大黑花芸豆凝集素的分离纯化及温度、pH、色氨酸修饰对其活性的影响

摘要

大黑花芸豆(Phaseolus multiflorus.Wiud)种子经匀浆、浸取、硫酸铵分级沉淀、阴离子交换层析(DEAE-Sepharose)、阳离子交换层析(CM-Sepharose)和Sepllacryl S-200分子筛层析得到凝集素样品(PML).经SDS-PAGE检测为一分子量约为28k的单一条带,Sephacryl S-100凝胶过滤测得其表观分子量约为56 kD表明PML是由两个相同亚基组成的蛋白.温度低于60℃时,PML较为稳定,当温度达80℃时,其凝血活性完全丧失;pH为5.6～9对活性影响不大,pH为12时,活性大部分丧失;高温和强碱对荧光光谱有较大影响.NBS修饰Trp结果表明,在天然状态下有3个色氨酸分子被修饰,其中第二和第三个色氨酸分子对其活性至关重要.%A lectin ( Phaseolus multiflorus. Willd lectin,PML) was purified from the seeds of P. multiflorus by extraction, precipitation with (NH4)2 SO4, anion-exchange chromatography on DEAE-Sepharose column, cation-exchange chromatograpby on CM-Sepharose column,followed by gel filtration on a Sephacryl S-200 column. PML was a homodimer with molecular mass of 56 kDa and composed of two identical subunits which were 28 kDa. The change of agglutinating activity in different temperature and pH indicated that the hemagghtinating activity of PML was steady below 60 ℃ and at pH 5.6 to 9.0. The effect of chemical modification of amino add residues upon hemagglutination was investigated. There was a definite decrease of fluorescence intensity which corresponds to the oxidation of tryptophan. Under native conditions 3 tryptophan residues were available for modification by N-Bromosuccinimide (NBS) ,which had crucial effect on the hemagghtinating activity.