西兰苔凝集素的分离纯化及性质研究

Purification and Partial Characterization of Lectin from Broccolini

摘要:

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。

Abstract:

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.