Rajesh Singh R, Chang Jui Yoa
Department of Biochemistry and Molecular Biology, The University of Texas, Houston, TX 77030, USA.
Biochim Biophys Acta. 2003 Sep 23;1651(1-2):85-92. doi: 10.1016/s1570-9639(03)00238-3.
Human alpha-thrombin is a very important plasma serine protease, which is involved in physiologically vital processes like hemostasis, thrombosis, and activation of platelets. Knowledge regarding the structural stability of alpha-thrombin is essential for understanding its biological regulation. Here, we investigated the structural and conformational stability of alpha-thrombin using the techniques of disulfide reduction and disulfide scrambling. alpha-Thrombin is composed of a light A-chain (36 residues) and a heavy B-chain (259 residues) linked covalently by an inter-chain disulfide bond (Cys(1)-Cys(122)). The B-chain is stabilized by three intra-chain disulfide bonds (Cys(42)-Cys(58), Cys(168)-Cys(182), and Cys(191)-Cys(220)) (Chymotrypsinogen nomenclature). Upon reduction with dithiothreitol (DTT), alpha-thrombin unfolded in a 'sequential' manner with sequential reduction of Cys(168)-Cys(182) within the B-chain followed by the inter-chain disulfide, generating two distinct partially reduced intermediates, I-1 and I-2, respectively. Conformational stability of alpha-thrombin was investigated by the technique of disulfide scrambling. alpha-Thrombin denatures by scrambling its native disulfide bonds in the presence of denaturant [urea, guanidine hydrochloride (GdmCl) or guanidine thiocyanate (GdmSCN)] and a thiol initiator. During the process, cleavage of the inter-chain disulfide bond and release of the A-chain from B-chain was the foremost event. The three disulfides in the B-chain subsequently scrambled to form three major isomers (designated as X-Ba, X-Bb, and X-Bc). Complete denaturation of alpha-thrombin was observed at low concentrations of denaturants (0.5 M GdmSCN, 1.5 M GdmCl, or 3 M urea) indicating low conformational stability of the protease.
人α-凝血酶是一种非常重要的血浆丝氨酸蛋白酶，它参与止血、血栓形成和血小板激活等生理重要过程。了解α-凝血酶的结构稳定性对于理解其生物学调节至关重要。在此，我们使用二硫键还原和二硫键重排技术研究了α-凝血酶的结构和构象稳定性。α-凝血酶由一条轻A链（36个残基）和一条重B链（259个残基）组成，它们通过链间二硫键（Cys(1)-Cys(122)）共价连接。B链由三个链内二硫键（Cys(42)-Cys(58)、Cys(168)-Cys(182)和Cys(191)-Cys(220)）稳定（胰凝乳蛋白酶原命名法）。用二硫苏糖醇（DTT）还原后，α-凝血酶以“顺序”方式展开，B链内的Cys(168)-Cys(182)先顺序还原，然后是链间二硫键，分别产生两个不同的部分还原中间体I-1和I-2。通过二硫键重排技术研究了α-凝血酶的构象稳定性。在变性剂[尿素、盐酸胍（GdmCl）或硫氰酸胍（GdmSCN）]和硫醇引发剂存在下，α-凝血酶通过重排其天然二硫键而变性。在此过程中，链间二硫键的断裂和A链从B链的释放是首要事件。B链中的三个二硫键随后重排形成三种主要异构体（分别命名为X-Ba、X-Bb和X-Bc）。在低浓度变性剂（0.5 M GdmSCN、1.5 M GdmCl或3 M尿素）下观察到α-凝血酶完全变性，表明该蛋白酶的构象稳定性较低。
