Poster 154 - Purification and characterization of a coagulant thrombin-like enzyme from the venom

Poster 154. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.513.

PURIFICATION AND CHARACTERIZATION OF A COAGULANT THROMBIN-LIKE ENZYME FROM THE VENOM OF BOTHROPS LEUCURUS

Magalhães, A¹; Richardson, M¹; Magalhães, H.P.B²; Gontijo,S.S¹; Bello, C. A¹; Hermógenes, . L. N¹; Ferreira, R. N¹.; Sanchez, E. F¹.

1 Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, MG 30510-010, Brasil; 2 Faculdade de Farmácia, Universidade Federal de Minas Gerais, Brasil

A coagulant trombin-like enzyme (leucuroxobin) was purified from Bothropsleucurus snake venom by a combination of gel filtration on Sephacryl S-200 affinity chromatography on Sepharose-agmatin and ion-exchange chromatography on DEAE-Sepharose CL-6B with an overall yield of 37% and a purification factor of 11.As revealed by SDS-PAGE under reducing conditions, the enzyme is a monomeric glycoprotein with a Mr of 34 kDa, which decreased to 27 kDa after deglycosilation with N-glycosidase F (PNGase F).Approximately 98% of the protein sequence was determinated by sequencing the various fragments derived from digestion with endoproteases. The proteinase shares 100% of sequence identity with batroxobin from Bothropsatrox venom, only minor difference was found in the amino acid sequence at position 223, where approximately 50% of glutamine was found in addition to arginin (leucuroxobin). The purified proteinase split off fibrinopeptide A rapidly from human fibrinogen and fibrinopeptide B very slowly. In addition, it released the N-terminal peptide (Mr:4572) containing the first 42 residues from the Bb-chain. The proteinase does not activate factor XIII. The specific clotting activity was 198 NIH thrombin units/mg on human fibrinogen. The amidase activity of the enzyme towards Bz-L-Arg-pNA was inhibited by PMSF and competitively inhibited by benzamidine with Ki=2.41±0.207 x 10^-3 M. Kinetic properties of the enzyme were determined on representative synthetic chromogenic substrates for serine proteinases (glandular and plasmatic kallikrein, thrombin and plasmin). The enzyme showed higher catalytic efficience, measured by the kcat/Km relation for glandular kallikrein compared with that for thrombin or plasmin.