西兰苔凝集素的分离纯化及性质研究

Purification and Partial Characterization of Lectin from Broccolini

本文采用硫酸铵沉淀法,分别以生理盐水,蒸馏水,pH=7.2的Tris-HCl缓冲液,pH=7.2的磷酸缓冲液为浸提液,对西兰苔凝集素进行提取,然后依次利用DEAE-52阴离子交换层析和SephadexG-75对凝集素粗品进行分离纯化,并对纯化后的西兰苔凝集素的部分性质进行研究。结果表明：pH=7.2的Tris-HCl缓冲液的提取率最高,为3.57±0.02%;纯化后的西兰苔凝集素分子量约为27 ku,糖含量为7.60±0.03%,其凝血活力为28;糖抑制试验表明,西兰苔凝集素的凝集活性受D-甘露糖和阿拉伯糖的抑制;热稳定性实验表明90℃以下凝集素的凝集活性不受温度影响,有较好的热稳定性。此外,西兰苔凝集素的最适pH范围是7-8,强酸强碱环境中其凝血活性明显下降。在含有Ca2＋的溶液中其凝集活性有所增强,表明西兰苔凝集素活性受Ca2＋影响较大。

Broccolini lectin（BL） was extracted from Broccolini with normal saline, distilled water, Tris-HCl buffer and phosphate buffered saline（PBS）, followed by ammonium sulphate precipitation, and finally purified by chromatography DEAE-52 and Sephadex G-75.Partial characterization of BL was also studied. The results showed that Tris-HCl buffer had the highest extraction rate of 3.57±0.02%. Molecular mass of BL was estimated as 27 ku by SDS-PAGE. The BL contained 7.60±0.03% sugar and its clotting activity was 28. The sugar inhibition test showed that its hemagglutinating activity of erythrocyte could be inhibited by D- mannose and arabinose. Moreover, the lectin remained stable at 20-90 ℃and its optimum pH was 7-8. Its clotting activity was significantly decreased in strong acid and strong alkali environment. The lectin reaction was also influenced by divalent cations Ca2＋, in which its hemagglutinating activity was increased.