ISOLATION AND CHARACTERIZATION OF A NEW L-AMINO ACID OXIDASE FROM Crotalus durissus cascavella VENOM

TOYAMA, M.H.(1), OLIVEIRA, D.G.(1), SANTOS, L.M.B.(2), PEREIRA, R.(3), ANTUNES, E.(3), MONTEIRO, H.S.A.(4), MARTINS, A.M.C.(4), BERIAM, L.O.S.(5), NOVELLO, J.C.(1) MARANGONI, S.(1)

(1)Departamento de Bioquímica, (2)Departamento de Imunologia e Microbiologia, Instituto de Biologia, (3)Departamento de Farmacologia, Faculdade de Ciências Médicas, UNICAMP; (4)Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará. (5)Laboratório de Bacteriologia Vegetal, Centro Experimental do Instituto Biológico de Campinas, São Paulo, S.P., Brasil.

Objectives: We purified a novel L-amino acid oxidase (LAO) from Crotalus durissus cascavella snake venom and molecular, platelet aggregating, bactericidal and cytotoxic characterization was made.

Method and Results: This novel LAO was purified by two chromatographic steps: molecular mass and SP 5PW HPLC.  The purified enzyme (CDCE-LAO) is present as a dimer on gel filtration, with an apparent M(r) of 60 kDa for the monomer as estimated by Tricine SDS-PAGE.  CDCE-LAO showed high enzymatic specificity against L-leucine (56U/mg) and induce platelet aggregation of whole and washed platelets using doses of 5,3 and 1µg/ ml.  This novel protein inhibited around 85% and 35% of gram-positive and gram-negative bacterial growth rate, respectively and induced a significant decrease of viable B-cell number after stimulation with Con A in vitro.  This novel protein shows high content of acidic amino acids and its n-terminal  amino acid sequence was determined to be ADDRNPLAECFQENDYEEFLEIARNGLKATSNPKHVVIVGA

Conclusion: This novel LAO represents around 7.5% of whole Crotalus durissus cascavella venom.  In the three other samples of Crotalus durissus cascavella venom this LAO represent around 2.3 - 3.5% of whole venom.  This novel protein showed significant bactericidal, platelet and haemagglutination activities and this suggests that this protein plays an important role in of blood homeostasis.

CORRESPONDENCE TO:

Daniela Garcia de Oliveira, Rua Antônio Jose Silva Martelinho 470 Apto. 12, Campinas, SP, CEP: 13031-580, Brasil, Email: gaveira@yahoo.com.br