Comparative studies on three rattlesnake toxins

Abstract

Toxins from the venoms of Crotalus durissus terrificus, Crotalus s. scutulatus and Crotalus viridis concolor were compared using gel filtration, ion-exchange chromatography on DEAE-Sephacel and denaturing and non-denaturing polyacrylamide gel electrophoresis. The three heterodimeric native toxins behaved similarly on each of the separation media, except that the C. d. terrificus toxin displayed a pronounced tendency to dissociate on DEAE-Sephacel, even in the absence of urea. In the presence of 6M urea, subunit dissociation was quantitative for all three toxins. Recombination of purified subunits resulted in toxins which eluted from the gel filtration column in identical fashion to native toxins. Non-denaturing polyacrylamide gel electrophoretic patterns of recombined toxins actually showed greater band resolution than did the native toxins. Six hybrid toxins were generated on polyacrylamide gels from cross-combinations of purified subunits, each with different mobilities than the parental toxins. Mobilities of the hybrid toxins depended principally upon the mobilities of the basic subunits. All three purified native toxins showed comparable LD50's in female mice (0.039-0.061 micrograms/g). The C. d. terrificus acidic X C. s. scutulatus basic hybrid toxin showed toxicity identical to that of the C. s. scutulatus recombined toxin. Phospholipase activity is associated with the basic subunit in all three toxins. Intact toxins show a distinctive lag in phospholipase activity which is not seen with purified basic subunits alone. These results indicate that the principal toxins in these three venoms are homologous.