理化因素对白芸豆凝集素生物学活性影响

Effects of physico-chemical factors on biological activity of lectin from Phaseolus vulgaris L. seeds

[目的]研究物理与化学因子对白芸豆凝集素生物学活性的影响,为白芸豆凝集素开发利用奠定理论基础和提供技术参考.[方法]白芸豆（Phaseolus vulgarisL）种子经硫酸铵分级沉淀、DEAE-Sepharose离子交换和Sephadex G-100凝胶柱层析得到凝集素（PVL）;然后以不同温度和pH梯度、不同金属离子、不同变性剂处理样品,通过倍比稀释法测定凝集素的生物学活性.[结果]白芸豆凝集素能使兔血红细胞发生凝集,在20～70℃红细胞凝集活性不受影响,当温度达到80℃时凝集素样品仅有25％凝血活性,85℃加热30 min凝血活性全部消失.用EDTA溶液处理后,白芸豆凝集素失去凝血活性,金属离子Mg2＋和K＋能恢复凝血活性,Mn2＋能完全恢复凝血活性,Ba2＋、Cu2＋、Fe3＋、Ca2＋不能使凝血活性恢复.白芸豆凝集素在缓冲液pH低于4.0时凝血活性逐步降低,在pH 3.0时,活性仅有45％;在缓冲液pH高于7.2时活性逐步降低,pH 9.0时凝血活性仅有44％.3种变性剂变性试验中,脲能使凝集素凝血活性降低70％,盐酸胍能降低30％,SDS能降低18％.[结论]白芸豆凝集素具很强的热稳定性和广谱酸碱度适应能力;凝血活性依赖Mn2＋等金属离子,且仅能被变性剂6 mol/L脲部分抑制.在实际应用中,可根据适宜条件控制其物理和化学因素,防止凝集素变性失活.

[Objective ]The present investigation was done to determine the effects of phyico-chemical factors on bio- logical activities of the lectin fi＇om Phaseolus vulgaris L. seeds to provide theoretical references for application and develop- ment of Phaseolus vulgaris L. lectin. [Method]The Phaseolus vulgans L. lectin （PVL）was obtained by ammonium sulphate precipitation, ion exchange chromatography on diethylaminoethyl-Sepharose and carboxymethyl-Sepharose. The gel filtra- tion chromatography on Sephadex G-IO0 from bulb of the edible Phaseolus vulgaris L. was treated with EDTA and metal ions, various temperatures, pH and denaturants to investigate the effects on hemagglutinating activity. [Result]The lectin from Phaseolus vulgaris L. could agglutinate rabbit red blood cell. The results showed that the hemagglutinating activity of PVL was stable from 20 ℃ to 70 ℃. The hemagglutinating activity of PVL declined （only 25%） markedly at 80 ℃, and was totally inactivated at 85 ℃ after 30 rain. The procoagulant activity disappeared after the lectin from Phaseolus vulgaris L. was treated by EDTA solution. The activity was restored with Mg2＋ and K＋, and totally restored in the presence of Mn2＋. But Ba2~, Cu2＋, Fe3＋ and Ca2＋ failed to restore the activity of PVL. In the buffer solution at lower than 4.0 pH, the activity of PVL progressively decreased. At pH 3.0, the activity was only 45%. With pH for buffer solution above pH 7.2, its ac- tivity gradually dropped, while at pH 9.0, the activity was only 44%. Concerning the three denaturants, urea, Gu-HC1 and SDS brought down PVL activity by 70%, 30% and 18%, respectively. [Conclusion]The lectin is a thermal-stable protein and has higher acid- and alkali-ability of the hemagglutinating activity. PVL requires metal ions such as Mn2＋ for its hemagglutinating activity and hemagglutinating activity could be repressed partially by the denaturant urea at 6mol/L. In actual application, the lection activity could be kept under proper controlled physical and chemical conditions.