蓝隐藻藻蓝蛋白的分离纯化及结构与性质的分析-isolation and purification of phycocyanin from cyanobacteria and analysis of its structure and properties.docxVIP

摘要以自行培养的蓝隐藻（Chroomonas placoidea）为材料，经过压力破碎和高速离心分离去 除膜成分，得到的上清液分别采用 70%、80%、90%和 100%的硫酸铵分级沉淀藻蓝蛋白（PC）， 经两次分子筛层析纯化，PC 的 A646/A2807，经中性 PAGE 和 HPLC 检测其纯度，发现不含 杂蛋白。通过研究 PC 及其亚基在不同 pH 条件和尿素变性剂中的稳定性，摸索亚基变性条件， 采用SephacrylS-100 柱层析分离得到了富含β亚基的蓝色SG 组分和富含α亚基的绿色SG 组分。 同时结合尿素-SPS、二维电泳、聚酰胺薄膜层析等技术，对各级 PC 沉淀以及分离后的 亚基组分的吸收光谱、荧光谱、亚基组成、亚基等电点以及亚基 N-末端氨基酸组成等进行了 测定和分析。实验结果显示，纯化的蓝隐藻 PC 至少含有 4 种发光亚基，分别命名为 β1（20.3kDa），β2（16.7kDa），α1（9kDa）和α2（8.3kDa），其中微酸性的 β2 发光亚基是文献中没有报道的。β1和 β2 亚基的等电点处于 6.0 和 5.7 附近的微酸性区；α2 亚基等电点在 7.1 附近；而 α1 亚基则是一个等电点为 9.1 的碱性亚基。因此这些亚基可借助静电力彼此结合，在水溶液中形成分子量 大小不等、亚基比例不尽相同的 PC 复合物形式；此外二维电泳及 N-末端结果提示蓝隐藻 PC 中可能还结合有一些低丰度的无荧光蛋白，可能在 PC 的亚基组装和复合物形成中担负一定的 作用。关键词：蓝隐藻；藻蓝蛋白；亚基拆分； 分子筛；二维电泳AbstractPhycocyanin (PC) are separated and purified from Chroomonas placoidea cultured in laboratory by ammonium sulfate fractional precipitation and two steps of gel filtration chromatography. The purified PC samples are confirmed to contain almost no impurities in HPLC and native PAGE, and show an A645/A280 ratio up to 7. The denaturing conditions of PC are investigated by studying the stability of PC and its subunits in different pH conditions and in the denaturant urea. The β subunits fraction (SB) and the green fraction enriched in α subunits are then isolated by SephacrylS-100 column chromatography. The absorption and fluorescence spectra as well as extinction coefficient of PC and its separated subunits are determined. Combined with urea-SDS, two-dimensional electrophoresis, polyamide thin layer chromatography, the subunits composition, isoelectric point and the N-terminal amino acid are also analyzed.The results show that extinction coefficient of the purified PC of C. placoidea is about 3.7 ~ 4.1ml/ (mg.cm). It contains at least four different fluorescent subunits, which are named as β1 (20.3kDa, pI6.0), β2 (16.7 kDa, pI≈5.7), α1（9kDa，pI9.1）and α2（8.3kDa，pI7.1）. The β2 subunit is confirmed to have a smaller molecular weight and lower isoelectric point than reported β1 subunit, and possiblyp