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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 k Da。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-Glc N)二糖,且其绝对分子质量(64.3 k Da)远高于标准品HS(17.3 k Da)。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(Glc NH2),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 from porcine lung tissue (remove the trachea) and to determine its type and structure. Methods: Glycosaminoglycan was extracted by enzymatic hydrolysis of trypsin and papain, and purified by Q-Sepharose Fast Flow anion exchange chromatography. The types of polysaccharides were analyzed by cellulose acetate film electrophoresis. The absolute molecular mass was determined by high performance gel permeation chromatography and octahedron laser light scattering. The SAX-HPLC method was used to analyze the structure of the disaccharide. Results: Four kinds of glycosaminoglycans, hyaluronic acid (HA), heparan sulfate (HS), chondroitin sulfate B (CSB) and chondroitin sulfate A (CSA) 490, 480 and 370 mg · kg-1, and the absolute molecular weights were 40.7, 64.3, 28.6 and 26.5 kDa, respectively. Among them, the molar ratio of 4-sulfated disaccharide to 6-sulfated disaccharide was higher and the charge density was higher in CSA compared with standard CSA (tracheal source), while HS contained a novel structure Uronic acid-glucosamine (IV-H, △ UA-Glc N) disaccharide, and its absolute molecular mass (64.3 k Da) is much higher than that of standard HS (17.3 kDa). CONCLUSIONS: The glycosaminoglycan in pig lung mainly contains HA, HS, CSA and CSB, which is a good resource for the development of glycosaminoglycan. Among them HS contains high content of free glucosamine (Glc NH2), the structure is relatively new, it is possible Developed into a new drug.