Two isoforms of a thrombin-like enzyme designated TLE-B and TLE-P were purified from the venoms of Lachesis muta muta (bushmaster) snakes captured in two different geographical localities, Manaus (Brazil) and Pucallpa (Perú). TLE-B and TLE-P showed Mr values of 44000 and 43000 under reducing conditions on SDS-PAGE, which decreased to 27000 after deglycosylation with N-glycosidase F (PNGase F). The purified proteinases split off fibrinopeptide A rapidly from human fibrinogen and fibrinopeptide B more slowly. In addition, both enzymes released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bβ-chain. Their specific clotting activities were equivalent to 1000 and 900 NIH thrombin units/mg on human fibrinogen and 526 and 606 NIH thrombin units/mg on bovine fibrinogen for TLE-B and TLE-P, respectively. Kinetic properties of these enzymes were determined using representative chromogenic substrates. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Purified rabbit IgG against TLE-B reacted with both enzymes forming a continuous precipitin line on immunodiffusion. Furthermore, Western blot and indirect ELISA were used to compare the antigenic cross-reactivity for both enzymes as well as the venoms of L. muta muta and Bothrops snakes. Incubation of human α2-macroglobulin (α2-M) with each enzyme at molar ratios of 1:1, 1: 2 and 1:4 enzyme:inhibitor resulted in retarding their clotting activities by approximately 12 times, whereas their amidolytic activities were not affected. However, the Mr 180000 subunits of α2-M were not cleaved by these enzymes, suggesting that α2-M inhibits TLEs by steric hindrance. Similarly, inhibitions of their clotting activities were obtained using high concentrations of rabbit IgG (40 μg, corresponding to molar ratio enzyme:inhibitor of 1:2) against TLE-B.
|Number of pages||12|
|Journal||Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology|
|State||Published - Oct 2003|
Bibliographical noteFunding Information:
This work was supported by the Brazilian agencies, Conselho Nacional de Desenvolvimento Cientı́co e Tecnológico (CNPq) and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), by grants CBB 2359/97 and EDT-24.000/01.
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- Clotting enzymes
- Snake venoms