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

Abstract

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® 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 (IC 50 ) 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 Dforms of amino acids, to produce H 2 O 2 . The enzyme is important for the purification and determination of certain amino acids and for the preparation of α-keto acids.

Purification, Characterization and Antibacterial Activity ofl-amino Acid Oxidase fromCerastes cerastes

“…The LAAO activity of CcLAAO tested with l ‐leucine as the substrate was optimal at 20°C and pH 8, unlike Bungarus caeruleus LAAO conditions (37°C and pH 4.5) . CcLAAO was inactive at extreme temperatures (4°C and 60°C).…”

Snake Venom L-Amino Acid Oxidases: Trends in Pharmacology and Biochemistry

“…Furthermore, the correlation between the production of LAAOs and their utilization in metabolic pathways involving nitrogen, as well as the production of hydrogen peroxide, opens perspectives for new applications of these enzymes as bactericidal, antiviral, and antitumor agents, making them a promising biotechnological agent. Thus various research groups have studied LAAOs isolated from different snake species [ 12 , 14 , 15 , 20 , 21 , 23 , 24 , 29 , 30 , 32 – 35 , 38 – 48 ].…”

“…Kinetic studies suggest that LAAOs present preferential catalytic specificity for hydrophobic and aromatic L-amino acids ( Table 1 ), whereas their affinity for polar and basic amino acids is low [ 12 , 15 , 16 , 21 , 24 , 28 , 30 , 32 , 33 , 35 , 44 , 46 , 47 ]. Positively charged amino acids such as L-lysine and L-arginine present unfavorable electrostatic interactions with the catalytic site of the enzyme [ 50 ].…”

MipLAAO, a new L-amino acid oxidase from the redtail coral snake Micrurus mipartitus

“…The abundance of this enzyme in venoms varies among species, from traces (0.15%) as in Naja oxiana ( Samel et al, 2008 ) to major proportions (25%) as in Bungarus caeruleus ( More et al, 2010 ). In Micrurus venoms, it represents a low abundance component, with a range of 0.7–4.0% of the total proteins.…”