Poster 36 - Purification, specificity and three-dimensional structure model of the giroxin (LMR-47)

Poster 36. 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.395.

Purification, Specificity and Three-dimensional Structure Model of the Giroxin (LMR-47) Isolated from Lachesis muta rhombeata venom

Silva, Jr F.P¹;Guedes, H.L.M.¹; Correa-Netto, C.²; Aguiar, A.²; Gimenez, A.R²; De-Oliveira, C.L.³; Torreani, I.L.³and De-Simone, SG^1,4

1 Departamento de Bioquímica e Biologia Molecular, IOC, FIOCRUZ.; 2 Instituto Vital Brazil, Niterói,RJ ; 3 Laboratório Nacional de Luz Síncrotron,Campinas, SP; 4 Departamento de Biologia Celular e Molecular, Instituto de Biologia, UFF, Niterói, RJ, Brazil

Serine peptidases from some Viperidae venoms are able to convert fibrinogen into fibrin by releasing fibrinopeptides A and/or B. Nevertheless, in most cases, these fibrin monomers are unable to be cross-linked by Factor XIII, resulting in aberrant clots that are independent of plasmin activity for dissolution. The in vivo significance of this activity is dependent upon the plasmatic concentration of the enzyme but in common envenomations it usually causes an anticoagulant effect. Although thrombin has many activities these proteinases have been termed thrombin-like enzymes (TLE) since they mimic the fibrinogen clotting, a central process in mammalians. Thus, in view of the potential biotechnological and clinical application of SVTLEs, we have performed structural studies of the LMR-47.

Lachesismuta rhombeata giroxin (LMR-47) was purified using two chromatography steps (affinity and HPLC gel filtration) and after concentration analyzed by mass spectrometry (MALDI-TOF), 1D and 2D-gel electrophoresis, circular dichroism and SAXS (small-angle X-ray scattering). LMR-47 is homologous to the mammalian serine proteases trypsin (40 % identity) and thrombin (30% identity), is inhibited by several trypsin-like inhibitors and contains 22% of carbohydrate. The Kratly plot indicated that the protein possess flexible chains which is in accordance with the Fourier function and dichroism analysis. After ten sets of SAXS measurements a model was generated using the GASBOR program. The LMR-47, present flexile domains and a refined abinitio model of the three-dimensional structure was obtained (data were collected at 4 mg/mL, and 10°C).