凤凰木种子毒蛋白的高效液相色谱研究

本文介绍用HPGFC对凤凰木种子中提取的三种毒蛋白分别进行色谱分析。并对分离的蛋白峰进行紫外光谱扫描来确认蛋白的纯度。根据标准分子量曲线,分别得到它们的分子量并与SDS-PAGF所得分子量进行比较,用柱后衍生法测定了三个蛋白各自的氨基酸组成。

A Study on the Proteins from Seeds of Delonix regia by HPLC

From the seeds of Delonix regia, three novel toxic proteins constituents named R1, R2, and R3 have been isolated and purified by extraction with 20mmol/L phosphate saline (0. 9% NaCl) buffer (PBS) at pH7. 2, and DEAE-52 cellulose column (1. 6×25cm) chromatography eluted with a gradient cosisting of salt-free 10mmol/L Tris/HCl buffer, pH7. 2 v. s. 0. 24mol/L NaCl Tris/HCl buffer. Three proteins were further separated on protein-pak60+protein -pakl25 7. 5mm (ID)×30cm, 10μm] columns. The mobile phase was 0. 2mol/L phosphate buffer (pH6-5). The flow rate was kept constant at 0. 8ml/min by YSB-2tpye high press pump. The effluent was monitored at a wavelength of 280nm on photodide array monitor. These three proteins are proved to be homogeneous by SDS-PAGE, IEF and HPGFC experiments. The molecular weights of the three proteins are estimated to be 28,700D, 25,700Dand 22,980D by SDS-PAGE and 28,860D, 26,100D, 23,500D by HPGFC analyses respectively. The isoelectic points of R1, R2, R3 are around 5. 2. The difference between their isoelectric points are within 0. 05 pH unit and somehow difficult to distinguish. This is in agreement with their chromatographic behaviors on ion-exchange column. The amino acid compositions of the three proteins were determined with OPA postcolumn derivatization fluorescence detection.