一步柱层析纯化B-藻红蛋白及其毛细管电泳结合激光诱导检测

One-step Chromatography for B-phycoerythrin Purification and Detection by Capillary Electrophoresis Assay with Laser-induced Fluorescence

本研究采用硫酸铵沉淀结合DEAE-SepharoseFF离子交换柱层析技术分离纯化紫球藻中的B-藻红蛋白。试验结果表明,藻红蛋白粗提物经65%硫酸铵沉淀2d后过柱层析,B-藻红蛋白的纯度为A545nm/A280nm=3.0;而用65%硫酸铵沉淀2个月后,只需采用一步柱层析,B-藻红蛋白的纯度可提高到5.37,即达到纯品要求(纯度大于4.5)。说明65%硫酸铵对B-藻红蛋白具有高度选择性,其工艺效果对分离纯化有很大影响。特征吸收光谱和荧光光谱证实纯化后的产物符合B-藻红蛋白的性质,Native-PAGE电泳只出现单一染色带,表明获得的B-藻红蛋白是均一的;SDS-PAGE电泳出现2条染色带,一条着色较深带为α和β亚基,分子量为16～18kD左右,另一条较浅为γ亚基,分子量大约为31kD。同时建立了涂层毛细管柱结合激光诱导荧光电泳技术快速检测B-藻红蛋白纯度的分析方法,结果出现单一峰,表明毛细管电泳用于B-藻红蛋白的纯度检测高效可靠。

Ammonium sulphate sedimentation combined with DEAE Sepharose Fast Flow chromatography for B-phycoerythtin （B-PE） purification from the red alga Porphyridium cruentum was established in this paper.The rusults showed that after precipitation with 65% saturated ammonium sulphate for two days and purification with chromatography, the purity of B- phycoerythrin reaches A545nm/A280nm=3.0. However after precipitation for two months, the high purity of B-phycoerythrin can be obtained by only one-step chromatography to reach 5.37 and can fulfill the purity standards as 4.5. This suggests that 65% ammonium sulphate has a high selectivity on B-PE. The effect of precipitation process intensely affects influenced the purity of B-phycoerythrin. The absorption spectrum and the fluorescence spectrum of producetion are both in accordance with characteristics of B-phycoerythrin. Native-PAGE showed only one single dark ribbon which means that B-phycoerythrin is homogeneous. SDS-PAGE appeared two ribbons, one is α- and β-subunit with molecular weight about 16-18kD, while the other one is γ -subunit with molecular weight about 31kD. At the same time, capillary electrophoresis and laser induced fluorescence method are both established for fast analysis on B-phycoerythrin purity. The result shows one single peak that means capillary electrophoresis method is highly effective for analysis on purity of B-phycoerythrin.