猪肝组织中硫酸乙酰肝素的分离纯化及二糖组成分析

Isolation,purification and disaccharide composition analysis of heparan sulfate from porcine liver

目的从少量猪肝组织中分离纯化硫酸乙酰肝素(HS)并进行二糖组成分析。方法分别采用胰蛋白酶及中性蛋白酶对猪肝组织进行酶解,再分别采用AG 50W-X2强阳离子交换色谱、Sephadex G-10凝胶过滤色谱、Q-Sepharose Fast Flow强阴离子交换色谱法纯化。将纯化的HS分别采用肝素酶Ⅰ和Ⅲ降解,所得到的二糖经2-氨基苯甲酰胺标记后,采用PA03色谱柱分析其二糖组成。结果经色谱和电泳分析,所得到的HS不含蛋白质及其它糖胺聚糖,其二糖组成以非硫酸化和低硫酸化二糖为主。结论该方法可以有效分离、纯化猪肝组织中的HS,并适用于少量组织中HS二糖组成分析。

Purpose To isolate and to purify heparan sulfate（HS）from a small amount of porcine liver tis- sue and to analyze its disaccharide composition. Methods The HS was digested with trypsin and neutrase, and then purified by chromatography with AG 50W-X2, Sephadex G-10 and Q-Sepharose Fast Flow columns, respectively. The purified HS was treated with heparinase Ⅰ and Ⅲ , the resulted disaccharides were labeled with 2-aminobenzamide （2-AB） and analyzed by HPLC on a PA03 column. Results The chromatography and electrophoresis data showed that the product was free of proteins and other glycosaminoglycans. Non-sulfated and low sulfated disaccharides were major components in porcine liver HS. Conclusion The method can be efficiently used to extract, purify and analyze disaccharide composition of HS from a small amount of liver tissue.