Coagulant thrombin-like enzymes from the venoms of Brazilian and Peruvian bushmaster (Lachesis muta muta) snakes

Arinos Magalhaes, Rodrigo N. Ferreira, Michael Richardson, Silea Gontijo, Armando Yarleque, Henrique P.B. Magalhaes, Carlos Bloch, Eladio F. Sanchez

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29 Scopus citations

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 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. © 2003 Elsevier Science Inc. All rights reserved.
Original languageAmerican English
Pages (from-to)255-266
Number of pages12
JournalComparative Biochemistry and Physiology - B Biochemistry and Molecular Biology
DOIs
StatePublished - 1 Jan 2003

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