猪肺组织糖胺聚糖的分离纯化及其结构鉴定

目的:从猪肺组织(去除气管)中提取分离糖胺聚糖并确定其种类和结构特点。方法:采用胰蛋白酶和木瓜蛋白酶联合酶解提取糖胺聚糖,利用Q-Sepharose Fast Flow强阴离子交换柱层析进行分离纯化。采用醋酸纤维素薄膜电泳分析多糖的种类,高效凝胶渗透色谱与十八角激光散射仪联用法测定其绝对分子质量,采用SAX-HPLC法对酶解二糖进行细微结构分析。结果:从猪肺组织中获得透明质酸(HA)、硫酸乙酰肝素(HS)、硫酸软骨素B(CSB)和硫酸软骨素A(CSA)4种糖胺聚糖,其含量分别为690、490、480和370 mg·kg^-1,绝对分子质量分别为40.7、64.3、28.6和26.5 kDa。其中,CSA组分与标准品CSA(牛气管来源)相比,4-硫酸化二糖和6-硫酸化二糖的摩尔比更大,电荷密度更高;而HS中含有结构新颖的△己糖醛酸-氨基葡萄糖(IV-H,△ UA-GlcN)二糖,且其绝对分子质量(64.3 kDa)远高于标准品HS(17.3 kDa)。结论:猪肺组织中糖胺聚糖主要含有HA、HS、CSA和CSB,是糖胺聚糖开发的良好资源;其中HS含有高含量的自由氨基葡萄糖(GlcNH₂),结构较为新颖,有可能开发成为一种新的药物。

Objective: To extract and isolate glycosaminoglycans(GAGs) from tissues of porcine lung(removing lung trachea), and characterize the structural properties of purified GAGs.Methods: The tissues of porcine lung were firstly digested with trypsin and papain, and the sulfated polysaccharides were then isolated and purified by anion exchange chromatography(Q-Sepharose Fast Flow).The species of polysaccharides were identified by cellulose acetate membrane electrophoresis.Meanwhile, the absolute molecular mass of purified GAGs was determined by high performance gel permeation chromatography and laser scattering instrument.Following enzymatic digestion, the disaccharide compositions were deter mined by SAX-HPLC.Results: Four GAGs were obtained from tissues of porcine lung including hyaluronic acid(HA), heparan sulfate(HS), chondroitin sulfate B(CSB) and chondroitin sulfate A(CSA), and the contents of the corresponding fractions were 690, 490, 480, and 370 mg·kg^-1, respectively.The absolute molecular mass was 40.7, 64.3, 28.6, and 26.5 kDa, respectively. A bigger mole ratio of 4-sulfate disaccharide to 6-sulfate disaccharide and higher charge density were observed in our isolated CSA compare with the standard CSA(purified from bovine trachea);In addition, the isolated HS showed a special disaccharide unit(△UA-GlcN), and its absolute molecular mass(64.3 kDa) was higher than that of standard HS(17.3 kDa).Conclusion: HA, HS, CSA and CSB were obtained from tissues of porcine lung, which can be a good source for glycosaminoglycans exploitation.Specifically, a novel HS containing high content of GlcNH₂ was discovered, which could be developed as a new drug in future studies.