadherin was down-regulated in OVM-treated HSF cells. The effect of OVM was silenced after forming a complex with trypsin, suggesting that the protease inhibitory ability of OVM is important for its effect on cell migration. CONCLUSION These results suggested that promotion of OVM on cell migration was achieved by enhancing cell adhesion to extracellular matrix, reducing intercellular aggregation, and strengthening cytoskeleton. The finding of the promotion effect of OVM on cell migration is important for understanding its role in wound healing and cancer pathological processes.

N-Glycoproteomic Analysis of Chicken Egg Yolk

Posttranslational N-glycosylation of food proteins plays a critical role in their structure and function. However, the N-glycoproteome of chicken egg yolk (CEY) has not been studied yet. Glycopeptides hydrolyzed from CEY proteins were enriched, with deglycosylation occurring using PNGase F, and then were identified using a shotgun glycoproteomics strategy. A total of 217 N-glycosylation sites and 86 glycoproteins were identified in CEY, and these glycoproteins are mainly involved in the binding, biological regulation, catalytic activity, and metabolic processes. Among the identified CEY glycoproteins, 22 were recognized as proteases and protease inhibitors, suggesting that a proteinase/inhibitor regulation system exists in CEY; further, 15 were members of the complement and immune systems, which provide protection against potential threats during hatching. The study provides important structural information about CEY glycoproteins and aids in the understanding of the underlying mechanism of embryo development as well as changes in CEY functional characteristics during storage and processing.