【作者】 刘超；

【导师】 鲍锦库；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 川泽泻（Alisma plantago-aquatica）球茎经磨碎、匀浆、浸取、硫酸铵分级沉淀得到粗品，将粗品用于阴离子交换柱（DEAE-Sepharose）、甲壳素亲和层析和SephacrylS-100分子筛层析得到凝集素纯品。在变性剂巯基乙醇存在和不存在的条件下经SDS-PAGE检测均为单一蛋白带，分子量约为22.4KD，SephacrylS-100凝胶过滤测得其表观分子量约为22.5KD，表明APL是由单亚基组成的蛋白。APL能凝集人ABO血红细胞，不凝集兔血红细胞，其凝集人血细胞的效价为1.8ug／mL。泽泻凝集素凝血活性依赖于Ca²⁺、Mg²⁺。糖抑制实验结果表明，N-已酰葡萄糖胺能抑制APL的凝血活性。中性糖含量的测定，含有4.26％的中性糖，表明泽泻凝集素是一种糖蛋白。 APL的荧光光谱研究在激发光为280nm时，其最大荧光发射峰在328nm处，荧光发射光谱未见有酪氨酸（Tyr）残基的发射峰，表明Tyr残基的荧光通过能量转移到Trp上，使荧光强度增强；在激发光谱为295nm时，其最大荧光发射峰也在328nm处。比游离Trp的最大荧光发射峰（348nm）蓝移了近20nm，说明Trp周围的极性较弱，处于疏水的非极性微环境，并未暴露于周围的溶剂中。研究不同温度、pH和基团特异性化学修饰后APL凝血活性的变化和荧光光谱的变化，当温度达60℃以上时，活性开始下降，到70℃时活性仅有42％保留，到80℃时凝血活性全部消失；当pH为4时，活性仅保留20％，pH为6-8对活性的影响不大，最适的pH值为6.8左右，pH9.0时凝血活性只有40％。由此表明更多还原

【Abstract】 The studies on Alisma plantago-aquatica lectin （APL） mainly included the purification , characterization of APL, and the relationship between structure and bioactivity of APL. APL was isolated from the bulbs of Alisma plantago-aquatica by extraction, precipitation with （NH₄）₂SO₄, ion-exchange chromatography on DEAE-Sepharose, chitin-aff inity chroma-tography column and followed by gel filtration on Sephacryl S-100. The purified lectin showed both a single protein band on SDS-PAGE under the presence or not of the β-mercaptoethanol, and the molecular weight was 22. 4KD. The molecular weight was also 22. 5KD determined further by gel filtration on Sephacryl S-100. The results implied that APL was a single protein. APL can agglutinate all human ABO system groups erythrocyte at 1.8ug/mL and can not agglutinate rabbit erythrocyte. The results of carbohydrate inhibition assay showed that N-Acetyl-D-glucosamine can inhibit the agglutinating activity of APL. Test of the neutral carbohydrate indicated the lectin APL was a glycoprotein which contained 4. 26% saccharide.The native APL exhibited a characteristic circular dichroic （CD） spectrum, double negative bands centered at 195nm and 200nm. It was estimated that APL was a high α- helix protein. The fluorescence spectrum （FLS） of APL excited at 280nm and 295nm showed a maximum peak both at更多还原