Carlson T H, Babcock T, Atencio A C, Levinson C, Mora H R
Department of Pathology, School of Medicine, University of New Mexico, Albuquerque 87131.
J Biol Chem. 1988 Feb 15;263(5):2187-94.
Antithrombin III exists in plasma as major and minor isoforms differing in affinity for heparin. The nature of the binding of each purified isoform to immobilized heparins was investigated. Unfractionated, mixed-affinity heparin bound each isoform with both high affinity and concentration-dependent low affinity. The isoforms were resolved when filtered through low-affinity heparin (heparin repeatedly passed over immobilized antithrombin III) columns. Following chemical modification of a specific tryptophan residue required for heparin binding, each isoform failed to bind to either low-affinity or mixed-affinity heparin-agarose, but elution of the modified higher-affinity isoform was retarded on both gels. Because the modified lower-affinity isoform eluted with the similarly sized bovine serum albumin in these experiments, the difference in isoform affinity for heparin appears to be the result of a unique, secondary heparin-binding site in the higher-affinity isoform that can bind a heparin site with low affinity for antithrombin III. This interpretation was supported by the chromatographic behavior of the isoforms on mixed-affinity agarose during reverse gradient elution. Two other populations of each of the tryptophan-modified isoforms were identified. Since these isoforms bound tightly to mixed-affinity heparin-agarose but eluted at lower salt concentrations than the corresponding unmodified isoforms, both isoforms may contain additional secondary sites that interact weakly with heparin. A general model of heparin-antithrombin III interaction is proposed in which a high-affinity heparin site initially interacts with a primary site on antithrombin III. The subsequent conformational change leads to a cooperative, entropy-driven association between secondary sites on the protein and low-affinity sites on heparin, stabilizing antithrombin III in its activated form.
抗凝血酶III在血浆中以对肝素亲和力不同的主要和次要异构体形式存在。研究了每种纯化异构体与固定化肝素的结合性质。未分级的混合亲和力肝素以高亲和力和浓度依赖性低亲和力结合每种异构体。当通过低亲和力肝素（反复通过固定化抗凝血酶III的肝素）柱过滤时，异构体得以分离。在对肝素结合所需的特定色氨酸残基进行化学修饰后，每种异构体都无法与低亲和力或混合亲和力肝素琼脂糖结合，但在两种凝胶上，修饰后的高亲和力异构体的洗脱均延迟。因为在这些实验中，修饰后的低亲和力异构体与大小相似的牛血清白蛋白一起洗脱，所以异构体对肝素亲和力的差异似乎是由于高亲和力异构体中存在一个独特的二级肝素结合位点，该位点可以与对抗凝血酶III亲和力低的肝素位点结合。异构体在反向梯度洗脱过程中在混合亲和力琼脂糖上的色谱行为支持了这一解释。还鉴定出了每种色氨酸修饰异构体的另外两个群体。由于这些异构体与混合亲和力肝素琼脂糖紧密结合，但在比相应未修饰异构体更低的盐浓度下洗脱，所以这两种异构体可能都含有与肝素弱相互作用的额外二级位点。提出了一个肝素-抗凝血酶III相互作用的通用模型，其中高亲和力肝素位点首先与抗凝血酶III上的一个主要位点相互作用。随后的构象变化导致蛋白质上的二级位点与肝素上的低亲和力位点之间发生协同的、熵驱动的缔合，从而使抗凝血酶III稳定在其活化形式。
