Thunberg L, Bäckström G, Lindahl U
Carbohydr Res. 1982 Mar 1;100:393-410. doi: 10.1016/s0008-6215(00)81050-2.
An octasaccharide with high affinity for antithrombin, isolated after partial deaminative cleavage of heparin and previously found to have the following predominant structure (see formula in text) has been studied further. High-voltage, paper electrophoresis of the 3H-labelled disaccharides obtained by deamination with HNO2 (pH 1.5) followed by reduction with Na[3H]BH4 showed approximately 25% of mono-O-sulfated components, in addition to L-iduronic acid(2-O-SO3)-2,5-anhydro-D-[3H]mannitol (6-O-SO3). The monosulfated disaccharides were identified by high pressure, ion-exchange chromatography as L-iduronic acid(2-O-SO3)-2,5-anhydro-D-[3H]mannitol, L-Iduronic acid-2,5-anhydro-D-[3H]mannitol(6-O-SO3). and D-glucuronic acid-2,5-anhydro-D-[3H]-mannitol L, iduronic acid 2,5-anhydro-D-[3H]mannitol(6-O-SO3), and D-glucuronic acid-2,5-anhydro-D-[3H]-mannitol. These components originated from the reducing, terminal disaccharide residue (units 7 and 8), as indicated by selective labelling with Na[3H]-BH4. The structural variability within this region suggests that it is not part of the antithrombin-binding sequence. Neither enzymic removal of the non-sulfated L-iduronic acid unit 1 nor N-deacetylation (by hydrazinolysis) at unit 2 had any significant effect on the affinity of the octasaccharide for antithrombin. However, removal of the disaccharide corresponding to units 1 and 2, by selective deamination of the N-deacetylated octasaccharide, yielded a low-affinity hexasaccharide. In addition, a high-affinity deamination product was formed, presumably an octasaccharide containing a 6-sulfated 2-deoxy-2-C-formyl-D-pentofuranosyl unit due to ring contraction in unit 2. These results suggest that the 6-sulfate group in unit 2 may be involved in antithrombin binding. It is concluded that the antithrombin binding site in heparin is represented by the pentasaccharide sequence extending from unit 2 to unit 6 of the octasaccharide studied.
对一种对抗凝血酶具有高亲和力的八糖进行了进一步研究。该八糖是在肝素部分脱氨基裂解后分离得到的，之前发现其具有以下主要结构（见文中分子式）。用亚硝酸（pH 1.5）脱氨基，随后用硼氢化钠[³H]还原得到的³H标记二糖进行高压纸电泳，结果显示除了L-艾杜糖醛酸（2-O-硫酸酯）-2,5-脱水-D-[³H]甘露糖醇（6-O-硫酸酯）外，约25%为单-O-硫酸化成分。通过高压离子交换色谱法鉴定出单硫酸化二糖为L-艾杜糖醛酸（2-O-硫酸酯）-2,5-脱水-D-[³H]甘露糖醇、L-艾杜糖醛酸-2,5-脱水-D-[³H]甘露糖醇（6-O-硫酸酯）、D-葡萄糖醛酸-2,5-脱水-D-[³H]甘露糖醇、L-艾杜糖醛酸2,5-脱水-D-[³H]甘露糖醇（6-O-硫酸酯）以及D-葡萄糖醛酸-2,5-脱水-D-[³H]甘露糖醇。如用硼氢化钠[³H]选择性标记所示，这些成分源自还原端二糖残基（单元7和8）。该区域内的结构变异性表明它不是抗凝血酶结合序列的一部分。无论是酶法去除非硫酸化的L-艾杜糖醛酸单元1，还是对单元2进行N-脱乙酰化（通过肼解），对八糖与抗凝血酶的亲和力均无显著影响。然而，通过对N-脱乙酰化八糖进行选择性脱氨基，去除对应于单元1和2的二糖，得到了一种低亲和力的六糖。此外，还形成了一种高亲和力的脱氨基产物，推测是一种八糖，由于单元2中的环收缩，其含有一个6-硫酸化的2-脱氧-2-C-甲酰基-D-戊呋喃糖基单元。这些结果表明单元2中的6-硫酸基团可能参与抗凝血酶结合。得出的结论是，肝素中的抗凝血酶结合位点由所研究的八糖中从单元2延伸至单元6的五糖序列表示。
