Nienaber V L, Berliner L J
Department of Chemistry, Ohio State University, Columbus 43210.
Thromb Haemost. 1991 Jan 23;65(1):40-5.
The primary structures of bovine and human alpha-thrombins are highly homologous yet their x-ray structures are not yet complete enough to distinguish differences. In order to probe and compare their dynamic conformations in solution, we examined bovine and human alpha-thrombins with a series of active site directed fluorosulfonylphenyl spin labeled inhibitors and fluorophores which probe a region within 10-15 A of the catalytic serine residue. Overall, the nitroxide moieties were more immobilized in the bovine vs human derivatives reflecting either more apolar binding regions or steric obstructions to the motion of the nitroxide in bovine thrombin. Most of the labels which distinguish indole (apolar ligand) binding in human thrombin were found to display similar interactions in bovine thrombin, although slight differences in the general topography of this region were suggested. The two active site directed fluorophores, dansyl fluoride and p-nitrophenyl anthranilate showed differences in both lambda emmax and lambda exmax of the complexes with bovine and human-alpha-thrombin, respectively, Several of the effects observed i.e., ligand binding (indole or benzamidine) and the subtle hydrophobic interactions between the nitroxide moiety and the protein active site would be difficult to assess from an x-ray structure determination alone.
牛α-凝血酶和人α-凝血酶的一级结构高度同源，但其X射线结构尚不够完整，无法区分差异。为了探测和比较它们在溶液中的动态构象，我们用一系列活性位点导向的氟磺酰苯基自旋标记抑制剂和荧光团检测了牛α-凝血酶和人α-凝血酶，这些荧光团可探测催化丝氨酸残基10 - 15埃范围内的区域。总体而言，与人类衍生物相比，牛衍生物中的氮氧化物部分固定得更多，这反映出牛凝血酶中存在更多非极性结合区域或对氮氧化物运动的空间阻碍。发现大多数区分人凝血酶中吲哚（非极性配体）结合的标记在牛凝血酶中表现出相似的相互作用，尽管该区域的总体形貌存在细微差异。两种活性位点导向的荧光团，丹磺酰氟和对硝基苯基邻氨基苯甲酸分别与牛α-凝血酶和人α-凝血酶形成的复合物在最大发射波长（λemmax）和最大激发波长（λexmax）上均存在差异。仅通过X射线结构测定很难评估所观察到的几种效应，即配体结合（吲哚或苯甲脒）以及氮氧化物部分与蛋白质活性位点之间的微妙疏水相互作用。
