Biochemical And Pharmacological Study Of The Venom Of The Spider Heteropoda Venatoria

Heteropoda venatoria is a venomous spider species distributed worldwide and has a characteristic habit of feeding on insects.The venom was analyzed by SDS-PAGE using standard protocols,except for polypeptides with molecular weights below 10 kDa,the venom proteins were mainly distributed in three bands on SDS-PAGE,corresponding to the molecular weights of approximately 35 kDa,50 kDa and 70 kDa,respectively.A total of 137 mass peaks were detected and multiple mass peaks are present in most HPLC fractions by HPLC and MALDI-TOF MS analysis,indicating a single HPLC fractions actually contains various peptides and a great diversity of the crude venom.The venom peptides followed a single-modal distribution,approximately 90%of masses localized in the mass range of 3000-4500 Da.This distribution modal is different from typical double-modal distribution from other spider venoms analyzed previous.It was found that the venom had potent inhibitory effect on voltage-gated sodium channels(VGSCs)in Periplaneta americana cockroach dorsal unpaired median(DUM)neurons with an the IC50 values of 6.25±0.02 ?g/mL(n=5)by path-clamp analysis.100 ?g/mL venom led to complete inhibition of the peak currents of DUM VGSCs.However,100?g/mL venom only partially blocked the VGSC currents in rat dorsal root ganglion cells,a much lower inhibitory effect than that on DUM VGSCs.Application of 10 ?g/mL and 100 ?g/mL venom only resulted in 10.2%±2.1%(n=5)and 27.0%±3.7%(n=5)reduction of peak currents,respectively.10 ?g/mL venom could reduce the inward peak currents at all the depolarized voltages tested.It did not change the activation and reverse voltages,but it actually caused a positive shift of maximum activation voltage from 0 mV to+10 mV.Intrathoracical administration of the venom immediately led to symptoms of envenomation including flaccid paralysis,lethargy and ataxia,while no such symptoms were observed in the cockroaches receiving injections of water.High doses of the venom could cause death.The LD50 was 28.18 ?g/g of body weight in the cockroach.The second chapter of this paper focused on the study of interaction between HpTx-I and Nav1.7.HpTx-I is a 33-residue peptide toxin isolated from Heteropoda venatoria venom with a molecular weight of 3921.5425 Da.It contains six cysteines which form three disulfide bond and belongs to the ICK motif.The amino acid sequence of HpTx-I was determined to be DCGTIWHYCGTDQSECCEGWKCSRQLCKYVIDW by the MALDI-TOF/TOF analysis.Among the tested four VGSC subtypes,hNav1.7 was the most sensitive to HpTx-I(IC50=0.282±0.035 ?M),which was 5 fold sensitive compared with hNavl.5,and the toxin had very low inhibitory effect on rNavl.3 and rNavl.4.The related experimental results indicated that HpTx-I had little or no effect on steady-state inactivation of the hNav1.7,despite it has capacity to inhibit the peak currents of the channel.The application of 0.5 uM HpTx-I did not change the activation and reverse voltages,but it actually caused a positive shift of maximum activation voltage from 0 mV to+10 mV.These data indicated that it might be more difficult to activate hNavl.7 current in the presence of HpTx-I by a stronger depolarization voltage.Moreover,it has not capacity to inhibit the peak currents of reconstruction hNavl.8 and hNavl.5.