Purification of an L-amino acid oxidase from Bungarus caeruleus (Indian krait) venom

Snake venoms are rich in enzymes such as phospholipase A(2), proteolytic enzymes, hyaluronidases and phosphodiesterases, which are well characterized. However, L-amino acid oxidase (LAO EC.1.4.3.2) from snake venoms has not been extensively studied. A novel L-amino acid oxidase from Bungarus caeruleus venom was purified to homogeneity using a combination of ion-exchange by DEAE-cellulose chromatography and gel filtration on Sephadex (R) G-100 column. The purified monomer of LAO showed a molecular mass of 55 +/- 1 kDa estimated by SDS-PAGE. The specific activity of purified LAO was 6,230 +/- 178 U/min/mg, versus 230 +/- 3.0 U/min/mg for the whole desiccated venom, suggesting a 27-fold purification with a 25% yield. Optimal pH and temperature for maximum purified enzyme activity were 6.5 and 37 degrees C, respectively. Platelet aggregation studies show that purified LAO inhibited ADP-induced platelet aggregation dose-dependently at 0.01 to 0.1 mu M with 50% inhibitory concentration (IC50) of 0.04 mu M, whereas at a 0.08 mu M concentration it did not induce appreciable aggregation on normal platelet-rich plasma (PRP). The purified protein catalyzed oxidative deamination of L-amino acids while the most specific substrate was L-leucine. The purified LAO oxidizes only L-forms, but not D-forms of amino acids, to produce H2O2. The enzyme is important for the purification and determination of certain amino acids and for the preparation of alpha-keto acids.