The Purification, Glycosylation Structure And Properties Of Hen Egg White Ovomacroglubulin

Ovomacroglobulin（OVM） is a glycoprotein from chicken egg white, accounts for 0.5% of the total egg white proteins, and possesses kinds of bioactivities. However, there is lace of study about OVM in China. The present study aims to the separation and purification of OVM and other egg white proteins, and then characterizes the glycosylation structure of OVM, investagets the properties of OVM interact with metal ions and mediate cell migration. The main findings were as follows:（1） The effect of polyethylene glycol（PEG） on egg white proteins were studied, based on this, a co-purification method of chicken egg white proteins was established. Hen egg white solution was prepared and then separated to four fractions using PEG precipitation. Then ovomucin was obtained from precipitation A by further purification using sodium chloride solution. All the other fractions were further purified through anion exchange chromatography, and lysozyme, ovotransferrin, ovalbumin and ovoflavoprotein were obtained respectively. The purity of ovomucin, lysozyme, ovotransferrin, ovalbumin and ovoflavoprotein were 82.4%, 91.8%, 94.6%, 96.5% and 88.2%, extraction ratio were 63.6%, 30.1%, 77.8%, 88.6% and 53.2%, respectively. The activity of lysozyme was 18,500 U/mg, and the biological activities of ovotransferrin and ovoflavoprotein were also maintained. This method has the advantage of simple and efficient, and is suitable for industrial-scale production, will benefit for the applications of bioactivity egg white proteins.（2） A simple two-step chromatographic method for the purification of OVM was created by the optimization of PEG fractional precipitation process and improvement of the lucid chromatography workflow. OVM-rich egg white fraction which obtained from PEG precipitation was successively separated using anion-exchange chromatography and gel filtration chromatography. The optimized PEG precipitation concentration range is 4-8%, anion-exchange chromatography elution was performed at sodium chloride concentration of 0.18 mol/L with flow rate of 2.5 m L/min. The elution which contained OVM was concentrated by ultrafiltration, then was purified by gel filtration chromatography. The sample loading volume was 5 m L, elute flow rate was 1.0 m L/min. The purified OVM shown a single band in SDS-PAGE. The purified sample was identified as “gi|45382565|ovostatin（Gallus gallus）” by LC-ESI-LTQ. This two-step chromatographic method save at least one third of time, and the extraction ratio is increased about 120%.（3） The purification processes of OVM were further optimized. PEG precipitation concentration range was 5-9%, the obtained precipitation was purified directly using a Sephacryl S-200 HR column. The column was eluted with PBS buffer（p H 7.4）, and the optimal flow rate is 0.75 m L/min, sample loading volume is 3 m L. OVM was obtained with a purity of 97.0%. The SDS-PAGE analysis results showed that OVM was maintained collagenase inhibition activity. The atomic force microscope（AFM） analysis results demonstrated that OVM was disc-shaped nanoparticle with a diameter of about 38±4 nm and height of about 2.7±0.5 nm, and the molecular was integrated. This protocol has an advantage of rapidity, the purified OVM can be obtained in 10-12 h, and the extraction ratio was increased to 62.5%. This methold will facilitate the studies and the development of application of OVM.（4） The glycosylation structure of OVM was characterized by mass spectrometry and two-dimensional electrophoresis. The molecular weight of OVM subunit is measured using MALDI-TOF-MS as 183.3 k Da, therefore the molecular weight of carbohydrate moieties is 21.1 k Da, and accounts for 11.5% of the whole OVM molecule. OVM was deglycosylated by PNGase F and subsequently digested by proteases, and N-glycosylation sites were identified using HPLC-ESI-MS/MS. The results of MS/MS showed that 11 sites were glycosylated, 1 site（N 1221） was in both glycosylated and non-glycosylated forms. Comparing with its homologous proteins, OVM N-glycosylation sites were lost. On the two-dimensional electrophoresis gel, a series of OVM spots horizontally distributed at 170 k Da with an isoelectric point range of 5.03-6.03, indicating the heterogeneity of glycosylation of OVM. These results provided important information for understanding of structure, function of OVM.（5） The interactions between OVM and metal ions was studied by spectroscopy. Fluorescence spectroscopy showed that the quenching rate constants of Zn²⁺, Cu²⁺ and Fe³⁺ to OVM were 6.22×109 L/（mol?s）, 4.13×1011 L/（mol?s） and 1.16×1012 L/（mol?s）, respectively. The results indicated Zn²⁺ was dynamic quenching, while Cu²⁺ and Fe³⁺ were static quenching. The binding constants of Cu²⁺ and Fe³⁺ to OVM were 5.27×102 L/mol and 9.57×105 L/mol, and the binding sites were 0.8 and 1.2, respectively. The synchronous fluorescence spectrometry results showed that the maximum emission wavelength of tryptophan residues was red-shifted by Cu²⁺. CD and FT-IR spectroscopy indicated that β-sheet structure was translated to disordered structure after Cu2 + and Fe3 + binding to OVM. Based on these results, a hypothesis was proposed that OVM not only inhibits metalloproteinases by "trapping" effect, but also by competitively combining with the metal ions.（6） The effects of OVM on fibroblast proliferation and migration were investigated. The in vitro model of mouse embryonic fibroblasts（3T6） and human primary skin fibroblasts（HSF） were used. The cell number, cell viability, and cell cycle, as well as the expression of FGF and TGF-β were not significantly altered under the treatment with OVM, indicated that OVM has no effect on the proliferation of fibroblasts. Scratch assays showed that OVM could promote the migration of 3T6 cells（25%） and HSF cells（28%）. As well, the adhesion of HSF cells to the collagen matrix was also enhanced 78% by OVM treatment. Meanwhile, β1-integrin, β-tubulin, and β-actin were up-regulated, while E-cadherin was down-regulated in OVM-treated HSF cells. These results suggested that the promotion of OVM on cell migration was achieved by enhancing cell adhesion to extracellular matrix, reducing intercellular aggregation, and strengthening cytoskeleton.