Atha D H, Lormeau J C, Petitou M, Rosenberg R D, Choay J
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1987 Oct 6;26(20):6454-61. doi: 10.1021/bi00394a024.
The role of 3-O- and 6-O-sulfated glucosamine residues within the heparin octasaccharide critical for biological activity, iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, was determined by comparing its ability to bind antithrombin, induce a conformational change in this protease inhibitor as monitored by the enhancement of intrinsic fluorescence, and accelerate (at saturation) the interaction of this protein with human factor Xa. The octasaccharide produced a maximum 48% increase in intrinsic fluorescence at 37 degrees C and a rate of factor Xa inhibition of 6 X 10(5) M-1 s-1 as measured by stopped-flow fluorometry at 25 degrees C. The basal rate of the antithrombin-factor Xa interaction observed in the absence of oligosaccharide was 2 X 10(3) M-1 s-1. The synthetic pentasaccharide, consisting of residues 2-6, produced fluorescence enhancement and rate of inhibition equal to those of the octasaccharide. However, a similar pentasaccharide, identical in all respects except that it lacked the 3-O-sulfate on residue 4, produced less than a 5% fluorescence enhancement and a rate of factor Xa inhibition of 8 X 10(3) M-1 s-1. The tetrasaccharide consisting of residues 2-5 produced a 35% fluorescence enhancement and a rate of factor Xa inhibition of 3 X 10(5) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)
肝素八糖中对生物活性至关重要的3 - O - 和6 - O - 硫酸化葡糖胺残基，即艾杜糖醛酸----N - 乙酰葡糖胺6 - O - 硫酸酯----葡糖醛酸----N - 硫酸化葡糖胺3,6 - 二 - O - 硫酸酯----艾杜糖醛酸2 - O - 硫酸酯----N - 硫酸化葡糖胺6 - O - 硫酸酯----艾杜糖醛酸2 - O - 硫酸酯----脱水甘露糖醇6 - O - 硫酸酯的作用，是通过比较其结合抗凝血酶的能力、诱导该蛋白酶抑制剂构象变化（通过内在荧光增强监测）以及加速（在饱和时）该蛋白与人因子Xa的相互作用来确定的。八糖在37℃时使内在荧光最大增加48%，在25℃通过停流荧光法测量，其对因子Xa的抑制速率为6×10⁵M⁻¹s⁻¹。在无寡糖时观察到的抗凝血酶 - 因子Xa相互作用的基础速率为2×10³M⁻¹s⁻¹。由2 - 6位残基组成的合成五糖产生的荧光增强和抑制速率与八糖相同。然而，一种类似的五糖，除了4位残基上缺少3 - O - 硫酸酯外其他方面都相同，其荧光增强小于5%，对因子Xa的抑制速率为8×10³M⁻¹s⁻¹。由2 - 5位残基组成的四糖产生35%的荧光增强，对因子Xa的抑制速率为3×10⁵M⁻¹s⁻¹。（摘要截短于250字）
