Lindahl U, Thunberg L, Bäckström G, Riesenfeld J, Nordling K, Björk I
J Biol Chem. 1984 Oct 25;259(20):12368-76.
Oligosaccharides with different affinities for antithrombin were isolated following partial deaminative cleavage of pig mucosal heparin with nitrous acid. The smallest high-affinity component obtained was previously identified as an octasaccharide with the predominant structure: (Formula: see text). The interaction of this octasaccharide, and of deca- and dodecasaccharides containing the same octasaccharide sequence, with antithrombin was studied by spectroscopic techniques. The near-ultraviolet difference spectra, circular dichroism spectra, and fluorescence enhancements induced by adding these oligosaccharides to antithrombin differed only slightly from the corresponding parameters measured in the presence of undegraded high-affinity heparin. Moreover, the binding constants obtained for the oligosaccharides and for high-affinity heparin were similar (1.0-2.9 X 10(7) M-1 at I = 0.3). In contrast, two hexasaccharides corresponding to units 1-6 and 3-8, respectively, of the above sequence showed about a 1000-fold lower affinity for antithrombin, and also induced considerably different spectral perturbations in antithrombin. Since the 1-6 hexasaccharide contains a reducing-terminal anhydromannose residue instead of the N-sulfated glucosamine unit 6 of the intact sequence, these results strongly support our previous conclusion that the N-sulfate group at position 6 is essential to the interaction with antithrombin. The low affinity of the hexasaccharide 3-8 provides further evidence that a pentasaccharide sequence 2-6 constitutes the actual antithrombin-binding region in the heparin molecule. Structural analysis of the various oligosaccharides revealed natural variants with an N-sulfate group substituted for the N-acetyl group at position 2. The preponderance of N-acetyl over N-sulfate groups at this position may be rationalized in terms of the mechanism of heparin biosynthesis, assuming that the D-gluco configuration of unit 3 is an essential feature of the antithrombin-binding region.
用亚硝酸对猪黏膜肝素进行部分脱氨基裂解后，分离出了对抗凝血酶具有不同亲和力的寡糖。所得到的最小的高亲和力组分先前已被鉴定为一种主要结构为（化学式：见原文）的八糖。通过光谱技术研究了这种八糖以及含有相同八糖序列的十糖和十二糖与抗凝血酶的相互作用。将这些寡糖添加到抗凝血酶中所诱导的近紫外差光谱、圆二色光谱和荧光增强，与在未降解的高亲和力肝素存在下测得的相应参数相比，仅有轻微差异。此外，寡糖和高亲和力肝素的结合常数相似（在离子强度I = 0.3时为1.0 - 2.9×10⁷ M⁻¹）。相比之下，分别对应于上述序列的1 - 6单元和3 - 8单元的两种六糖对抗凝血酶的亲和力低约1000倍，并且在抗凝血酶中也诱导出明显不同的光谱扰动。由于1 - 6六糖含有一个还原端脱水甘露糖残基，而不是完整序列中的N - 硫酸化葡糖胺单元6，这些结果有力地支持了我们先前的结论，即6位的N - 硫酸基团对于与抗凝血酶的相互作用至关重要。3 - 8六糖的低亲和力进一步证明了五糖序列2 - 6构成了肝素分子中实际的抗凝血酶结合区域。对各种寡糖的结构分析揭示了在2位存在N - 硫酸基团取代N - 乙酰基团的天然变体。假设单元3的D - 葡糖构型是抗凝血酶结合区域的一个基本特征，那么从肝素生物合成机制的角度来看，该位置N - 乙酰基团相对于N - 硫酸基团的优势可能是合理的。
