1.Purification And Identification Of A Natural Mutant Of Huwentoxin-â…  From The Venom Of Ornithoctonus Huwena 2.Biochemical Studies On The Crude Venom Of Spider Latrodectus Tredecimguttatus

1. Purification and Identification of a Natural Mutant of Huwen toxin - I from the venom of Ornithoctonus huwenaThe Chinese bird spider Selenocosmia huwena(=Ornithoctonus huwena), distributed in the hilly areas of Yunnan and Guangxi in the south of China, was recently identified as a new species .The crude venom of O.huwena contains a mixture of compounds with different types of biological activities, of which neurotoxins have been widely and deeply studied. Huwentoxin- I , a neurotoxic peptide, is the most abundant toxic component in the crude vemon of the Chinese bird spider Ornithoctonus huwena. It contains 33 amino acid residues, including six cysteine residues that form three pairs of disulfide bonds. Being a blocker of N-type Ca²⁺ channel, the toxin can block neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation with its action site located on the foresynaptic membrane.In this study, a natural mutant of Huwentoxin- I was purified and identified from the venom of the spider Ornithoctonus huwena by ion exchange chromatography and reversed phase HPLC. The molecular weight of the mutant is 3, 636 Da determined by MALDI-TOF mass spectrometry and the amino acid sequence is H₂N-ACKGV FDACT PGKNE CCPNR VCSDK HKWCK WQ-COOH determined by Edman degradation in combination with tandem mass spectrometry and aminoacid composition analysis. The mutant contains 32 amino acid residues, including six cysteine residues that form three pairs of disulfide bridges. The mutant is derived from HWTX- I by truncating one amino acid residue from its C-terminus and replacing its residue lysine32(K32) by glutamine(Q).The biological activity of the mutant is obviously different from that of HWTX- I .The natural mutant can cause the death of adult mouse after intracerebroventricular injection, but can not block the neuromuscular transmission of the isolated mouse phrenic nerve-diaphragm preparation. This result indicates that the residue K32 plays a key role in the biological activity of HWTX-I and the mutant is of significance to the study on the structure-function relationship of spider toxins.2. Biochemical Studies on the Crude Venom of Spider Latrodectus tredecimguttatusThe spider Latrodectus tredecimguttatus, "black widow spider", is one of the most venomous spiders in the world. Since 2000, seriously injury accidents caused by black widow spider have taken place in Harmi area of Xinjiang Province, which attracted national-wide attention. In July, 2002, we gathered the spiders and their eggs in Harmi area of Xinjiang Province, and then the spider was successfully bred and propagated in our lab. In the present work, the indoor ecology ^ venom collection and venom biochemistry of Latrotectus tredecimguttatus were systematically studied.The indoor ecology study of Latrotectus tredecimguttatus includes the durations of spider egg bags, spiderling and adult spider, and the survival rate of spiders. The effects of the mating of female with male spiders hatched in the same or different periods on the rates of egg-laying spiders and fertilized egg bags are also included. The results show that the durations of spider egg bags laid in summer and autumn are about 50d and 230d respectively; The durations of female spiderling and male spiderling of type A(hatched from the egg bags laid mainly in summer) are about 120d and 100d respectively, while those of type B(hatched from the egg bags laid mainly in autumn) are about 300d and 280d respectively, The female baby spiders grow into adults after 6 peels, while male spider after 4 peels. In general, the duration of adult female spider is 50-60d, andthat of male spider is about 30d. The ages when baby spiders start to be bred individually have obvious effect on the survival rate, the percentage of adult spiders to baby spiders that are bred individually. The spiders bred individually from the 4th age have the highest survival rate (58.09%). The results also show that when the female spider mates with the male spider that is hatched about 30d later, the laying egg rate (82.61%) and fertilized egg bag rate (89.82%) are higher than those when the female spider mating with the male spider hatched in the same period.We have developed a new electrical stimulation method for collecting venom from the medium-sized spider. A comparision of biological activities and peptide components of crude venoms collected by electrical stimulation and by the traditional method (extrating toxin from the isolated venomous gland) was investigated. Results show that the crude venom of Latrodectus tredecimguttatus have strong biological activities to block the neuromuscular transmission of the isolated phrenic nerve-diaphragm preparation of mice. The intraperitoneal LD50 in mice of the venom collected by electrical stimulation is 0.163mg/Kg, and the venom can block the neuromuscular transmission in mouse phrenic nerve-diaphragm preparation completely in average 23.3 + 2.2 min at the concentration of 3.2ug/ml. The LD50 of the venom collected from the isolated venomous gland is 0.393mg/Kg, and the venom can block the neuromuscular transmission completely in average 25.0 ± 2.2min at...