Preparation, Structure Characterization And Function Of Phosvitin From Hen Egg Yolk

Phosvitin is the major phosphoprotein from hen egg yolk. Due to its unique amino acid composition and highly phosphorylation, phosvitin possess many excellent physical and biological properties. And phosvitin was speculated to possess regulation properties of biological mineralization through the bioinformatics analysis. In our research, highly purified phosvitin was firstly prepared, then characterized its structure, identified the phosphorylation sites, and excavated the potential function with combing the bioinformatics analysis, lastly investigated the regulation mechanism of phosvitin in the mineralization and clarified its active sites. This study will lay a foundation for the application of phosvitin in biomimetic mineralization. The main research contents and results are as follows:An attempt was made to develop a new protocol for preparing phosvitin with high efficiency and simplicity in a mild condition. Hen egg yolk sequentially diluted with equal mass of distilled water and0.17M NaCl, to obtain yolk granules after removing the yolk plasma by centrifugation. Granules was dissolved with10%1.74M NaCl, added3%w/w PEG6000and the solution was adjusted to pH4.0, desalted by dialysis after centrifugation and lyophilized. The purity of phosvitin (Pvti) was99%, which identified by SDS-PAGE, and yield was47%. Based on this protocol, phosvitin was prepared from egg yolk of Hy-Line Brown and White Leghorn, the purity examined by HPLC was93.14%and91.80%, respectively; and the yield was63.93%and58.89%, respectively. Further study showed that there had hardly significant between Hy-Line Brown and White Leghorn in the content, composition and secondary structure of phosvitin, they were all consistent with Sigma. Furthermore, Polyvalent metal-free phosvitin was purified through ion-exchange chromatography, the optimal condition was as follows: Pvti treated with Na2EDTA was eluted with an increasing gradient of NaCl (0,0.35and0.5mol/L) in Tris-HCl buffer (0.05mol/L, pH7.5) by DEAE column, the eluate was collected, polyvalent metal-free phosvitin (Pvtf) was obtained through dialysis against distilled water and lyophilized.(2) Systematic analysis the structure of phosvitin, the studies showed that amino acid composition of phosvitin contain up to30%Ser, rich in acidic amino acids (Asp, Glu) and basic amino acids (Arg, His), and contain a very small amount of the aromatic amino acids (Tyr, Cys and Trp). The conformation of phosvitin was mainly random coil and (3-sheet structure while metal-free phosvitin increased the β-turn content, by using Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD). Phosvitin possessed87phosphorylation sites (83P-Ser and4P-Thr), which were identified by LC-ESI-MS/MS (LTQ) and combined the prediction results by bioinformatics analysis. In addition, bioinformatics analyzed the11types of phosvitin from9different species and5types of phosphorylated protein from human through amino acid compositions, multiple sequence alignment and phylogenetic analysis, the results revealed that they all rich in acidic amino acids. Phosvitin and dentin phosphophoryn (DPP) have similar structure and properties, meanwhile the evolutionary relationships was close, this suggested phosvitin may have similar biological functional activity with DPP, can play an important regulation role in the process of bio mineralization.(3) Comprehensive study of the interaction of phosvitin with calcium ions from the chemical, thermodynamic and structural aspects, by using calcium ion selective electrode (ISE), isothermal titration calorimetry (ITC), CD and fluorescence spectroscopy, respectively. The results showed that, under neutral and alkaline conditions, there existed two classes of binding sites in the interaction between phosvitin and calcium: high affinity site and weak affinity site. The binding constant of high affinity is about104mol-1, and binding sites were nearly30mol of calcium per mole of phosvitin. This reaction was driven by enthalpy, the conformation of phosvitin increased random and (3-sheet structure, while decreased β-turn structure, the main interaction force was electrostatic, hydrogen bonds or Van der Waals. Meanwhile, the binding constant and the binding site of low affinity were not constant, since a large amount of calcium ions accumulated in the vicinity of the calcium saturation region or the molecule aggregation, the binding reaction was driven by entropy, the conformation of phosvitin increased β-sheet structure, and main interaction force was hydrophobic force. However, under acidic condition, the interaction between phosvitin and calcium was entropy-driven endothermic reaction, and the main interaction force was weak hydrophobic force.