【作者】 邓乾春；

【导师】 谢笔钧；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2007， 博士

【摘要】 活性蛋白质（active proteins）是一类具有特殊生理活性的蛋白质，广泛分布于动植物组织和植物种子等部位中。银杏种仁含有约10％的蛋白质，但目前为止仅有少量关于银杏种仁抗菌蛋白的研究，对于这个巨大的宝库还有待我们去挖掘。实验室前期研究得到了具有较强抗氧化活性的银杏种仁清蛋白，新蛋白GAP2a为银杏种仁清蛋白主要的抗氧化活性功能因子之一，分子量为29248Da，由两条分子量接近和氨基酸序列相似的肽链连接而成，在得到该蛋白肽质量酶谱的基础上，测定了其中肽段（2470）和肽段（1911）两个肽段的氨基酸序列；蛋白质二级结构主要以β-折叠或卷曲结构为主，a-螺旋结构含量较少，分子中的Trp残基暴露于分子表面或处于疏水内核。本文旨在尝试利用现代色谱分离方法和近代分析鉴定技术，在实验室前期研究基础上，研究了银杏种仁清蛋白中各级分的特性和抗氧化活性，以进一步确定银杏种仁蛋白中功能因子。功能因子GAP2a纯化方法的优化及鉴定、一级结构的进一步测定和生物信息学分析、溶液构象及与其抗氧化活性的构效关系等则是本研究的三个方面重点。同时还对GAP2a的生物活性进行了研究。利用现代免疫学和药理实验方法，从整体动物水平、细胞水平以及分子水平全面了解GAP的免疫调节药理作用，并从体外评价了了GAP的抗氧化活性。主要研究结果如下：1．GAP的级分分析及活性成分筛选通过DEAE-52纤维素柱，并控制洗脱条件从GAP中分级得到了GAP1-1、GAP1-2、GAP1-3和GAP2四个级分，经SDS-PAGE电泳、RP-HPLC和理化特性分析表明，四个级分中的蛋白差异较大，非蛋白成分集中在缓冲溶液洗脱级分GAP1-1中，GAP2中蛋白含量最高，组分最少，分子量主要分布在14.4kD-20.1kD范围，且巯基和二硫键含量也最高，表面疏水性最小，蛋白具有良好的亲水性和结构稳定性，同时GAP2还具有理想的氨基酸组成和较强的体外抗氧化活性，因此通过分级得到了一种可利用性好的蛋白级分GAP2，其次为GAP1-3。2．GAP2纯化方法的优化及理化特性与生物活性的研究经过GAP2纯化方法的优化，减少了蛋白质在DEAE-Sephadex A50层析柱上的停留时间，最大程度的保持了蛋白质的生物活性和天然结构，得到了级分GAP2a和GAP2b。通过SDS-PAGE电泳、Native-PAGE电泳、Sephadex G50凝胶过滤色谱、反相高效液相色谱和紫外扫描谱图鉴定表明GAP2a为单一蛋白组分，分子量大小和氨基酸组成与黄文得到的GAP2a为同一种蛋白质。研究结果表明，GAP2a在GAP中的含量为3.8％，具有优良的溶解性，纯度高达94.53％，完全可用于理化特性的研究和序列测定；GAP2a分子中含有2对二硫键，两条肽链由二硫键连接而成。其体外抗氧化活性优于GAP和GAP2，初步研究表明GAP2a还具有较好的体外免疫调节功能，体外对S180肿瘤细胞生长有一定的抑制作用。论文同时还对GAP2b的理化特性和体外抗氧化活性也进行了分析。3．GAP2a一级结构研究和生物信息学软件分析本论文从GAP2a两条肽链的拆分、N端氨基酸序列、部分酶解肽段的氨基酸序列的研究，进一步丰富了GAP2a一级结构的新内容。GAP2a经过SDS-PAGE和PVDF转模后拆分为两条肽链，采用蛋白质测序仪得到的N端氨基酸序列，轻链为（K，N，D）-A-D-S-V-T-V-A-F-（V，F），重链为（D，H，S）-A-（A，V）-（N，T）-V-（G，V）-（T，I）-V-（F，L，A）-（V，F）；采用ESI-MS／MS方法测定了蛋白酶解物中四条肽段的氨基酸序列，质量数2353.98Da的肽段为两条链共有，氨基酸序列为：AVVVDNSTWTSRNVPMNDGHR；轻链中质量数为1486.66Da的肽段序列为：STEMNTGESLQYK；重链中质量数为1574.04Da的肽段序列为：LVGNAAELGNPTCTSK。