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

Snake venoms are rich in enzymes such as phospholipase A2, 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® 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 37ºC, respectively. Platelet aggregation studies show that purified LAO inhibited ADP-induced platelet aggregation dose-dependently at 0.01 to 0.1 µM with 50% inhibitory concentration (IC50) of 0.04 µM, whereas at a 0.08 µM 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 α-keto acids.(AU)

Preliminary studies with a neurotoxin obtained from Bungarus caeruleus venom

The neurotoxin purified from the venom of Bungarus caeruleus causes a neuromuscular blockade on acetylcholine-induced muscle twitch response in isolated frog rectus abdominis muscle preparation. Neuromuscular blockade produced by d-tubocurarine on acetylcholine-induced muscle twitch response in an isolated frog rectus abdominis muscle preparation was reversed to normal muscle twitch response in presence of neostigmine. Whereas the purified neurotoxin produced an irreversible neuromuscular blockade in presence of the same strength of neostigmine. As it is already known, botulinum toxin, which also brings about neuromuscular blockade, is effectively used as a drug in the treatment of painful movement disorders. Since the purified toxin also causes paralysis of the muscle, we propose its possible efficacy in the treatment of neuromuscular disorders.(AU)