Zhaoxia Cai

Proteomic analysis of egg white proteins during the early phase of embryonic development

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development.

Ultrasensitive and rapid lead sensing in water based on environmental friendly and high luminescent l-glutathione-capped-ZnSe quantum dots

Water-soluble and high luminescent l-glutathione-capped-ZnSe quantum dots (QDs) were applied for ultrasensitive Pb(II) detection. l-glutathione-capped-ZnSe QDs were synthesized by a facile colloid aqueous phase route. It is characterized by transmission electron microscopy, X-ray powder diffraction spectrometry, Fourier transform infrared spectrometry, Ultraviolet-visible spectrometry and fluorescence spectrometry. Quantum yields of l-glutathione-capped-ZnSe QDs could reach 50.1% without post irradiation. The fluorescence quenching of the l-glutathione-capped-ZnSe QDs depended on the concentration, pH and reaction time of the Pb(II) solution. The possible mechanism of fluorescence of l-glutathione-capped-ZnSe QDs quenched by Pb(II) was discussed in detail. Under optimal conditions, the quenched fluorescence intensity increased linearly with the concentration of Pb(II) in the range of 1.0×10(-8)∼8.0×10(-7)mol/L. The limit of detection for Pb(II) was 0.71 nmol/L. This developed method is demonstrated to contain improved sensitive and selective characteristics for the detection of trace Pb(II) in water.

Affinity adsorption and separation behaviors of avidin on biofunctional magnetic nanoparticles binding to iminobiotin

Knowing the adsorption behavior of target proteins on biofunctional magnetic nanoparticles is of great importance for the separation and purification of proteins. Adsorption behaviors of avidin on biofunctional magnetic nanoparticles binding to iminobiotin were investigated under different conditions of temperature, pH, ionic strength, and feed avidin concentration. Biofunctionalization of the non-functional nanoparticles was performed, coupled with iminobiotin. Characterization of the particles was carried out using transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). The results showed the avidin adsorption behaviors were mainly dependent on affinity interaction between avidin and iminobiotin coupled with the nanoparticles, which exhibited temperature, pH, ionic strength, and feed avidin concentration sensitivity. Maximum avidin adsorption capacity was achieved as 225 mg avidin/g biofunctional nanoparticles. Results were well fitted to the Langmuir isotherm model with the feed avidin concentration of less than 45 μg/ml. Based on the experiments above, the biofunctional magnetic nanoparticles were used to separate avidin from treated egg-white solution containing large amounts of other proteins. The avidin was isolated in 92% yield with an optical purity of more than 98.5% according to the HPSEC data. The regeneration of these nanoparticles was also studied and almost 87.3% of avidin could still be recovered by these regenerated nanoparticles.

The inhibition of fluorescence resonance energy transfer between multicolor quantum dots for rapid and sensitive detection of Staphylococcus aureus.

In this paper, we constructed the fluorescence resonance energy transfer (FRET) system between two multi-color quantum dots (QDs) of two sizes for rapid and sensitive detection of Staphylococcus aureus. In this system, green-emitting QDs conjugated with rabbit anti-S. aureus antibodies were used as energy donors while orange-emitting QDs conjugated with goat-anti-rabbit IgG were used as energy acceptors to form FRET system. Pre-binding of Staphylococcus aureus (S. aureus) on the donor occupied the binding sites and thus blocked resonance energy transfer between two colors QDs, leading to the quenching fluorescence of the acceptor. The fluorescence of acceptor has a linear calibration graph with the concentrations of S. aureus from 52 to 2.6×10(5) CFU mL(-1). The low detection limit was 10 CFU/mL. It was worth mentioning that the detection method of S. aureus had been applied to the analysis of apple juice and milk samples, which could potentially be developed into a sensor in the further study.

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.

Systematic evaluation of avidin-biotin interaction by fluorescence spectrophotometry.

The avidin-biotin interaction was evaluated systematically by fluorescence spectroscopy under different conditions of temperature, pressure, pH, metal ions, incubation time and initial avidin concentration. The binding constant was calculated according to the modified Stern-Volmer equation, which deduced the existence of static quenching mechanism. The data obtained revealed that avidin-biotin interaction exhibited temperature, pH, metal ions, incubation time and initial avidin concentration sensitivity. The binding constants decreased with increase in temperature, while the binding sites were independent of temperature. The values of thermodynamic parameter ΔH (-149.85 kJ mol(-1)) and ΔS (-284.26 J mol(-1) K(-1)) suggested hydrogen bonds and van der Waals played a major role in the reaction. The binding constants between avidin and biotin increased firstly and then decreased gradually with the increase of pH values. Metal ions can also affected the binding constants between avidin and biotin. The association kinetics firstly acquired by the combination of the change in fluorescence per unit time and the modified Stern-Volmer equation indicated that the reaction time required to reach equilibrium was 2200 s, and the average reaction rate for the binding process was very high in the first 180 s. Reaction of the avidin in the first 180 s was more than 40% of the total avidin involved in the whole process.

