Hileman R E, Smith A E, Toida T, Linhardt R J
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City 52242, USA.
Glycobiology. 1997 Mar;7(2):231-9. doi: 10.1093/glycob/7.2.231.
Porcine intestinal mucosal heparan sulfate was exhaustively depolymerized on a large scale using heparin lyase II (heparinase II) or heparin lyase III (heparitinase, EC 4.2.2.8). The oligosaccharide mixtures formed with each enzyme were fractionated by low pressure gel permeation chromatography. Size-uniform mixtures of disaccharides, tetrasaccharides, and hexasaccharides were obtained. Each size-fractionated mixture was then purified on the basis of charge by repetitive semipreparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 13 homogenous oligosaccharides. The purity of each oligosaccharide was demonstrated by the presence of a single peak on analytical strong-anion-exchange high-performance liquid chromatography and reversed polarity capillary electrophoresis. The structures of these oligosaccharides were established using 500 MHz one- and two-dimensional nuclear magnetic resonance spectroscopy. Three of the thirteen structures that were solved were novel while the remaining 10 have been previously described. All of the structures obtained using heparin lyase III contained a delta UAp residue (where delta UAp is 4-deoxy-alpha-L-threo-hex-4-eno-pyranosyluronic acid) at their nonreducing termini. Structures obtained using heparin lyase II contained both delta UAp and delta UAp2S (where S is sulfate) at their nonreducing termini. These results are consistent with the reported specificity of both enzymes.
使用肝素酶II（肝素酶II）或肝素酶III（类肝素酶，EC 4.2.2.8）对猪肠粘膜硫酸乙酰肝素进行大规模彻底解聚。用低压凝胶渗透色谱法对每种酶形成的寡糖混合物进行分级分离。获得了二糖、四糖和六糖的大小均匀的混合物。然后通过重复的半制备强阴离子交换高效液相色谱法，根据电荷对每个大小分级的混合物进行纯化。这种方法已导致分离出13种均一寡糖。通过分析型强阴离子交换高效液相色谱法和反相毛细管电泳上出现的单峰证明了每种寡糖的纯度。使用500 MHz的一维和二维核磁共振光谱确定了这些寡糖的结构。所解析的13种结构中有3种是新的，其余10种先前已有描述。使用肝素酶III获得的所有结构在其非还原末端都含有一个δUAp残基（其中δUAp是4-脱氧-α-L-苏-己-4-烯-吡喃糖醛酸）。使用肝素酶II获得的结构在其非还原末端同时含有δUAp和δUAp2S（其中S是硫酸根）。这些结果与两种酶报道的特异性一致。
