ISOLATION AND PARTIAL CHARACTERIZATION OF THE L-AMINO ACID OXIDASE FROM Bothrops alternatus SNAKE VENOM

Stábeli, R.G., Oliveira, E.B.

Faculty of Medicine of Ribeirão Preto, University of São Paulo, SP, Brazil

L-amino acid oxidase (LAO, EC 1.4.3.2), one of the major components of many snake venoms (up to 30%), catalyzes the oxidative deamination of L-amino acids producing the corresponding a-ketoacids, hydrogen peroxide and ammonia. The aim of the present work is to isolate and characterize the LAO of the B. alternatus venom. The enzyme was purified by sequential affinity chromatography on Sepharose – Zn⁺⁺, SephacrylS-200 gel filtration and isoeletric focusing. This latter step yielded three active forms of the enzyme with pIs4.2, 4.6 and 5.2. The homogeneity of the enzyme with pI5.2 (LAO-5.2) was demonstrated by SDS-PAGE and reverse-phase HPLC over C-4 column, thus, LAO-5.2 was used for further characterization. The enzyme seems to be a dimer based on the MW determination by SDS-PAGE (66,000) and gel filtration on S-200 column (120,000). Substrate specificity toward different amino acids was determined by measuring the rate of degradation of individual amino acids present in the incubation mixture. Tyr, Phe, Met and Leu were, by far, the most rapidly oxidized substrates while Ile and Cys-Cys were degraded at rates 10-20 times slower.

LAOs have been purified from various snake venoms and most of their biological effects are believed to be, at least in part, mediated by the hydrogen peroxide released during the enzymatic reaction. We trust that highly purified LAO preparation, such as the one derived from B. alternatus venom here described, are important tools to further our understanding of the participation of LAO in promoting apoptosis and others effects such as cytotoxicity, platelet aggregation, haemorrhage, hemolysis and edema.

CORRESPONDENCE TO:

Rodrigo GuerinoStábeli, Rua: Domingos Russo 81, Ribeirão Preto, SP, CEP: 14061200, Brasil, Email: rstabeli@rbi.fmrp.usp.br