To investigate the geographic variations in venoms of two medically important pitvipers, we have purified and characterized the phospholipases A2 (PLA2s) from the pooled venoms of Calloselasma rhodostoma from Malaysia, Thailand, Indonesia, and Vietnam, as well as the individual venom of Trimeresurus mucrosquamatus collected from both North and South Taiwan. Enzymatic and pharmacological activities of the purified PLA2s were also investigated. The complete amino acid sequences of the purified PLA2s were determined by sequencing the corresponding cDNAs from the venom gland and shown to be consistent with their molecular weight data and the N-terminal sequences. All the geographic venom samples of C. rhodostoma contain a major noncatalytic basic PLA2-homolog and two or three acidic PLA2s in different proportions. These acidic PLA2s contain Glu6-substitutions and show distinct inhibiting specificities toward the platelets from human and rabbit. We also found that the T. mucrosquamatus venoms from North Taiwan but not those from South Taiwan contain an Arg6-PLA2 designated as TmPL-III. Its amino acid sequence is reported for the first time. This enzyme is structurally almost identical to the low- or nonexpressed Arg6-PLA2 from C. rhodostoma venom gland, and thus appears to be a regressing venom component in both of the Asian pitvipers.
The major l-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78μg/mL (0.006μM) and 1.56μg/mL (0.012μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50μg/mL (0.2-0.4μM). Catalase at the concentration of 1mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme.
A thrombin-like enzyme, purpurase, was purified from the Cryptelytrops purpureomaculatus (mangrove pit viper) venom using high performance ion-exchange and gel filtration chromatography. The purified sample (termed purpurase) yielded a homogeneous band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 35,000. The N-terminal sequence of purpurase was determined to be VVGGDECNINDHRSLVRIF and is homologous to many other venom thrombin-like enzymes. Purpurase exhibits both arginine ester hydrolase and amidase activities. Kinetic studies using tripeptide chromogenic anilide substrates showed that purpurase is not fastidious towards its substrate. The clotting times of fibrinogen by purpurase were concentration dependent, with optimum clotting activity at 3mg fibronogen/mL. The clotting activity by purpurase was in the following decreasing order: cat fibrinogen>human fibrinogen>dog fibrinogen>goat fibrinogen>rabbit fibrinogen. Reversed-phase HPLC analysis of the products of action of purpurase on bovine fibrinogen showed that only fibrinopeptide A was released. Indirect ELISA studies showed that anti-purpurase cross-reacted strongly with venoms of most crotalid venoms, indicating the snake venom thrombin-like enzymes generally possess similar epitopes. In the more specific double-sandwich ELISA, however, anti-purpurase cross-reacted only with venoms of certain species of the Trimeresurus complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.