Purification And Characterization Of Active Bio-molecules From Chinese Leek Seeds

As a traditional herb, Chinese leek seeds possess multiple physiological active functions. The thesis focused on the purification and characterization of natural active peptides and proteins from Chinese leek seeds.Two active peptides were purified and characterized. The Chinese leek seed extract were isolated and purified by a Gel filtration chromatography and RP-HPLC. Two novel peptides were obtained, and the sequences were identified to be Gly-Ser-Gln (GSQ) and Ser-Asn-Ala (SNA), respectively, by LC-ESI-MS/MS. GSQ could effectively quench free radicals and exert antimicrobial activity. Moreover, GSQ could also protect LO2 cells from H2O2-induced apoptosis. The other peptide, namely SNA, showed strong antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria.An active protein, designated as Pro-1, with lectin-like activity was extracted with PBS buffer (pH 5.0) and purified through ion exchange chromatography and Gel filtration chromatography. Pro-1 was a glycoprotein with a molecular weight of 23.6 kDa and sugar content of 3.6 ± 0.25%. Pro-1 was a dimmer comsisting of two same subunits through disulfide bonds, and its N-terminal 7 residues were determined to be CVIAPVA. The secondary structure contained 35.8% α-helices,8.6% β-sheets,18.3% turns and 37.3% random coils. Pro-1 showed strong thermostability and could survive incubation at 100 ℃ for 1 h. In terms of pH stability, although the hemagglutinating activity showed no significant change at pH values below pH 6.0, and the protein was quickly inactivated above pH 6.0. The hemagglutinating activity could be inhibited by fructose, mannitol and sorbose, indicating that Pro-1 was a complex lectin. Furthermore, the activity of Pro-1 could be completely inhibited by Ba2+ at the concentration of 10 mmol/L. And Pro-1 displayed selective inhibition on cell proliferation. Pro-1 could effectively inhibit the proliferation of HepG2 cells at the concentration of 40 μmol but only slightly affected the proliferation of LO2 and Caco-2 cells.The mechanism of Ba-+-mediated inactivation of Pro-1 was investigated by Fluorescence and Circular Dichroism (CD) spectra. Fluorescence quenching spectra demonstrated that Ba2+ could quench Pro-1 endogenous fluorescence in both static and dynamic states. Interaction between Ba2+ and Pro-1 increased the hydrophobicity of the Tyr structural domain and changed he conformation of Pro-1. Meanwhile, Circular Dichroism spectra also found that content of a-helical structure of Pro-1 increased but that of β-sheet structure decreased with increasing concentrations of Ba2+. Therefore, it was speculated that Ba2+ changed the conformation of Pro-1, leading to conformational changes of glycosylation domains and inhibition of Pro-1 activity. It was also likely that by direct binding to glycosylation domains, Ba2+ hindered the recogniton and interaction between Pro-1 and sugar side chains on cell surface.