高效凝胶过滤色谱法分离测定豆薯种子蛋白

Isolation and Determination of the Seeds of Pachyrrhizus Errosis Protein by High Performance Gel Filtration Chromatography (GFC)

用高效凝胶蛋白往分离豆薯种子蛋白租提液，结合光电二极管阵列检测器对分离的蛋白峰进行紫外光谱扫描来确认蛋白的纯度，测定了3种蛋白的分子量，采用邻苯二甲醛（OPA）柱后衍生法测定了豆薯种子蛋白的氨基酸含量。

From the seeds of Pachyrrhas errsus, three protein constituents, namel PE1,PE2 and PE3, have been isoated and purified by extraction with 5mmol/L phosPhate saline(0. 9 % NaCl) buffer(PB) at pH 7. 2,and S-Sepharose Fast Flow Column (2. 6cm × 15cm) chromatography which eluted with 5mmol/L phosphate buffer (pH 7. 0) containing 1mmol/L NaCl. Three proteins were burther separated on two connected Protein-Pak 60+Protein-Pak 125[7. 5mm× 39cm, 10μm] columns with mobile phase of 0. 2mol/L phosPhate buffer (pH 6.5). The flow rate was kept constant at 0. 8mL/min by YSB-2 type high press Pump. The efnuent was monitored at a wavelength of 280nm on photediode array detector. These three proteins are proved to be homogeneous by SDS-PAGE, IEF and HPGFC experiments, and all present the typical absorption spectra in ultraviolet region. The moleculer weights of the three proteins are approxiamtely 33 000D, 14 500D and 14 000D respectively by SDS-PAGE. But as using HPGFC analysis, the MW va1ue of PE2 is 28 000D. This indicates PE2 may be composed of two chains joined by disulfide bond, which is further proved from the latter amino acid composition analysis. The isoe1ectric points of three proteins are 4. 5, 6. 5 and 7. 5 respectively by using IEF. The amion acids compositions of the three proteins were determined with OPA post-column de rivatisation /fluoresc ence detection.