Studies On The Micro-Environment Effects Of Active Conformation And Primary Structure Of Functional Factor GAP2a From Ginkgo Seeds

Active proteins, which possess special physiological activities, are widely distributed in tissues of propagation, plant seeds etc. There are about 10% protein in Ginkgo seeds, but until recently, little research was done except the antifungal components in it. In the former works of our lab, Ginkgo seeds albumin (GAP) with strong antioxidant activity was isolated by modern methods, and GAP2a, as a novel protein, was a main functional factor with antioxidant activity in GAP, GAP2a was consisted of two peptides with similar molecular mass and similar amino acid sequence, the molecular mass of the protein is 29248Da. Amino acid sequence of peptide (2470) and peptide (1911) was determined on the basis of peptides mass finger (PMF). Belta-sheet was the main secondary structure in the protein with a little content of a-helical, Trp residuals located in the hydrophobic core or the surface of this protein.In the paper, the following were studied using chromatographic separation theory and identification technique on the basis of former research in our lab. At first, characteristics and antioxidant activity of the fractions in GAP were studied to confirm function factors in GAP. And then, optimizing purifying methods of GAP2a from GAP, its identification of its primary structure, the solution conformation and relationship with its antioxidant activity were studied. Furthermore, the bio-functions of GAP2a were also studied. At last, the immune regulation activity of GAP was studied at animal level, cell level and molecular level, and antioxidant activity of GAP was evaluated in vitro.Main conclusions in the paper:1. Fractional analysis and screening of active componentsGAP1-1 GAP1-2 GAP1-3 and GAP2 were gained from GAP in DEAE-52 column by controlling elution conditions. SDS-PAGE RP-HPLC and physical and chemical properties analysis showed that there were differences in the four fractions. There were mainly non-protein components in GAP1-1. Of the 4 fractions, GAP2 had the highest protein content and relatively less components with molecular weight between 14.4kD and 20.1kD. Also, the high sulfhydryl and disulfide bond content in GAP2 showed that the protein structure was stable, and GAP2 possessed a good hydrophilic property for its small surface hydrophobicity. Furthermore, GAP2 with idea amino acids composition possessed strong antioxidant ability. So GAP2 with the best availability was gained in the paper, and in the next place was GAP1-3.2. Optimizing purifying method, physical and chemical properties, and also bioactivities of GAP2a Retention time of GAP2 protein in DEAE-Sephadex A50 column was reduced by optimizing purified methods to retain bioactivity and natural structure of protein at the greatest extent. GAP2a and GAP2b were obtained in this step. SDS-PAGE, Native-PAGE, Sephadex G50 gel filtration chromatography, RP-HPLC and UV spectrum results showed that GAP2a was a single protein component, the molecular mass and the amino acids composition were as the same as GAP2a obtained by Huangwen. The content of GAP2a in GAP was 3.8% and GAP2a was highly solubility in water. GAP2a with 94.53% purity satisfied with determination of physical and chemical properties and amino acids sequence. There were two S-S bonds in GAP2a and its two peptides were linked by S-S bond. GAP2a possessed an ideal amino acids composition and good bioactivitities. Antioxidant activity of GAP2a was better than GAP and GAP2. Primary studies also showed that GAP2a had better immune regulation function in vitro and could inhibited growth of S180 cells in vitro. At the same time, physical and chemical properties and antioxidant activity were determined too.3. Primary structure study and bioinformatics analysis of GAP2aSeparation of two peptides, amino acids sequences of N terminal and part hydrolyzed peptides of GAP2a were studied. For N terminal determination, proteins were run on a SDS-polyacrylamide gel, and then transferred on PVDF membrane directly for protein sequencing determination. Light chain sequences was (K, N, D)-A-D-S-V-T-V-A-F-(V, F), heavy chain sequences was (D, H, S)-A-(A, V)-(N, T)-V-(G,V)-(T, I)-V-(F, L, A)-(V, F). Amino acids sequences of four peptides from hydrolysates of GAP2a were determined by using ESI-MS/MS. Peptide (2353.98Da) with AVVVDNSTWTSRNVPMNDGHR sequence existed in two chains. Sequence of peptide (1486.66Da) in light chain and peptide (1574.04Da) in heavy chain were STEMNTGESLQYK and LVGNAAELGNPTCTSK respectively. S-S bond of two peptides were sheared by reduction methods and then separated by RP-HPLC. Bioinformatics soft in ExPASy web was used to predict the physical and chemical properties and secondary structure. The results showed that GAP2a was a novel protein and located at cytoplasmic or nuclear of cells. GAP2a wasn't a membrane protein with a high hydrophilic and stable structure. Beta-sheets were main secondary structure in GAP2a with a little content of alpha-helix. 