1. The biological properties of nine venom samples from six taxa of Micrurus were investigated. The venoms exhibited low protease, phosphodiesterase and 5'-nucleotidase activities, moderate to strong phospholipase A and hyaluronidase activities, variable L-amino acid oxidase activity and were devoid of arginine ester hydrolase and thrombin-like activities. Some venom samples exhibited strong acetylcholinesterase activity. Venoms of M. c. dumerili and M. frontalis exhibited exceptionally high alkaline phosphomonoesterase activity while two of the M. f. fulvius venom samples tested exhibited strong hemorrhagic activity in mice. 2. The polyacrylamide gel electrophoretic patterns of the venoms indicate that most of the Micrurus venom proteins are basic proteins. All Micrurus venoms tested exhibited similar SDS-polyacrylamide gel electrophoretic patterns, with an intense low mol. wt protein band. 3. The Micrurus venoms appear to exhibit biological properties similar to other elapid venoms found in Asia and Africa. There are, however, no common characteristics in the biological properties of the venoms examined at the generic level.
The genera Ophiophagus and Naja comprise part of a clade of snakes referred to as cobras, dangerously venomous front-fanged snakes in the family Elapidae responsible for significant human mortality and morbidity throughout Asia and Africa. We evaluated venom enzyme variation for eleven cobra species and three N. kaouthia populations using SDS-PAGE venom fingerprinting and numerous enzyme assays. Acetylcholinesterase and PLA2 activities were the most variable between species, and PLA2 activity was significantly different between Malaysian and Thailand N. kaouthia populations. Venom metalloproteinase activity was low and significantly different among most species, but levels were identical for N. kaouthia populations; minor variation in venom L-amino acid oxidase and phosphodiesterase activities were seen between cobra species. Naja siamensis venom lacked the α-fibrinogenolytic activity common to other cobra venoms. In addition, venom from N. siamensis had no detectable metalloproteinase activity and exhibited an SDS-PAGE profile with reduced abundance of higher mass proteins. Venom profiles from spitting cobras (N. siamensis, N. pallida, and N. mossambica) exhibited similar reductions in higher mass proteins, suggesting the evolution of venoms of reduced complexity and decreased enzymatic activity among spitting cobras. Generally, the venom proteomes of cobras show highly abundant three-finger toxin diversity, followed by large quantities of PLA2s. However, PLA2 bands and activity were very reduced for N. haje, N. annulifera and N. nivea. Venom compositionalenzy analysis provides insight into the evolution, diversification and distribution of different venom phenotypes that complements venomic data, and this information is critical for the development of effective antivenoms and snakebite treatment.
The biological properties of adult and juvenile inland taipan (Oxyuranus microlepidotus) snake venoms were examined. The enzymatic activities, intravenous median lethal dose and procoagulant activity of the juvenile venom samples were not significantly different from those of the adult venom samples. Also, the juvenile and adult venoms exhibited similar electrophoretic patterns, indicating that they possessed similar protein composition.
King cobra (Ophiophagus hannah) venom L-amino acid oxidase (LAAO), a heat-stable enzyme, is an extremely potent antiproliferative agent against cancer cells when compared with LAAO isolated from other snake venoms. King cobra venom LAAO was shown to exhibit very strong antiproliferative activities against MCF-7 (human breast adenocarcinoma) and A549 (human lung adenocarcinoma) cells, with an IC50 value of 0.04±0.00 and 0.05±0.00 μg/mL, respectively, after 72-hr treatment. In comparison, its cytotoxicity was about 3-4 times lower when tested against human non-tumourigenic breast (184B5) and lung (NL 20) cells, suggesting selective antitumour activity. Furthermore, its potency in MCF-7 and A549 cell lines was greater than the effects of doxorubicin, a clinically established cancer chemotherapeutic agent, which showed an IC50 value of 0.18±0.03 and 0.63±0.21 μg/mL, respectively, against the two cell lines. The selective cytotoxic action of the LAAO was confirmed by phycoerythrin (PE) annexin V/7-amino-actinomycin (AAD) apoptotic assay, in which a significant increase in apoptotic cells was observed in LAAO-treated tumour cells than in their non-tumourigenic counterparts. The ability of LAAO to induce apoptosis in tumour cells was further demonstrated using caspase-3/7 and DNA fragmentation assays. We also determined that this enzyme may target oxidative stress in its killing of tumour cells, as its cytotoxicity was significantly reduced in the presence of catalase (a H2O2 scavenger). In view of its heat stability and selective and potent cytotoxic action on cancer cells, king cobra venom LAAO can be potentially developed for treating solid tumours.
Four homologous single chain phospholipases A2 (Pa-1G, Pa-5, Pa-12C and Pa-15) were tested for neuromuscular effects on chick biventer cervicis and mouse hemidiaphragm nerve-muscle preparations. The four isozymes blocked directly elicited (mouse hemidiaphragm) and indirectly elicited (mouse and chick nerve-muscle preparations) twitch responses in concentrations of 1-30 micrograms/ml. The order of potency seen in both types of preparations was Pa-1G = Pa-5 greater than Pa-12C much greater than Pa-15. All four isozymes caused slow-onset, sustained contractures and reduction of muscle membrane potentials. In the chick preparation, responses to acetylcholine, carbachol and KCl were reduced by exposure to the toxins. It is concluded that the toxins act primarily postsynaptically to depress muscle contractility, perhaps by directly damaging muscle fibres. The order of potency agrees with their phospholipase A2 activity. Pa-1G is unusual because it is an acidic molecule, most toxic phospholipases being basic.
cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized. One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2). The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA. Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively. The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.