红藻条斑紫菜凝集素的纯化及其色氨酸化学修饰对其活性的抑制作用

为了探索条斑紫菜凝集素(Porphyra yezoensis Ueda lectin, PYL)的作用机理，对其进行了分离和纯化．条斑紫菜经磷酸盐缓冲液浸泡、20%～75%硫酸铵分级、DEAE 纤维素52离子交换层析和Sephadex G-200凝胶过滤层析，得到PYL纯品. Sephadex G-200分子筛层析测得其分子量为63.2 kD，在非还原SDS-PAGE上显示1条蛋白染色带，分子量为63.1 kD，还原SDS-PAGE显示1条蛋白染色带，亚基分子量为15.8 kD．PYL在对兔、大鼠、鸡、羊、狗血细胞的凝集作用中，对大鼠红细胞的凝集活性最高.PYL在pH 6.50～10.53范围内均有活性，在pH 8.40～8.91活性最高．经42 ℃热处理10 min后，仍然对大鼠红细胞血凝活性保留12.5%，其活性最大温度范围为4 ℃～20 ℃, 48 ℃加热10 min后，其活性完全丧失．EDTA对PYL的凝集活性有抑制作用，最小抑制浓度为156 mmol/L，而 Ca2+和Mg2+未发生凝集抑制现象．PYL凝集大鼠红细胞的作用不被D 果糖、葡萄糖、D-半乳糖、D-甘露糖、菊粉、γ球蛋白、牛甲状腺球蛋白等所抑制，但可被蔗糖和麦芽糖抑制，最小抑制浓度蔗糖为20 mmol/L，麦芽糖为40 mmol/L．用N 溴代丁二酰亚胺(NBS) 对PYL分子中的Trp残基进行化学修饰，有2.1个Trp残基被修饰，修饰后PYL活性丧失, 表明Trp残基是PYL凝集活性所必需的基团．

Reduced Activity of Lectin Purified from Red Alga Porphyra yezoensis Ueda by Chemical Modification for Tryptophan Residues

Porphyra yezoensis Ueda lectin(PYL) was purified with PBS extraction,and fractionated by 20%～75% ammonium sulfate,then subjected to DEAE-cellulose 52 ion-exchange chromatography and gel filtration with Sephadex G-200.The molecular weight of the purified PYL was 63.2 kD by Sephadex G-200 and 63.1 kD in non-reduced SDS-PAGE.In reduced SDS-PAGE,a single 15.8 kD band was found,suggesting PYL was composed of four identical subunits.The purified PYL could hemagglutinate the erythrocytes from rat,rabbit,goat and chicken,especially rat.The optimum pH was between 8.40 to 8.91 within the range of 6.50 to 10.53.The maximum hemagglutination activity at was 4 ℃～20 ℃,while the activities reduced to12.5% at 42 ℃ for 10 minutes.No activity was observed after 48 ℃incubation for 10 minutes.EDTA of 1.56 mmol/L or higher concentration significantly inhibited the hemagglutination activity,and Ca2+ and Mg2+ had little effect on PYL activities.D-fructose,D-glucose,D-galactose,D-mannose,bovine thyroglobulin and human gamma-globulin did not alter the activity of PYL.Sucrose and maltose significantly inhibited the PYL activity at the concentrations of 20 mmol/L and 40 mmol/L,respectively.N-bromosuccinimide(NBS) modification resulted in 2.1 tryptophan(Trp) residue changes completely ablated the hemagglutination ability,indicating the Trp residues were essential for the PYL activity.