| Phosvitin is one of the most phosphorylated proteins in nature, whose amount of serine is nearly 55% and 80% of these serine are phosphorylated. According to the research, phosvitin phosphopeptide derived from phosvitin by trypsin has better biological activities such as antioxidant. Based on the previous research on phosvitin in our lab, this study is aimed at PPP4 which is one kind of phosvitin phosphopeptides. Firstly, the interaction between PPP4 and calcium was researched, and then we studied the effect of PPP4 on both collagen biomineralization and osteoblastic MC3T3-E1 cells matrix mineralization. In the end, in order to develop PPP4 as functional food we found out the proper technology to prepare the PPP4-calcium complex. The contents and results are as follows:Three classes of binding sites are found between PPP4 and calcium with ITC. According to the change of △H, we found that the process between the binding of PPP4 and calcium was exothermal, then became endothermic and became exothermal finally. The Thermodynamic parameters △G was below zero, which suggested that this process was spontaneous.Both binding constants reached 104 mol-1, and the high affinity binding constant was 7.875×104, while the low affinity binding constant was 2.942×104. The number of binding sites in the class of high affinity(2.833) is much higher than that in the class of low affinity(1.587).With the increase of calcium, the secondary structure of PPP4 became rigid, the ratio of its β-sheet increased by 27.8%, while the β-turn decreased by 37.5%. Its activity of binding calcium was gradually decreased and fluorescence quenching ability of calcium to PPP4 fitted to exponential model.Using collagen film to study the role which PPP4 and PV played in collagen biomineralization according to PILP theory. The morphology, crystallization and mineral distribution were studied with SEM/EDS, TEM/SAED and XRD. We found that PV and PPPV can induce intrafibrillar mineralization and promote the transformation from ACP to HAP.After 2d, the ratio of calcium and phosphate was 1.67 in control group, suggesting that hydroxyapatite has formed, while the ratio was 1.93 in PPP4 group, suggesting that it was amorphous calcium phosphate. After 14 d, both PV and PPP4 had obvious characteristic peaks of hydroxyapatite. There is no significant change in the secondary structure of PPP4 in reaction solution. Totally speaking PV and PPP4 induced slow and well-organized collagen biomineralization.Using MC3T3-E1 to test the function of PPP4 on cell proliferation and differentiation. With CCK-8 assay to test cell proliferation and flow cytometry analysis to test cell apoptosis. Treated with 100 and 200μg/m L PPP4 respectively, cell proliferation activity increased by 32% and 45% comparing with control group. The apoptosis was all below 10% in all treatment group. These results suggested that PPP4 can significantly promote osteoblast proliferation and has no significant toxicity to cells. PPP4 can promote ALP activity and matrix mineralization, which are osteoblast early differentiation and terminal differentiation symbols respectively. However PPP4 cannot promote cell differentiation when it was added in high concentration and long period.To develop its as functional food, we prepared PPP4-Ca complex. Mass ratio of PPP4 and calcium chloride was 3:1, reaction time was 50 min, temperature was 50℃ and pH was 7.5 were the optimal conditions. FITR showed that-COOH,-NH2 and phosphate radical participated in the binding of calcium. DSC showed that the heat stability of PPP4 had no obvious change after binding with calcium and its denaturation temperature mounted to 122℃. The calcium holding rate of the complex was 98% and 73.45% after freeze thawing and digestion respectively. |
PDF Full Text Request