Structural stability of human alpha-thrombin studied by disulfide reduction and scrambling.
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.
D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D019791:Guanidine / Q000737:chemistry; D006490:Hemostatics / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008623:Mercaptoethanol / Q000737:chemistry; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
D004220:Disulfides; D006490:Hemostatics; D007527:Isoenzymes; D008623:Mercaptoethanol; D013917:Thrombin; D019791:Guanidine