Chemiluminescence evaluation of antioxidant activity and prevention of DNA damage effect of peptides isolated from soluble eggshell membrane protein hydrolysate.

A new kind of soluble eggshell membrane protein (SEP) was prepared from eggshell membrane (ESM). The extraction rate of SEP could rise to 90% by two times, basically accomplishing the complete utilization of the whole ESM. Five proteases were employed as hydrolytic enzyme for the preparation of antioxidative peptides from SEP, and the antioxidative activities of the hydrolysates were investigated using a chemiluminescence method. Among the hydrolysates, alcalase hydrolysates with the highest free radical scavenging activity were further separated into three fractions using Sephadex G-25 gel filtration chromatography of the 4 h hydrolysate (SP1, SP2, and SP3). Among these three fractions, SP2 with an average molecular weight of 618.86 Da possessed the strongest fraction of scavenging activity. The IC50 values of the superoxide radicals, hydroxyl scavenging activities, and protective effect on DNA damage caused by hydroxyl radicals generated were 0.10, 0.18, and 0.95 mg/mL, respectively. These results demonstrate that inexpensive ESM waste could be a new alternative in the production of antioxidative peptides.

Hydroxyapatite nucleation and growth on collagen electrospun fibers controlled with different mineralization conditions and phosvitin

AbstractIn a tenfold-concentrated simulated body fluid, a strategy for rapid deposition of a biomimetic calcium phosphate layer on the scaffolds of electrospun collagen nanofiber membranes was developed. The aim of this study was to explore the effects of mineralization conditions and phosvitin (PV) on hydroxyapatite nucleation and growth. The mineralization model, the pH of the environment, and the deposition time were optimized. Scanning electron microscopy (SEM) images demonstrated that homogeneous and well-crystallized inorganic mineral layers were generated in the dynamic mineralization model system after incubating 3 h at pH 5.7. PV, which possesses the highest level of phosphorylation among egg proteins, was used as a model protein to investigate the contribution of PV in the mineralization process. The morphological structure and composition of the collagen/calcium phosphate composite nanofibers were also characterized by energy dispersive spectroscopy, scanning photoelectron spectrometer, X-ray diffraction (XRD), and Fourier transform infrared spectroscopy. XRD results showed the transformation process of mineralization materials from dicalcium phosphate dihydrate (DCPD) to HA through the changes of characteristic peaks at approximately 11° of DCPD and 31.8° of HA. 1.0 mg/mL. Phosvitin significantly promoted the phase transformation from DCPD to hydroxyapatite. High performance liquid chromatography results indicated that PV induced the mineralization rather than being the part of the hydroxyapatite. The minerals formed on electrospun collagen nanofiber membranes were identified to be from hydroxyapatite. These findings extended the potential application field of PV to biomimetic material.

Fluorescence switch biosensor based on quantum dots and gold nanoparticles for discriminative detection of lysozyme

It is important to detect lysozyme (LYZ) in a simple and rapid manner because of its potential application in the treatment of diseases and in the food industry. In this study, it was observed that the strong fluorescence of LYZ-modified Quantum Dots (QDs-LYZ) could be effectively quenched by gold nanoparticle(AuNPs) modified with antibodies against LYZ (anti-LYZ) due to fluorescence resonance energy transfer (FRET) between QDs-LYZ and anti-LYZ-AuNPs. The fluorescence can be reversibly recovered by LYZ (on state) owing to specific competitive interactions between LYZ, QDs-LYZ and anti-LYZ-AuNPs. The interaction of QDs-LYZ with anti-LYZ-AuNPs was studied by absorption, fluorescence spectroscopy and transmission electron microscopy (TEM). Under optimal conditions, LYZ can be detected with a linear range of 50-1000ng/mL and a detection limit (LOD) of 33.43ng/mL. This rapid and selective QD-based sensor was successfully applied for quantitation of LYZ in actual egg products. Furthermore, the strategy described in this report could be followed conveniently to establish similar immunosensors for the rapid detection of other proteins using corresponding antibodies.

The impact of N-glycosylation on conformation and stability of immunoglobulin Y from egg yolk

Immunoglobulin Y (IgY) is a new therapeutic antibody, and its applications in industry are very broad. To provide insight into the effects of N-glycosylation on IgY, its conformation and stability were studied. In this research, IgY was extracted from egg yolk and then digested by peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase. SDS-PAGE and infrared absorption spectrum showed that carbohydrates were distinctly reduced after enzymolysis. The circular dichroism spectrum indicated that the IgY molecule became more flexible and disordered after removal of N-glycan. The fluorescence intensity revealed that Trp residues were buried in a more hydrophobic environment after disposal of N-glycan. Storage stability decreased with the removal of oligosaccharide chains based on size-exclusion chromatography analysis. Deglycosylated IgY exhibited less resistance to guanidine hydrochloride-induced unfolding. After deglycosylation, IgY was more sensitive to pepsin. Therefore, N-glycosylation played an important role in the maintenance of the structure and stability of IgY.