海刺猬体腔液凝集素的纯化、性质及其色氨酸的化学修饰  

Purification,properties and chemical modification of tryptophan residues in lectin from coelomic fluid of sea urchin Glyptocidaris crenularis

作　　者：李丹彤[1] 吴振海[2] 李悦[1] 王树敏[1] 朱莹[1] 

机构地区：[1]大连海洋大学农业部北方海水增养殖重点实验室,辽宁大连116023 [2]大连海洋大学辽宁省海洋生物资源恢复与生境修复重点实验室,辽宁大连116023

出　　处：《大连海洋大学学报》2012年第4期338-343,共6页Journal of Dalian Ocean University

基　　金：辽宁省教育厅团队项目(2008T021)

摘　　要：通过DEAE-纤维素52离子交换层析、Sephadex G-200凝胶层析,从海刺猬体腔液中分离纯化海刺猬Glyptocidaris crenularis凝集素(简称为GCL),并对其部分性质和色氨酸的化学修饰进行了研究。结果表明:在还原与非还原SDS-PAGE上,GCL都显示单一蛋白质条带,其相对分子量为94 462;GCL对人O型、兔、鸡、狗红细胞均具有血凝活性,对兔和狗红细胞的血凝活性最强,但对人A、B、AB型及鲫红细胞无血凝活性;GCL血凝活性在温度为4～33℃时最高,经36℃热处理10 min后,GCL对兔红细胞的血凝活性保留25%,经48℃加热10 min后,其血凝活性完全丧失;GCL血凝活性在pH为7.5～8.5时最高;Ca2+对GCL血凝活性无抑制作用,EDTA和Mg2+对其血凝活性有抑制作用;GCL血凝活性不被所测试的α-乳糖、D-果糖、D-半乳糖、D-甘露糖、D-葡萄糖和γ-球蛋白所抑制,但被蔗糖和麦芽糖所抑制,最小抑制浓度分别为40 mmol/L和20 mmol/L;用N-溴代丁二酰亚胺(NBS)对GCL分子中的色氨酸(Trp)残基进行化学修饰,有5.1个Trp残基被修饰,修饰后其血凝活性丧失75%,表明Trp残基是GCL血凝活性的重要组成部分。A lectin（GCL） was purified from coelomic fluid of sea urchin（Glyptocidaris crenularis） by DEAE-cellulose 52 ion exchange chromatography and gel filtration chromatography with Sephadex G-200.The lectin showed a single band on both reduced and non-reduced SDS-PAGE with molecular weight of 94 462,hemagglutinating the four tested erythrocytes（rabbit,dog,chicken and O type erythrocyte in human）,especially to rabbit and dog erythrocytes,without hemagglutinating erythrocytes of human（A,B,AB） and crucian carp（Carassius auratus）.The maximum hemagglutinating activity was observed at 4-33 ℃ and at pH 7.5-8.5,being reduced to 25% at 36 ℃ for 10 minutes.No activity was found 10 minutes after 48 ℃ incubation.The hemagglutination activity of GCL was significantly inhibited by divalent metal ion Mg2＋ and EDTA,except for Ca2＋.The hemagglutinating activity of GCL was significantly inhibited by sucrose at minimum inhibitory concentration of about 40 mmol/L and by maltose at minimum inhibitory concentration of 20 mmol/L.α-Lactose,D-Fructose,D-Galactose,D-Mannitol,D-Glucose and γ-Globulin showed little effect on the hemagglutination activity of GCL.Chemical modification of tryptophan（Trp） residues with N-Bromosuccinimide（NBS） showed that 5.1 tryptophan（Trp） residues modified in GCL,being accompanied by 75% loss of hemagglutinating activity,indicating that Trp residues played an important part in GCL hemagglutinating activity.

关 键 词：海刺猬 凝集素 纯化 化学修饰 

分 类 号：S917[农业科学—水产科学]