BINDING OF LOW-AFFINITY AND HIGH-AFFINITY HEPARIN TO ANTI-THROMBIN - COMPETITION FOR SAME BINDING-SITE ON PROTEIN
BINDING OF LOW-AFFINITY AND HIGH-AFFINITY HEPARIN TO ANTI-THROMBIN - COMPETITION FOR SAME BINDING-SITE ON PROTEIN
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DOI：
10.1111/j.1432-1033.1978.tb12568.x
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发表时间：
1978-01-01
期刊：
影响因子：
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通讯作者：
BJORK, I
中科院分区：
文献类型：
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作者：
DANIELSSON, A;BJORK, I
肝素制剂含有2种功能不同的多糖形式。两者都以1的化学计量比与抗凝血酶结合，但亲和力相差约1000倍。高亲和力部分几乎占据了原始制剂的所有抗凝活性，而低亲和力部分几乎没有活性。以前的研究无法确定这两种形式是否与蛋白质上的相同或不同位点结合。这里提出的实验旨在阐明这个问题。亲和层析实验表明，高亲和力肝素取代放射性标记的低亲和力肝素从基质连接的[牛]抗凝血酶，和125 I标记的抗凝血酶从基质连接的低亲和力肝素。还对低亲和力肝素从结合于微晶纤维素的抗凝血酶中置换放射性标记的高亲和力肝素的能力进行了定量分析。这些给出了一个值的结合常数的低亲和力肝素抗凝血酶接近的荧光测量所确定的反应物在溶液中。因此，实验表明，抗凝血酶上只有1个肝素结合位点，高亲和力和低亲和力肝素都与之结合。2种肝素形式的不同亲和力及其对抑制剂性质的不同影响必须完全归因于其结构的固有差异。
Heparin preparations contain 2 functionally distinct forms of the polysaccharide. Both bind to antithrombin with a stoichiometry of 1, but with affinities differing by about 1000-fold. The high-affinity fraction accounts for nearly all of the anticoagulant activity of the original preparation, while the low-affinity fraction is almost inactive. Previous investigations were not able to decide whether the 2 forms bind to the same or to different sites on the protein. The experiments presented here were designed to elucidate this problem. Affinity chromatography experiments showed that high-affinity heparin displaced radiolabeled low-affinity heparin from matrix-linked [bovine] antithrombin, and 125I-labeled antithrombin from matrix-linked low-affinity heparin. Quantitative analyses of the ability of low-affinity heparin to displace radiolabeled high-affinity heparin from antithrombin bound to microcrystalline cellulose were also made. These gave a value for the association constant for the binding of low-affinity heparin to antithrombin close to that determined by fluorescence measurements for the reactants in solution. The experiments thus suggest that there is only 1 heparin binding site on antithrombin, to which high-affinity and low-affinity heparin bind. The different affinities of the 2 heparin forms and their different effects on the properties of the inhibitor must be due solely to inherent differences in their structure.