Studies On Separation Purification And Structure Of Ginkgo Seed Protein And Its Biologic Activities

The significant biological and pharmacological activity of Ginkgo has been drawn scientist's much attention all over the world. The seed of the plant of Ginkgo biloba L. as a kind of traditional Chinese herb has been used for more than 600 years, however the main active component of activity and function is little known. Very few studies on the effects of anti-aging and anti-fatigue and relieving cough and asthma et al of Ginkgo seed has been reported. In order to ascertain the relationship between its biologic activities and its structure, this paper studied and discussed the preparation, purification and structure characterization as well as antioxidant, anti-aging of biologic activities of Ginkgo seed protein (GP) by using modem separation technology and modem chemical analysis method and medicinal experiments. The main results were shown as follows: 1.Extraction, isolation, purification and identification of Ginkgo seed proteinThe Ginkgo seed of Taixing were selected for experimental materials by its feature and chemical. Albumin (GAP), globulins (GPP) and salt-soluble protein (GGP) were isolated from Ginkgo seed by salting-in and salting-out methods for the first time and the isolation and preparation condition was studied.DEAE-cellulose ion-exchange chromatography was used to separate and purify GAP. It was successful to obtain two novel proteins-GAP I and GAP Ⅱ,by the combination of linear gradient and step gradient. GAP Ⅱ was further purified into GAP Ⅱ a by DEAE-Sephadex-A50 ion-exchange chromatography. Some methods such as spectrophotometer and chromatography and electrophoresis and mass spectrometry were used to identify the purity of GAP Ⅱ a. It was identified to be chromatography and electrophoresis grade pure. The data obtained from peptide mass fingerprinting (PMF) were used in protein database search and the result showed that GAP II a is an unknown protein. 2.The structure characterization of GAP Ⅱ a.The primary structure of GAP Ⅱ a was studied with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and non-denaturing ployacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate ployacrylamide gel electrophoresis (SDS-PAGE) by measuring the molecular weight of GAP Ⅱ a and its two peptides and their peptide mass fingerprinting. The results demonstrated that the measured molecular weight of GAP Ⅱ a was 29248Da and it consisted of two peptides which were connected by disulfide bond and were similar with their molecular weight and amino acid sequences. The results of gel staining of glycoprotein, chemical composition analysis, the peak width at half height of molecular ion peak and changing of amino acids content before and after alkali-β -elimination showed further that GAP Ⅱ a was a glycoproteinwith little glycosylation connected Ser or Thr with O-glucosidic bond.ESI-MS/MS method was applied to measure the amino acid sequence of peptide-2470 and peptide-1911. The sequence of peptide-2470 with 2470.0044Da MW was Ala-Val-Val-Val-Asp-Ser-Asn-Thr-Gly-Glu-Thr-Trp-His-Cys-Gly-Glu-Gly-Met-Pro-Asp-Pro-Asn - Arg and the sequence of peptide-1911 with 1910.9644Da was Leu-Va1-Gly-Asn-Thr-Ala-Asp-Leu-Gly-Asn-Trp-Gln-Ser-Met-His-Leu-Arg.As the result of Circular dichroism (CD) spectrum and Infra-red (IR) spectrum and raman spectrum shown, the secondary structure of GAP Ⅱ a was mainly 3 -sheet or coil and the content of a -helix was very small, it was coincident with secondary structure prediction by GOR and homologue method. It was shown by the differential UV-absorbance spectrum and fluorescence spectrum that there were both the Trp residues which lie outside the molecular and the Trp residues which lie in the inside hydrophilic structure in GAP Ⅱ a.GAP Ⅱ a's agglutination form was observed by scan electron microscope and transmission electron microscope. There were mainly empty and different size globoid form among other forms such as chip, needle and alveolus.The differential UV-absorbance spectrum and fluorescence spectrum...