Gettins P, Wooten E W
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Biochemistry. 1987 Jul 14;26(14):4403-8. doi: 10.1021/bi00388a032.
The denaturation of human and bovine antithrombin III by guanidine hydrochloride has been followed by 1H NMR spectroscopy. The same unfolding transition seen previously from circular dichroism studies [Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048-14053] at low denaturant concentration was detected here by discontinuous changes in the chemical shifts of the C(2) protons of two of the five histidines in human antithrombin III and of three of the six histidines in bovine antithrombin III. These two histidines in human antithrombin III are assigned to residue 1 and, more tentatively, to residue 65. Two of the three histidines similarly affected in the bovine protein appear to be homologous to residues in the human protein. This supports the proposal of similar structures for the two proteins. In the presence of heparin, the discontinuous titration behavior of these histidine resonances is shifted to higher denaturant concentration, reflecting the stabilization of the easily unfolded first domain of the protein by bound heparin. From the tentative assignment of one of these resonances to histidine-1, it is proposed that the heparin binding site of antithrombin III is located in the N-terminal region and that this region forms a separate domain from the rest of the protein. The pattern of disulfide linkages is such that this domain may well extend from residue 1 to at least residue 128. Thermal denaturation also leads to major perturbation of these two histidine resonances in human antithrombin III, though stable intermediates in the unfolding were not detected.
利用核磁共振氢谱对盐酸胍导致的人及牛抗凝血酶III的变性过程进行了跟踪研究。之前通过圆二色性研究[Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048 - 14053]在低变性剂浓度下观察到的相同的去折叠转变，在本文中通过人抗凝血酶III五个组氨酸中两个组氨酸的C(2)质子以及牛抗凝血酶III六个组氨酸中三个组氨酸的化学位移的不连续变化得以检测。人抗凝血酶III中的这两个组氨酸被确定为第1位残基，较不确定的是第65位残基。牛蛋白中同样受到影响的三个组氨酸中的两个似乎与人蛋白中的残基同源。这支持了两种蛋白质结构相似的推测。在肝素存在的情况下，这些组氨酸共振的不连续滴定行为向更高的变性剂浓度偏移，这反映了结合的肝素对该蛋白易于去折叠的第一个结构域的稳定作用。根据其中一个共振峰初步确定为组氨酸 - 1，推测抗凝血酶III的肝素结合位点位于N端区域，并且该区域与蛋白质的其余部分形成一个单独的结构域。二硫键连接模式表明该结构域很可能从第1位残基延伸至至少第128位残基。热变性也导致人抗凝血酶III中这两个组氨酸共振峰发生重大扰动，不过在去折叠过程中未检测到稳定的中间体。
