从大白芸豆种子中分离与表征一种新型凝集素WKBL

从大白芸豆种子中,经过缓冲液抽提、硫酸铵沉降、蓝胶亲和色谱(AF-Blue HC-650M) 、离子交换色谱(CM-Sephadex C-25)、凝胶过滤色谱(Sephadex G-50)等步骤,分离纯化了一种具有一定抗真菌活性和抗肿瘤活性的凝集素，命名为WKBL.经SDS-PAGE显示, WKBL分子量为25 kD，N端序列为DAVLYRGPGDLHTGS，过碘酸-雪夫染色(PAS)呈红色. 凝血活性实验显示, WKBL凝血效果明显，但只有在60 ℃以下才具有凝血活性,而pH 对其活性影响并不大. D-半乳糖、D-果糖、D-葡萄糖、甘露醇能抑制WKBL的活性.WKBL为金属依赖型凝集素，LiCl、SnCl2和K2Cr2O7能抑制WKBL活性，但CaCl2、BaCl2、ZnCl2、CuCl2、FeCl3和MgCl2对WKBL活性却无影响.WKBL对白血病细胞K562的生长显示出较强的抑制作用，其IC50为15 μmol/L，对HeLa细胞呈现抑制作用，对HepG2细胞的抑制作用不明显. 抑菌实验显示, WKBL对瓜果腐霉病菌(Pythium aphanidermatum)、苹果轮纹病菌(Physalospora piricola)、甜瓜枯萎病菌(Fusarium oxysporum f.sp.nelonis)和葡萄灰霉病菌(Botrytis cinerea)呈现出一定的抑制，但是其抑制率对浓度的依赖性并不高.

Isolation and Characterization of a Novel Lectin WKBL from Phaseolus lunatus Linn

A novel lectin with the molecular mass of 25 kD, designated WKBL, was isolated from seeds of white kidney beans. The purification steps involved ammonium sulfate precipitation, affi-gel blue chromatography, ion-exchange chromatography on CM-Sephadex C-25, gel filtration chromatography on Sephadex G-50. The lectin could be stained in red by periodic acid-Schiff (PAS) reagent and its N-terminal sequence was DAVLYRGPGDLHTGS. WKBL exhibited hemagglutinating activity in human erythrocytes. The hemagglutinating activity was mostly preserved up to 60℃. WKBL was stable over the pH 2~11. Among the carbohydrates tested, D-galactose, D-fructose, D-glucose, and mannitol were capable of inhibiting the hemagglutinating activity. WKBL was a metal dependent protein; the metallic compounds including LiCl, SnCl2 and K2Cr2O7 inhibited the activity whereas MgCl2, ZnCl2, BaCl2, CuCl2, FeCl3 and CaCl2 did not affect its activity. Proliferation of tumor cells K562 cells was inhibited strongly by WKBL with an IC50 of 15 μmol/L, whereas its inhibit is weaker to HeLa cells and slightly affected to HepG2. WKBL showed antifungal activity towards the growth of Pythium aphanidermatum, Physalospora piricola, Fusarium oxysporum f.sp.nelonis, Botrytis cinerea, but the inhibition rate was not dependent strongly on the concentration of WKBL.