COMPARATIVE STUDY OF COAGULANT THROMBIN-LIKE ENZYMES FROM THE VENOMS OF BRAZILIAN AND PERUVIAN BUSHMASTER (Lachesis muta muta) SNAKES

MagalhÃes, a.(1), Richardson, M.(1), Ferreira, R.N.(1), Gontijo, S.(1), Yarleque, A.(2), MagalhÃes, H.P.B.(3), Block, C.(4), Sanchez, E.F.(1)

(1)Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Brazil, (2)Departamento de Ciencias Biológicas, Universidad. Nacional Mayor de San Marcos, Perú, (3)Faculdade de Farmacia, Universidade Federal de Minas Gerais, Brazil, (4)Departamento de Bioquímica, Universidade de Brasilia, Brazil.

Objectives. The aim of this work was to conduct a comparative study of two thrombin-like enzymes (TLE-B and TLE-P) purified from the venoms of Lachesis muta muta  snakes captured in two different geographical localities, Manaus (Brazil) and Pucallpa (Perú). TLE-B and TLE-P showed Mr values of 40 and 41 kDa, under reducing conditions on SDS-PAGE, which were reduced to 27  kDa after treatment with PNGaseF. The proteinases split off fibrinopeptide A from human fibrinogen and fibrinopeptide B more slowly. In addition, the enzymes removed a 42-residue polypeptide from the NH₂-terminal end of the Bb-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. When injected into the tail veins of mice (15 – 120 ng/g mouse), both enzymes induced temporary episodes of opisthotonos and rapid rolling around the long axis of the animals. Kinetic properties of both 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 with the venoms of L. m. muta and Bothrops snakes. Incubation of human a2-macroglobulin with each enzyme at molar ratios of 1:1 and 1:2 enzyme:inhibitor, resulted in retarding their clotting activities by about 12 times, whereas their amidolytic activities were not affected. Similarly, inhibition of their clotting activities was obtained using high concentrations of rabbit IgG  (40 mg, corresponding to molar ratio  enzyme:inhibitor of 1:2). We conclude that TLE-B and TLE-P are nearly identical clotting enzymes.

Financial support: CNPq and FAPEMIG.

CORRESPONDENCE TO:

EladioOswaldo Flores Sanchez, Rua Furtado Nunes 110-101, Belo Horizonte, MG, CEP: 30730-090, Brasil, Email: eladio@funed.mg.gov.br