Zhong Shu-Rong, Jin Yang, Wu Jian-Bo, Jia Yong-Hong, Xu Gui-Li, Wang Gen-Chun, Xiong Yu-Liang, Lu Qiu-Min
Department of Animal Toxinology, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang East Road, Kunming 650223, China.
Toxicon. 2009 Nov;54(6):763-71. doi: 10.1016/j.toxicon.2009.06.004. Epub 2009 Jun 11.
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.
通过离子交换、凝胶过滤和亲和色谱法从圆斑蝰蛇毒中纯化出一种新的L-氨基酸氧化酶（命名为DRS-LAAO）。DRS-LAAO是一种同二聚体酶，通过尺寸排阻色谱法测得其分子量为120.0 kDa，在非还原和还原条件下通过SDS-PAGE测得其单体分子量为58.0 kDa。DRS-LAAO的N端氨基酸序列（ADDKNPLEECFREDD）与其他蛇毒L-氨基酸氧化酶具有高度同源性，尤其是与从蝰蛇科蛇毒中分离出的那些酶。该酶对疏水性L-氨基酸表现出高特异性。DRS-LAAO的最佳底物是L-亮氨酸，其次是L-苯丙氨酸和L-异亮氨酸，而L-脯氨酸、L-天冬酰胺、L-甘氨酸、L-丝氨酸和L-半胱氨酸这五种底物未被氧化。DRS-LAAO的最适pH为8.8。即使在剂量为55.0微克时，该酶也没有出血活性。DRS-LAAO对ADP（83.33 microM）和TMVA（55.0 nM）诱导的血小板聚集具有剂量依赖性抑制作用，其IC50值分别为32.8微克/毫升和32.3微克/毫升。DRS-LAAO对金黄色葡萄球菌（ATCC 25923）、铜绿假单胞菌（ATCC 27853）和大肠杆菌（ATCC 25922）的最低抑菌浓度（MIC）分别为9.0、144.0和288.0微克/毫升。该酶对这些菌株的最低杀菌浓度（MBC）是MIC值的两倍。这些结果表明，在这三种国际标准菌株中，DRS-LAAO对金黄色葡萄球菌具有最强的抗菌活性。还测试了DRS-LAAO对八株临床耐甲氧西林金黄色葡萄球菌（MRSA）分离株的抗菌活性。DRS-LAAO对这些分离株的MIC范围为4.5至36.0微克/毫升。该酶对这些分离株的MBC范围为9.0至72.0微克/毫升。
