Biochemical AndEnzymatic Characterization Of A New Serine-Protease From Bothrops Jararacussu Snake Venom.

1Sant´Ana. C. D.; 4Marcussi, S.; 1Ticli, F. K.; 3Stábeli, R. G.; 1Mazzi, M. V.; 2Silveira L. B.; 1Da Silva, J. O.; 4Giglio, J. R.; 2Soares, A. M.; 1Sampaio, S. V.

1 Departamento de Análises Clínicas, Toxicológicas e Bromatológicas - FCFRP-USP, Ribeirão Preto, SP. 2 Unidade de Biotecnologia – UNAERP, Ribeirão Preto, SP. 3 Laboratório de Bioquímica – IPEPATRO, Porto Velho, RO. 4 Departamento de Bioquímica - FMRP-USP, Ribeirão Preto, SP, Brasil.

Snake venom proteins and peptides are known to activate or inactivate many interactions in the haemostatic system. Some of these venom proteins have potential for the treatment of diseases in humans. The great majority of naturally occurring substances which interfere in vitro or in vivo with blood clotting are derived from snake venoms, specially from the Viperidae family which contain abundant specific and non-specific proteases. This abstract describes the biochemical and enzymatic characterization of a new serine-protease (SERPROT-I) from B. jararacussu snake venom. Isolation of SERPROT-I was carried out in three steps: [i] gel filtration on Sephadex G-75, pH 8,1; [ii] affinity chromatography on Benzamidine-Sepharose, pH 7,4 to 3,2; [iii] ultrafiltration through a YM= 30,000 Amicon membrane, all steps being monitored for clotting activity and A280nm Clotting activity was determined in 200µL of human plasma using a dose-response curve. Minimum clotting activity (MCA), amount (µg) of enzyme able to clot plasma in 60 sec, was used to evaluate the stability of SERPROT-I under different conditions of pH (2,5 to 10), temperature (-70ºC to 100ºC), presence of divalent íons (Ca⁺⁺, Mn^{++, Fe++, Mg++, Zn++ and Ca++}) and inhibitors (EDTA; 1,10-phenantrolina; EGTA; B-mercaptoethanol; aprotinine; PMSF and leupetin). Proteolytic activity was assayed upon different substrates (bovine fibrinogen, caseína and BAPNA). The highly purified enzyme was confirmed by SDS-PAGE stained with Comassie Brilhant Blue and AgNO3, as well as by reverse phase HPLC. SERPROT-I is an acidic glycoprotein with Mr= 54,000, showing clotting activity upon the human plasma, its MCD being 1,5µg, in addition to its proteolytic activity upon fibrinogen, casein and BAPNA. The enzymatic activity was inhibited by PMSF and Leupetin. SERPROT-I is stable from pH 4,5 to 8,0, from –8ºC to 60ºC and in the presence of Co⁺⁺, Zn⁺⁺, Mn^{++ and Ca++}. Serine-proteases with fibrin(ogen)olytic activity are useful for treatment to lower fibrinogen levels or to solubilize coagulated plasma (thrombolysis).

Supported by: FAPESP, CNPq, CAPESand UNAERP

Correspondence to: cdalaqua@fcfrp.usp.br; suvilela@fcfrp.usp.br or andreimar@unaerp.br