Characterization and chemical modification of E toxin isolated from timber rattlesnake (Crotalus horridus horridus) venom

by Allen, Howell Raymond; Merchant, Michael Louis; Tucker, Randal K.; Fox, Jay W.; Geren, Collis R.

E toxin from Crotalus horridus horridus venom has a molecular mass as calculated from amino acid analysis of 4855 Da and consists of at least 42 amino acids as a single polypeptide chain. E toxin exists as multiple (5) isoforms. Isoforms off toxin were separated using cation exchange chromatography. Initial fractionation of lyophilized timber rattlesnake venom on CM 52 cation exchange resin provided a peak consisting of a mixture of E toxin isoforms. Subsequent fractionation off toxin isoforms on a strongly binding sulfoxypropyl cation exchange resin provided baseline separation (mass amounts of less than 100 mu g of protein). Five separate isoforms were isolated in sufficient quantities for further characterization. Al five isoforms off toxin were found to be lethal to BALB/c mice at a concentration of 1.3 mg/kg body weight i.p. in the presence of acetate. The acetate-requiring toxicity of E toxin to mice was nullified by different chemical modifications of specific amino acid residues. Nitration of the N-terminal tyrosine resulted in loss of lethality. Reductive methylation of 6 of the 8 lysines in E toxins had no effect on toxicity. Modification off toxins' three arginines also had no effect on toxicity. Reduction of one of the three disulfides had no effect, but reduction of two of the disulfides destroyed toxicity. Modification of carboxyl groups caused some loss of toxicity. Far uv circular dichroism spectroscopy off toxin isoforms revealed small differences in secondary structures among the isoforms in aqueous solution. The far uv CD spectrum off toxins (pooled isoforms) is different in aqueous buffer from that obtained in the more nonpolar solvent, methanol. Thermal denaturation circular dichroism spectroscopy indicates loss of alpha-helicity as monitored at 220 nm. Upon excitation at 294 nm, E toxin exhibits a fluorescence emission maximum at 355 nm,indicating that the two tryptophans are solvent exposed. Titration off toxin with a chemical denaturant (6 M guanidine HCl) does not induce a loss of fluorescent intensity, indicating that the tryptophans are fully solvent exposed and that the proteins are not susceptible to local unfolding around the tryptophans.

Journal of Natural Toxins
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