海萝凝集素的分离纯化及性质

Isolation,purification and characterization of lectin from Gloiopeltis furcata

海萝(Gloiopeltisfurcata)经PBS缓冲液抽提、硫酸铵分级、DEAE 52纤维素离子交换层析和SephadexG 200分子筛层析,从中纯化出海萝凝集素(GFL),在PAGE和等电聚焦电泳上各显示单一蛋白染色带,其等电点为9 0。用SephadexG 200分子筛层析测得其相对分子质量为8318。海萝凝集素除对人血型红细胞无凝集活性外,对单胞藻及兔、鲤、鲫的红细胞均表现出一定程度的凝集活性,对兔红细胞的凝集活性最强,且不被已测试的D-果糖、D-半乳糖、葡萄糖、蔗糖、乳糖所抑制,而被D-甘露糖、牛甲状腺球蛋白、鸡卵白蛋白、γ-球蛋抑制。该凝集素在pH4 0～10 2均有活性,而在pH6 5～9 1时活性较高。该凝集素在90℃加热1h,活力并未减弱,说明这种凝集素具有很强的耐热性。

Lectins are proteins or glycoproteins.They show specific in agglutination for many kinds of cells.They behave important regularization function and some other activation in organism,such as inhibiting the increment of tumor cell,activating lymphocyte and inhibiting the agglutination of blood platelet,and they are also concerned with the process of fetation and metabolization.Lectins from terrestrial plants and animals have been isolated,characterized and exploited extensively in many aspects of biochemistry and biomedicine.There has not been a comparable utilization of lectins from marine algae,mainly due to difficulties in their isolation and obtaining sufficient material for study.The real study of marine algal lectins began in the 1970s,and by the end of 1994,only 10 kinds of green marine algae and 13 kinds of red marine algae were purified.In addition,5 of the marine algae were partly purified,while only 2 of them were sold as purified commodity,relatively few algal lectins have been purified and characterized in detail.We screened out red marine algae Gloiopeltis furcata in the sea near to Dalian,which showed more potentiality than some other marine algae in hemagglutinating activity.There hadn't been any report about the isolation,purification and characterization of lectin from Gloiopeltis furcata(GFL)before.We purified it and examined part of its characterization in order to explore the method of purifying marine algae lectins for the purpose of development of marine medicine and the approach of prevention and cure of diseases in marine culture animals.GFL was purified by extraction with PBS,followed by 35%-85% ammonium sulfate fraction,dialysis against distilled water and a combination of ion-exchange chromatography on DEAE-52 cellulose and gel filtration on sephadexG-200.The columns of 1.6 cm×30 cm are suitable for both.One single band appeared on both PAGE and isoelectrofocusing electrophoretogram of the lectin.Its isoelectric point was 9.0,and its relative molecular weight was 8318 on sephadexG-200.GFL displayed some agglutination activity with Chlamydomonas,erythrocytes of rabbit,common carp and crucian carp,but not any with erythrocytes of human (A,B,AB,O).The hemaggluting activity followed the order as erythrocytes of rabbit (23)>erythrocytes of common carp (22)=erythrocytes of crucian carp (22)>Chlamydomonas (21).The agglutination for erythrocytes of rabbit was the highest and the activity could not be inhibited by D-fructose,D-galactose,glucose,sucrose,lactose,but could be inhibited by D-mannose,bovine-thyroglobulin,egg albumin and gamma-globulin,and the minimum inhibitory concentration were respectively 0.5mol/L by D-mannose,15.20mg/mL by bovine-thyroglobulin,1.56mg/mL by egg albumin and 0.45mg/mL by gamma-globulin.Like most lectins from marine algae,GFL was found to be active over a wide range of pH.The hemagglutinating activity of the lectin appeared at pH 4.0-10.2,and more active at pH 6.5-9.1.The hemagglutinating activity was not affected by heating for 60 min at 90℃,so it was shown that the lectin had higher heat resistance.