苦荞凝集素的纯化及性质鉴定

Purification and Identification of Tartary Buckwheat Lectin

目的:从苦荞麦种子中提取、纯化苦荞凝集素,对其凝血及酶学活性等性质进行初步研究。方法:采用缓冲液抽提、硫酸铵沉淀、透析及DEAE-纤维素阴离子交换层析(DEAE fast flow,DEAE FF纯化凝集素;高碘酸希夫(periodic acid-Schiff,PAS染色鉴定其蛋白种类,硫酸-苯酚法测定糖含量;凝血实验和糖抑制实验检测其凝血活性和结合糖特异性;以对硝基苯磷酸二钠为底物,测定磷酸酯酶活性。结果:从苦荞麦种子中获得了一种具有凝血功能的蛋白质——苦荞凝集素(tartary buckwheat lectin,TBL)。纯化的TBL在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE图谱上显示单一条带,根据SDS-PAGE计算其分子质量约为62 kD。PAS染色证明TBL为糖蛋白,糖含量为5.8%。凝血实验表明TBL对人的O型血红细胞具有特异性凝集作用,效价为15μmL对其他血型的红细胞没有凝血效应。血凝活性被D甘露糖和D-葡萄糖抑制,推测其是一种甘露糖结合凝集素。酶学性质鉴定显示,TBL具有磷酸酯酶活性,米氏常数Km=9.86×10-3 mol/L。结论:TBL可能是苦荞麦种子中的一种具有多种酶学功能的甘露糖凝集素。

Purpose: To purify lectin from tartary buckwheat(TBL) and identify its hemagglutination and enzymatic activity.Methods: TBL was extracted and puried by ammonium sulfate precipitation, dialysis, and anion exchange chromatography.Periodic acid-Schiff(PAS) staining was used to identify its glycoprotein nature and sulfuric acid-phenol method was used to measure the sugar contents. The hemagglutination activity and sugar binding specicity of the puried TBL were tested by hemagglutination reaction and hemagglutination inhibition assays, and the phosphatase activity was measured by using4-nitrophenyl phosphate disodium salt hexahydrate(pas the substrate. Results: The puried TBL displayed a single band and a molecular weight of 62 kD, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDSPAGE).PAS staining indicated that TBL was a glycoprotein with a sugar content of 5.8%.Hemagglutination reaction demonstrated that TBL could agglutinate the O type of human blood with titer of 15 μmL. The hemagglutination activity was inhibited by Dglucose and Dmannose, from which, TBL can be deduced as a mannose-binding lectin. Further experiments demonstrated that TBL had phosphatase activity with a Michaelis constant Km of 9.86×10-3 mol/L. Conclusions:The TBL obtained in this study was a mannose-binding lectin(MBL) with enzymatic functions.