黄精凝集素Ⅱ的纯化及部分性质研究

Purification and Characterization of the Polygonatum cyrtonema Hua. Lectin Ⅱ

囊丝黄精（ＰｏｌｙｇｏｎａｔｕｍｃｙｒｔｏｎｅｍａＨｕａ．）的根状茎，经浸取、用硫酸铵分级沉淀、猪甲状腺球蛋白－Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和层析、ＣＭ－Ｓｅｐｈａｒｏｓｅ柱离子交换层析和ＳｅｐｈａｄｅｘＧ－１００凝胶过滤，可以分离纯化出黄精凝集素Ⅱ（ＰＣＬⅡ）．纯化的ＰＣＬⅡ在聚丙烯酰胺凝胶电泳中显示单一蛋白染色带；在快速高效液相色谱中亦为单一蛋白峰，经分子筛层析测得分子量为１５．９ｋＤ，最大紫外吸收值在２７８ｎｍ，ＰＣＬⅡ只凝集兔红细胞，当浓度为０．２５μｇ／ｍｌ时，即可发生凝集反应，此凝集兔红细胞的能力可被Ｄ－甘露糖和猪甲状腺球蛋白所抑制．氨基酸组成分析表明ＰＣＬⅡ分子中富含酸性氨基酸，Ｎ末端为丙氨酸．经测定ＰＣＬⅡ分子中含有３个色氨酸和２．４％的中性糖．原子发射光谱分析表明，该凝集素分子中含有Ｍｇ和Ｃａ两种金属元素．

Polygonatum cyrtonema Hua. lectin(PCL Ⅱ ) was isolated from Polygonatum cyrtonema Hua. by extraction. fraction with (NH_4)_2SO_4, affinity chromatography on the porcine thyroglobulin-Sepharose 4B column, ion-exchange chromatography on CM-Sepharose column and followed by gel filtration on sephadex G-100. The purified PCLⅡ shows a single protein band on PAGE and also single peak on FPLC;Its molecular weight is 15. 9 kD on sephadex G-100. The maximum absorption of the purified PCL Ⅱ is at 278 nm. PCL Ⅱ can agglutinate rabbit red cells at the concentration of 0. 25 μg/ml, the reaction can be inhibited by D-Man and porcine thyroglobulin;The N-terminal residue of PCLⅡ is Ala and the lectin is rich in acidic amino acids. It contains 3 Trp and 2. 4% neutral saccharide in per molecule. It also contains Mg￣(2+) and Ca￣(2+) as studied by atomic emission spectrum.