Huang Zhi, Yang Fang, Zheng Wen-Jie
College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Wei Sheng Wu Xue Bao. 2006 Jun;46(3):401-5.
Three Se-containing allophycocyanins (Se-APC) with high purity were purified from Se rich Spirulina platensis (Se-sp.) by hydroxyapatite chromatography, DEAE-52 anion-exchange chromatography and native gel preparative electrophoresis. Their biochemicial properties were explored by spectral scanning and electrophoresis analysis of Native-PAGE, SDS-PAGE and IEF on thin slab gel. Protein molecular weight (MW) of APC aggregation was determined by gel filter on Sephadex G-200 column. Se content of native and denatured Se-APC was detected by 2, 3-DAN fluorocence method. According to visible and fluorescence spectral character, three purified fractions of APC were identified to be APCI, APCII and APCIII. Native-PAGE and SDS-PAGE analysis revealed that they all shaped trimer (alphabeta) 3 of alpha and beta subunit with molecular mass of 18.3kDa and 15.7kDa, whereas APCI contains gamma subunit (about 32kDa) visibly and APCIII maybe contain the linker peptide of L(C)(8 - 10 kDa) based on their MW to be determined of 130.9, 98.1 and 106.30 kDa. IEF detection showed that the pl of Se-APCs was 4.76, 4.85 and 5.02 respectively. Se content of three purified Se-APCs were 316, 273 and 408 microg/g, which decreased about 25% after deaggregation treatment by 0.50 mol/L NaSCN and decreased more than 50% after denaturation treatment by 2-mercaptoethanol and reached to a steady content of 132 microg/g on average. These results indicated that Se incorporation into APC had no influence on function of energy transfer as well as biochemical property of APCs, and Se binding with APCs was highly relevant to its aggregation states whereas Se integrated steadily with its subunits.
通过羟基磷灰石层析、DEAE - 52阴离子交换层析和天然凝胶制备电泳，从富硒钝顶螺旋藻（Se - sp.）中纯化出三种高纯度的含硒别藻蓝蛋白（Se - APC）。通过在薄平板凝胶上进行天然聚丙烯酰胺凝胶电泳（Native - PAGE）、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE）和等电聚焦电泳（IEF）的光谱扫描和电泳分析，探究了它们的生化特性。通过葡聚糖凝胶G - 200柱上的凝胶过滤法测定了别藻蓝蛋白聚集体的蛋白质分子量（MW）。采用2,3 - 二氨基萘荧光法检测天然和变性Se - APC的硒含量。根据可见光谱和荧光光谱特征，鉴定出三种纯化的别藻蓝蛋白组分分别为APCI、APCII和APCIII。Native - PAGE和SDS - PAGE分析表明，它们均形成由α和β亚基组成的三聚体(αβ)3，分子量分别为18.3kDa和15.7kDa，而APCI明显含有γ亚基（约32kDa），基于其待测定的分子量130.9、98.1和106.30kDa，APCIII可能含有L(C)连接肽（8 - 10kDa）。IEF检测显示，Se - APCs的等电点分别为4.76、4.85和5.02。三种纯化的Se - APCs的硒含量分别为316、273和408μg/g；经0.50mol/L硫氰酸钠解聚处理后，硒含量下降约25%；经2 - 巯基乙醇变性处理后，硒含量下降超过50%，平均稳定在132μg/g。这些结果表明，硒掺入别藻蓝蛋白对能量传递功能以及别藻蓝蛋白的生化特性没有影响，硒与别藻蓝蛋白的结合与其聚集状态高度相关，而硒与其亚基稳定结合。