（已完成蛋白58％质量数序列的测定）还原法拆开两条肽链后在RP-HPLC色谱柱上进行了分离，得到了较为理想的结果。利用ExPASy网站上的生物信息学软件对GAP2a的理化特性和二级结构进行了预测和鉴定；结果表明，GAP2a为一种新蛋白，可能位于细胞质或细胞核内，非膜蛋白，亲水性强，结构稳定，二级结构中主要为折叠结构，而螺旋结构含量较少；其三维结构同酸性蛋白酶和铁氧化还原蛋白较为相似。4．GAP2a溶液构象及与其抗氧化活性关系研究采用荧光光谱、圆二色谱和DSC手段研究了GAP2a的溶液构象，并对GAP2a在环境pH值和温度处理下的去折叠过程以及与抗氧化活性的关系进行了探讨。得到如下几点结论：（1） GAP2a的最佳激发波长为283nm和290nm，Trp残基微环境位于蛋白质中一个低介电常数的折叠分子结构区域中，其疏水区主要位于分子结构内部。二级结构主要以B折叠为主（65.9％），a螺旋结构含量较少（7.1％），无转角结构存在。GAP2a在固体状态下的热变性具有较大程度的可逆性，而液体状态是不可逆的，在液体状态和3K／min的加热速率下，变性温度为75.72℃，变性焓变为148.58kJ／mol，熵变为0.43 kJ·mol^-1·K^-1，其热变性为一熵驱动过程。（2）环境pH值对GAP2a溶液构象的影响在酸性条件下和碱性条件下有较大的差异。在酸性条件下，变性过程经历了以下3个阶段：N（pH7时的天然态）→U_A（pH5时的酸致变性态）→A（pH1-pH3范围内熔球体状态），而在碱性条件下的变性为一序变过程，在强碱性环境中，蛋白分子产生了较大程度的去折叠作用，诱导蛋白质分子形成了更多的a螺旋结构。GAP2a的热变性过程则遵循Lumry-Eyring三态模型：N→U→D（即天然态→可逆变性中间态→不可逆变性态）。（3）根据pH环境和温度对GAP2a溶液构象及体外抗氧化活性影响的研究可知，GAP2a的抗氧化作用主要由以下两点决定的：①具有抗氧化活性的芳香族氨基酸残基（Trp、Tvr和Phe等）和含硫氨基酸残基（Met和Cys），在分子去折叠后这些残基的暴露使蛋白质的抗氧化活性增强；②GAP2a的a螺旋结构含量与其抗氧化活性具有正相关性，提示a螺旋结构可能为该蛋白质的优势活性构象。上述两个因素不是独立起作用的，只有到达一个平衡点时GAP2a的抗氧化活性才能最大程度的发挥。5．GAP的免疫药理调节活性研究以正常小鼠、模型衰老小鼠、自然衰老小鼠、免疫功能低下小鼠和辐射损伤小鼠为研究对象，在特异性免疫、细胞免疫与体液免疫三个方面并结合抗氧化功能和造血功能评价指标，从体内动物水平、细胞水平和分子水平上，研究并探讨了GAP的免疫药理活性。结果表明，GAP能提高动物体内SOD和GSH-Px活性，降低MDA水平，增强巨噬细胞非特异性吞噬能力、T淋巴细胞和B淋巴细胞的增殖活性，刺激Con A活化的脾细胞分泌IL-2，并显著提高L₃T₄⁺细胞、Lyt₂⁺细胞百分率及L₃T₄⁺／Lyt₂⁺细胞比值，促进小鼠体内血象的恢复，从而较为全面的起到免疫调节作用，有望成为一种较好的天然植物蛋白免疫增强剂。6．GAP的体外抗氧化活性研究采用化学发光体系测定了GAP对活性氧自由基的清除作用，结果表明GAP对不同体系具有一定的选择性。GAP在邻苯三酚-鲁米诺、硫酸亚铁-鲁米诺、硫酸铜-邻菲啰啉-抗坏血酸-双氧水和硫酸铜-邻菲啰啉-抗坏血酸-双氧水-脱氧核糖核酸化学发光体系中表现出良好的清除活性氧作用和保护DNA免受损伤功能；但在硫酸亚铁-鲁米诺-双氧水和双氧水-鲁米诺化学发光体系中则表现出“促氧化”作用。这可能主要是由于样品和体系中氧化还原电位的差异而导致的。GAP经碱性蛋白酶酶解后其体外抗氧化活性增强。正是由于GAP具有优良的清除体外活性氧的作用，使之其在体内和体外水平上均对S₁₈₀肿瘤具有一定的抑制作用。更多还原

【Abstract】 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.更多还原