(4) Effect of phosvitin on the phase transformation of calcium phosphate mineral was investigated in the biomimetic system. The mineral structure, composition and surface morphology was characterized by FTIR, X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS). The results showed that the calcium phosphate mineral transformed from dicalcium phosphate dihydrate (DCPD) to hydroxyapatite (HAP), through the "dissolution-recrystallization" pathway in this system. Phosvitin significantly accelerated the phase transformation, since the transform process was shortened from6h to less than0.5h. Besides, The characteristic peaks of protein was occurred in the FTIR spectra and the XRD patterns of calcium phosphate mineral, and the phosvitin concentration in the filtrate during the mineralization was reduced to0.5%, indicated that phosvitin was involved in the phase transformation of calcium phosphate mineral and incorporated into the mineral. The results of the addition sequence of substrates on the phase transformation of calcium phosphate showed that, Phosvitin played its role only if adding phosvitin before DCPD formed, indicates that the interaction between phosvitin and the free calcium ions was the key to promote its regulation mineralization of calcium phosphate. BSA used as a control, also promoted the phase transformation. However, compared with phosvitin, its effect was weak and was involved in different modes. BSA involved in the later phase transformation stages of calcium phosphate mineral while phosvitin participate the initial phase transformation. The peculiar role of phosvitin in the calcium phosphate mineralization process was closely related to its degree of phosphorylation.(5) Different dephosphorylation degrees of phosvitin were accomplished by treating phosvitin (Pv) with0.1,0.2,0.3,0.4mol/L NaOH solution. T1, T2, T3and T4were obtained through ultrafiltration, the dephosphorylation degree was2.98%,19.46%,43.39%,71.07%, respectively. Their effect on the mineralization were studied by using a pH-stat system. Phosvitin promote mineralization depended on concentration-dose relationship. Effect of different dephosphorylation degrees of phosvitin on the mineralization was studied by CD, FTIR, XRD, SEM, fluorescence spectroscopy. The results showed that phosphorylation was very important in the mineralization reaction. In addition, the dephosphorylation phosvitin increased its P-sheet structure, providing the template of mineralization. Due to its random structure reduced, low dephosphorylated levels of phosvitin decreased its adsorption capacity on mineral, decreased the calcium binding capacity as less phosphorus, and thereby weakened the effect of promotion on the mineralization. However, phosvitin could hydrolyzed to phosvitin peptides by high concentration of alkaline solution, the active regions were exposed, thus increased the nucleation sites, and significantly promoted mineralization. In summary, efficiency of promoting mineralization order was T4﹥T3﹥Pv﹥T2﹥T1﹥Control.(6) Phosvitin was partially dephosphorylated by alkaline and then digested with trypsin. The phosvitin phosphopeptides (PPP) fractions were separated and purified through DEAE ion-exchange chromatography and Sepharose G-25gel filtration chromatography. SDS-PAGE and CD analysis showed that PPPO and PPP1were possible the small molecule fragments (N-or C-terminal) after enzymatic hydrolysis, while PPP3and PPP4had similar secondary structure, but PPP4had more compact and β-sheet structure, while PPP3contained10kDa peptides. Effect of phosvitin phosphopeptides fractions on the mineralization by means of FTIR, XRD, SEM, fluorescence spectroscopy in pH-stat system. The results showed that, as compared with Pv, PPPO, PPP1and PPP4decreased its promoting efficiency, because PPPO and PPP1were small peptides, and not involved in the mineralization; meanwhile, PPP4owed too much β-sheet structure resulted in more compact, thus weakened the promotion effect. PPP3exposed its active region, thus the promoted effect was the most significant. The mineralization reaction rate was as follows: PPP3﹥Pv﹥PPP4﹥PPP1﹥Control﹥PPPO. The active region of promotion mineralization was D1165-R1258in the core region of phosvitin by LTQ MS/MS identification.