3D structure of GAP2a was similar to Aspergillopepsin and ferredoxin-1.4. Solution conformation and relation with its antioxidant activitySolution conformation of GAP2a was studied by fluorescence spectrum, circular dichroism and DSC. Unfolding process and change of antioxidant activity of GAP2a under different pH and temperature conditions were studied and discussed. Three conclusions were made:(1) The best excited wavelengths of GAP2a were 283nm and 290nm. Trp residues located in regions of low effective dielectric constant, such as inside a compact folded protein. Hydrophobic core mainly located in inside of protein structure. 6-sheet was abound 65.9% in GAP2a, a-helix content was 7.1%, there was not turn structure Studies on thermal properties showed that thermal denaturizing of GAP2a was a reversible process at solid state, and irreversible at liquid state. At liquid state and heating rate of 3K/min, denatured temperature was 75.72℃, enthalpy and entropy change were 148.58kJ/mol and 0.43 kJ·mol^-1·K^-1, respectively. The thermal denaturizing of GAP2a was driven by entropy.(2) Effect of environment pH condition on solution conformation of GAP2a was different at acid condition and alkaline condition. At acid condition, denatured process experienced three stages: Natural state (at pH7)→denatured state (at pH5)→Molten globule state (at pH1-pH3). At alkaline condition, denaturizing of GAP2a was a gradual change process. GAP2a was undergoing unfolding process with formation of more a-helix content at strong alkaline environment. Thermal denatured process of GAP2a obeyed Lumry-Eyring three-state model: Natural state→Reversible intermediate state→Irreversible denatured state.(3) Effect of pH and temperature conditions on solution conformation and antioxidant activity of GAP2a was discussed. Antioxidant activity of GAP2a was determined by two factors: A. Aromatic amino acid residues (Trp Tyr and Phe etc) and sulphur-contained amino acid residues (Met and Cys) in proteins were the main factors responsible for antioxidant activity. Exposure of these residues during unfolding process increased antioxidant activity of GAP2a. B. There was a positive linear correlation between the a-helix content and antioxidant activity of GAP2a. So a-helix structure may be an active conformation. The two factors didn't act independently. Only a balance between was achieved, would GAP2a possess the strongest antioxidant activity.5. Immune regulation activity of GAPBy using Normal mice, natural aging mice, D-galactose induced subacute aging mice, immunosuppressive model mice andγ-ray irradiated mice, effect of GAP on specific immune, cell immune, humoral immune function, antioxidant activity and hematopoiesis was evaluated. Results showed that GAP could increased SOD activity, GSH-Px activity and decreased MDA content in mice. GAP showed good effects such as increasing thymus index and spleen index of model mice, promoting proliferation of activatied and inactivatied T, B lymphocyte in vitro, stimulating IL-2 secretion of lymphocyte, enhancing leucocyte number, enhancing phagocytosing ability of peritoneal macrophage and DTH reaction, promoting production of serum hemolysins immunized with SRBC. L₃T₄⁺ cells, Lyt₂⁺ cells percent and L₃T₄⁺/Lyt₂⁺ cells ratio also increased significantly in group of mice fed with GAP. Hemogram in immune system damaged mice recovered too. So GAP could regulate immune function of mice across-the-board, and it's hopeful that GAP are developed into a immunopotentiator as a natural plant protein.6. Antioxidant activity of GAP in vitroThe reactive oxygen species (ROS) scavenging ability of GAP in vitro, as well as protecting effect of GAP on damaged DNA was studied. Effect of GAP on removal of superoxide anion was determined by Pyrogallol-Luminol system. The scavenging ability of GAP on hydroxide radicals was determined by CuSO₄-Phen-Vc-H₂O₂, FeSO₄-Luminol-H₂O₂ and FeSO₄-Luminol system. Luminol-H₂O₂ system was used to measure scavenging effect on hydrogen peroxide. Preventing DNA damage effect of GAP was determined by CuSO₄-Phen-Vc-H₂O₂-DNA chemiluminescence's system. Results showed that GAP possessed a good scavenging potency on ROS, but promoted oxidation in the FeSO₄-Luminol-H₂O₂ and Luminol-H₂O₂ system. Not every chemiluminescence system was suitable for determining antioxidant activity of protein. Maybe it is related with redox potential of samples and system. GAP was hydrolyzed by alkaline protease and hydrolysate got better antioxidant ability. GAP could inhibit growth of S180 in vivo and in vitro by means of its good antioxidant ability.