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 37°c,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.