鳙鳃凝集素的分离纯化及理化性质的研究

Purification and Characterization of Lectin from Gill of Bighead Carp(Aristichthys nobilis)

利用DEAE-SepharoseFF离子交换和SephacrylS-200HR及Superdex20010／300GL分子筛层析技术，从鳙鳃组织中分离纯化到一种岩藻糖专一的凝集素，命名为GANL。在还原SDS-PAGE电泳上显示单一蛋白染色带，其亚基相对分子质量为37kD。经Superdex200凝胶过滤层析测得其天然相对分子质量为220kD。GANL的中性糖含量为13．4％。因此，GANL作为一种由相同亚基组成多亚基糖蛋白。GANL对兔红细胞有专一的凝集活性，其凝集活性不依赖Ca2＋。在被测的单糖、双糖及糖蛋白中，仅岩藻糖能抑制其凝集活性。氨基酸分析表明GANL的Asp、Glu、Leu、Val、Lys的含量较高，Cys—S的含量为0．81％。GANL的最适pH为8～9；具有很高的热稳定性，最适温度为50℃。

A fucose-binding lectin from gill of bighead carp（Aristichthys nobilis）was purified and characterized. The purification procedure consisted of extracting soluble proteins in 25 mmol/L Tris-HCl buffer （pH8.5）, separating on DEAE-Sepharose FF ion exchange columns followed by gel filtration chromatography on Sephacryl S-200 HR and Superdex 200 10/300 GL. The purified lectin designated GANL showed a single protein band with an apparent molecular mass of 37 kD when submitted to SDS-polyacrylamide gel electrophoresis under reducing conditions. The native molecular mass of GANL determined by gel filtration on a Superdex 200 column was 220 kD and its carbohydrate content was estimated to be 13. 4%. Therefore, GANL is a homomuhimeric glycoprotein. The purified lectin only agglutinated native erythrocytes from rabbit without the addition of Ca2＋. Its activity was not inhibited by any of the mono, disaccharities and glycoprotein tested except for fucose. Amino acid composition analysis revealed that the lectin contained high contents of Asp, Glu, Leu, Val, and Lys. GANL was stability with broad pH（5~11） and the optimum pH was 8.0-9.0. The lectin is thermostable with a temperature optima of 50 ℃ .