1. Coagulant thrombin-like enzymes from the venoms of Brazilian and Peruvian bushmaster (Lachesis muta muta) snakes.
- Author
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Magalhaes A, Ferreira RN, Richardson M, Gontijo S, Yarleque A, Magalhaes HP, Bloch C, and Sanchez EF
- Subjects
- Animals, Brazil, Cattle, Chromatography, Affinity, Chromatography, Gel, Cross Reactions, Crotalid Venoms immunology, Enzyme Inhibitors pharmacology, Fibrinogen chemistry, Fibrinogen metabolism, Humans, Immunohistochemistry, Isoenzymes chemistry, Isoenzymes metabolism, Kinetics, Metalloendopeptidases pharmacology, Peptide Fragments chemistry, Peptide Fragments metabolism, Peptide Mapping, Peru, Rabbits, Thrombin antagonists & inhibitors, Thrombin chemistry, Trypsin metabolism, alpha-Macroglobulins pharmacology, Crotalid Venoms enzymology, Thrombin isolation & purification, Thrombin metabolism, Viperidae
- Abstract
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 Bbeta-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 alpha2-macroglobulin (alpha2-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 alpha2-M were not cleaved by these enzymes, suggesting that alpha2-M inhibits TLEs by steric hindrance. Similarly, inhibitions of their clotting activities were obtained using high concentrations of rabbit IgG (40 microg, corresponding to molar ratio enzyme:inhibitor of 1:2) against TLE-B.
- Published
- 2003
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