Pavão Mauro S G
Laboratório de Bioquímica e Biologia Celular de Glicoconjugados, Programa de Glicobiologia, Instituto de Bioquímica Médica Leopoldo de Meis, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rua Professor Rodolpho Paulo Rocco, 255, 4° andar, sala 4A-08, Cidade Universitária, Ilha do Fundão, CEP 21941-913, Rio de Janeiro, Brazil,
Methods Mol Biol. 2015;1229:79-94. doi: 10.1007/978-1-4939-1714-3_9.
Sulfated polysaccharides with unique structures of the chondroitin/dermatan and heparin/heparan families of sulfated glycosaminoglycans have been described in several species of ascidians (Chordata-Tunicata). These unique sulfated glycans have been isolated from-ascidians and characterized by biochemical and spectroscopic methods. The ascidian glycans can be extracted by different tissues or cells by proteolytic digestion followed by cetylpyridinium chloride/ethanol precipitation. The total glycans are then fractionated by ion-exchange chromatography on DEAE-cellulose and/or Mono Q (HR 5/5) columns. Alternatively, precipitation with different ethanol concentrations can be employed. An initial analysis of the purified ascidian glycans is carried out by agarose gel electrophoresis on diaminopropane/acetate buffer, before or after digestion with specific glycosaminoglycan lyases or deaminative cleavage with nitrous acid. The disaccharides formed by exhaustive degradation of the glycans is purified by gel-filtration chromatography on a Superdex-peptide column and analyzed by HPLC on a strong ion exchange Sax-Spherisorb column. 1H or 13C nuclear magnetic resonance spectroscopy in one or two dimensions is used to confirm the structure of the intact glycans.
在几种海鞘（脊索动物-被囊动物）中发现了具有独特结构的硫酸化多糖，这些多糖属于硫酸化糖胺聚糖的软骨素/皮肤素和肝素/乙酰肝素家族。这些独特的硫酸化聚糖已从海鞘中分离出来，并通过生化和光谱方法进行了表征。海鞘聚糖可以通过蛋白水解消化，然后用十六烷基吡啶氯化物/乙醇沉淀，从不同的组织或细胞中提取。然后，通过在DEAE-纤维素和/或Mono Q（HR 5/5）柱上进行离子交换色谱法对总聚糖进行分级分离。或者，也可以采用不同乙醇浓度的沉淀法。在用特定的糖胺聚糖裂解酶消化或用亚硝酸进行脱氨基裂解之前或之后，通过在二氨基丙烷/醋酸盐缓冲液中进行琼脂糖凝胶电泳对纯化的海鞘聚糖进行初步分析。通过在Superdex-肽柱上进行凝胶过滤色谱法纯化聚糖彻底降解形成的二糖，并在强离子交换Sax-Spherisorb柱上通过HPLC进行分析。使用一维或二维的1H或13C核磁共振光谱来确认完整聚糖的结构。
