Fischer A M, Tapon-Bretaudiere J, Bros A, Boisson C, Gulino D, Jozefonvicz J
Département d'Hématologie, C.H.U. Necker-Enfants Malades, Paris, France.
Thromb Res. 1988 Apr 15;50(2):317-24. doi: 10.1016/0049-3848(88)90232-0.
Antithrombin III (AT III) inhibits thrombin via an arginine-serine interaction. Insoluble polystyrene resins grafted with arginyl methyl ester have been synthesized, and their interaction with thrombin tested. One of these resins was selected for its high affinity for thrombin. In this paper we report the characteristics of this thrombin resin interaction. Using this substituted polystyrene resin as a support for affinity chromatography, we have compared the binding of thrombin with that of other proteins (prothrombin, Factor IXa, trypsin and AT III). It was found that 0.7 mg of highly purified human thrombin (2,100 U/mg) was bound to 1 g of resin. This could only be eluted at high ionic strength (1.5 M) and the amidolytic and clotting activities of the eluted thrombin remained unchanged. The binding of thrombin to the resin involves the active site of the enzyme but also other residues since, when DIP thrombin was used, the inactive enzyme could be eluted at lower ionic strength (1.0 M). This resin seems to be specific for thrombin because it does not bind the other serine-proteases (trypsin or Factor IXa), prothrombin (the inactive precursor of thrombin) or AT III. The arginyl residues of the resin are important for the specificity of the interaction with Factor IIa since prolyl residues are totally ineffective. Chromatography performed on such a resin is a very efficient method of purifying thrombin, and may be very useful for the removal of thrombin as a contaminant of plasma protein fractions.
抗凝血酶III（AT III）通过精氨酸 - 丝氨酸相互作用抑制凝血酶。已合成了接枝精氨酰甲酯的不溶性聚苯乙烯树脂，并测试了它们与凝血酶的相互作用。其中一种树脂因其对凝血酶具有高亲和力而被选中。在本文中，我们报告了这种凝血酶 - 树脂相互作用的特性。使用这种取代的聚苯乙烯树脂作为亲和色谱的支持物，我们比较了凝血酶与其他蛋白质（凝血酶原、因子IXa、胰蛋白酶和AT III）的结合情况。发现0.7毫克高度纯化的人凝血酶（2100 U/毫克）与1克树脂结合。这只能在高离子强度（1.5 M）下洗脱，并且洗脱的凝血酶的酰胺水解和凝血活性保持不变。凝血酶与树脂的结合涉及酶的活性位点，但也涉及其他残基，因为当使用二异丙基磷酸酯（DIP）凝血酶时，无活性的酶可以在较低离子强度（1.0 M）下洗脱。这种树脂似乎对凝血酶具有特异性，因为它不结合其他丝氨酸蛋白酶（胰蛋白酶或因子IXa）、凝血酶原（凝血酶的无活性前体）或AT III。树脂的精氨酰残基对于与因子IIa相互作用的特异性很重要，因为脯氨酰残基完全无效。在这种树脂上进行的色谱是纯化凝血酶的一种非常有效的方法，并且对于去除作为血浆蛋白组分污染物的凝血酶可能非常有用。
