Poster 111 - Purification and characterization of the biological and enzymatic activity of Bjussu-LAAO,

Poster 111. 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.470.

Purification and Characterization of the Biological and Enzymatic activity of Bjussu-LAAO, an L-Amino acid Oxidase from Bothrops jararacussu snake venom

^{1Ticli, F.K.; ²Stabeli, R.G.; ¹Mazzi, M.V.; ¹Sant’Ana, C.D.; ¹Da Silva, J.O.; ³Giglio, J.R.; ⁴Soares, A.M. and ¹Sampaio, S.V.}

1 Depto. ACTB, FCFRP-USP, Ribeirão Preto. 2 Lab. Bioquímica, IPEPATRO, Porto Velho. 3 Depto. Bioquímica - FMRP-USP, Ribeirão Preto. 4 Un. Biotecnologia,UNAERP, Ribeirão Preto.

L-amino acid oxidases (LAAO) usually displaying a wide range of biological activities depending on the snake species from whose venom they are extracted. Only one LAAO was so far isolated from a Bothropsvenom (B. moojeni) and the isolated enzyme was leishmanicide. We report now the isolation of an LAAO (Bjussu-LAAO) through three chromatographic steps: Gel Filtration on Sephadex G-75; Affinity chromatography in Benzamidine Sepharose and Hydrophobic chromatography an Phenyl Sepharose. Gel filtration resolved the crude venom into 4 fractions, PI to PIV. Fraction PI was concentraded and disalted by ultra filtration using a YM 10,000 Amicon membrane and then resolved into 3 new fractions, API to APIII in Benzamidine Sepharose, APII, showing LAAO activity, was again concentraded by ultra filtration and resolved into 5 final fractions HAPI to HAPV, HAPI being named Bjussu-LAAO. The enzymatic assay using a conjugated enzyme able to generate H2O2 from the oxidative deamination of L-leu by the LAAO and then oxide o-phenylenediamine by radish peroxide, was monitored at 490 nm. The purification procedure is relatively simple and a affords a highly purified LAAO as assayed by SDS-PAGE. BjussuLAAO shows relatively milder toxic effects as evaluated in 18-22 g Swiss mice. Bjussu-LAAO induced edema corresponding to a 93% increase of the mouse paw volume at a high dose 100ug/50uL and clotted 200 uL of human plasma in 60 sec. at 12.5 ug/25uL besides displayng these relatively low activities, Bjussu-LAAO did not show any myotoxic (25 to 100 ug/50uL), hemhorragic (50 to 200 ug/50uL) or phospholipasic (1 to 25 ug/25uL) activity.

Supported by: FAPESP, CNPq and USP.

Correspondence to: maumazzi@fcfrp.usp.br