化学修饰对川木通凝集素活性及构象的影响

Effects of chemical modification on the hemagglutination activity and conformation of Clematis montana Buch.-Ham lectin

川木通干燥茎蛋白粗提液经DEAE-Sepharose离子交换柱和Sephacryl S-100分子筛分离后得到川木通凝集素(命名为CML)。Tricine SDS-PAGE分析表明CML的分子量为12.5 kDa,Sephacryl S-100分子筛分析CML的表观分子量为25.8 kDa,表明CML是由两个亚基组成的同源二聚体。过碘酸-希夫(periodic acid Schiff reacition,PAS)染色结果表明CML为糖蛋白,糖含量为12.2%。氨基酸残基修饰色氨酸(Trp)、精氨酸(Arg)和-SH CML凝血活性的必需基团。每分子CML中含有6个Trp。荧光光谱显示这些基团修饰后荧光强度都有不同程度的下降,最大发射峰位置也相应变化,表明这些残基修饰导致蛋白质构象变化。酪氨酸(Tyr)、苏氨酸(Ser/Thr)和组氨酸(His)修饰后,活性并没有变化,但荧光强度显著减弱,说明修饰后CML分子的活性中心的必需构象并没有受到破坏,但氨基酸的修饰导致色氨酸的微环境发生变化。

A novel lectin was isolated from Clematis montana Buch.-Ham stem （Ranunculaceae） using ion exchange and gel filtration chromatographies. The purified Clematis montana lectin （CML） is a homodimer of 12. 5 kDa subunits. It was a glycoprotein and had 12.2% neutral sugar. Modification of CML with NBS resulted in the modification of about 6 tryptophan residues and a complete loss of its activity. While modification of arginine and -SH residues led to considerable loss of the hemagglutinating activity. It indicated that these residues are essential for the hemagglutinating and involved at carbohydrate-binding site. Fluorescence evidence also verified the assumption. Modification of tyrosine, serine/ threonine and histidine did not affect hemagglutination activity, but led to the decrease of relative intensity at 295 nm. This decrease is mostly due to the modification of these residues. The results indicated that tyrosine, serine/threonine and histidine are required for the structural/conformational properties of the lectin.