Purification and characterization of a new l-amino acid oxidase from Daboia russellii siamensis venom

• DOI: 10.1016/j.toxicon.2009.06.004
• Publication Date: 2009-06-12T12:10:11Z
• Authors: Shu-Rong Zhong; Yang Jin; Jian-Bo Wu; Yong-Hong Jia; Gui-Li Xu; Gen-Chun Wang; Yu-Liang Xiong; Qiu-Min Lu
• Keywords: 0301 basic medicine; Staphylococcus aureus; 0303 health sciences; Platelet Aggregation; Molecular Sequence Data; Microbial Sensitivity Tests; Viper Venoms; L-Amino Acid Oxidase; Substrate Specificity; 3. Good health; 03 medical and health sciences; Pseudomonas aeruginosa; Chromatography, Gel; Escherichia coli; Humans; Electrophoresis, Polyacrylamide Gel; Amino Acid Sequence

A new L-amino acid oxidase (designated as DRS-LAAO) was purified from Daboia russellii siamensis venom by ion-exchange, gel filtration and affinity chromatographies. DRS-LAAO is a homodimeric enzyme with a molecular weight of 120.0 kDa as measured by size exclusion chromatography and the monomeric molecular weight of 58.0 kDa as measured by SDS-PAGE under both non-reducing and reducing conditions. The N-terminal amino acid sequence (ADDKNPLEECFREDD) of DRS-LAAO shares high identity with other snake venom L-amino acid oxidases, especially with those isolated from viperid venoms. The enzyme displayed high specificity towards hydrophobic L-amino acids. The best substrate of DRS-LAAO was L-Leu followed by L-Phe and L-Ile, while five substrates--L-Pro, L-Asn, L-Gly, L-Ser and L-Cys were not oxidized. Optimal pH of DRS-LAAO was 8.8. The enzyme showed no hemorrhagic activity even at a dosage of 55.0 microg. DRS-LAAO dose-dependently inhibited platelet aggregation induced by ADP (83.33 microM) and TMVA (55.0 nM) with an IC(50) value of 32.8 microg/ml and 32.3 microg/ml, respectively. The minimum inhibitory concentrations (MICs) of DRS-LAAO against Staphylococci aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were 9.0, 144.0 and 288.0 microg/ml, respectively. The minimum bactericidal concentrations (MBCs) of the enzyme for these strains were twice of the MIC values. These results showed that DRS-LAAO had the strongest antimicrobial activity against S. aureus among these three international standard stains. Antibacterial-activities of DRS-LAAO against eight clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were also tested. The MICs of DRS-LAAO against these isolates ranged from 4.5 to 36.0 microg/ml. And the MBCs of the enzyme against these isolates ranged from 9.0 to 72.0 microg/ml.