BINDING OF LOW-AFFINITY AND HIGH-AFFINITY HEPARIN TO ANTI-THROMBIN - ULTRAVIOLET DIFFERENCE SPECTROSCOPY AND CIRCULAR-DICHROISM STUDIES
BINDING OF LOW-AFFINITY AND HIGH-AFFINITY HEPARIN TO ANTI-THROMBIN - ULTRAVIOLET DIFFERENCE SPECTROSCOPY AND CIRCULAR-DICHROISM STUDIES
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DOI：
10.1021/bi00609a026
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发表时间：
1978-01-01
期刊：
影响因子：
2.9
通讯作者：
BJORK, I
中科院分区：
文献类型：
--
作者：
NORDENMAN, B;BJORK, I
肝素通过与血浆蛋白酶抑制剂抗凝血酶结合而作为抗凝剂，从而增加抑制剂使许多凝血丝氨酸蛋白酶失活的速率。两种形式的肝素对基质连接的[牛和人]抗凝血酶的亲和力不同，可通过亲和色谱法分离。高亲和性肝素级分具有高抗凝活性，而低亲和性级分几乎没有这种活性。这2个馏分的结合，以抗凝血酶在溶液中的紫外差光谱和圆二色性[CD]，其特征在于。肝素组分均不影响抗凝血酶的远紫外CD光谱，表明肝素结合不会导致蛋白质二级结构的显著变化。这两个馏分改变了近紫外吸收和CD光谱的抗凝血酶，这表明局部扰动的环境中的一些芳香族氨基酸的蛋白质肝素结合。高活性，高亲和力肝素馏分造成更大的光谱变化比相对不活跃，低亲和力馏分。这与高亲和力组分诱导抗凝血酶的构象变化的概念是相容的，该构象变化与抑制剂的活化有关。定量研究表明，2肝素馏分结合抗凝血酶的摩尔比为1：1，可能在蛋白质上的相同的网站，但他们的亲和力差异很大。在生理离子强度下，高亲合性部分的结合常数为107 M-1，而低亲合性部分的结合常数约为5 × 107 M-1。104 M-1
Heparin acts as an anticoagulant by binding to the plasma protease inhibitor antithrombin, thereby increasing the rate at which the inhibitor inactivates a number of coagulation serine proteases. Two forms of heparin, differing in their affinity for matrix-linked [bovine and human] antithrombin, may be separated by affinity chromatography. The high-affinity heparin fraction has a high anticoagulant activity, while the low-affinity fraction is virtually devoid of such activity. The binding of these 2 fractions to antithrombin in solution was characterized by UV difference spectroscopy and circular dichroism [CD]. Neither heparin fraction affected the far-UV CD spectrum of antithrombin, indicating that heparin binding does not lead to significant changes of the secondary structure of the protein. Both fractions changed the near-UV absorption and CD spectra of antithrombin, suggesting local perturbations of the environment of some aromatic amino acids of the protein on heparin binding. The highly active, high-affinity heparin fraction caused much larger spectral changes than the relatively inactive, low-affinity fraction. This is compatible with the concept that the high-affinity fraction induces a conformational change of antithrombin, related to the activation of the inhibitor. Quantitative studies showed the 2 heparin fraction to bind to antithrombin in a molar ratio of 1:1, possibly to the same site on the protein, but their affinities differed widely. The binding constant of the high-affinity fraction at physiological ionic strength was of the order of 107 M-1, while that of the low-affinity fraction was about 5 .times. 104 M-1.