New Proteases from Crotalus Atrox Venom

Abstract Rattlesnake envenomation gives rise to many dramatic symptoms in the victim. Included among these are hypotension and hemorrhage. In this paper we present the results of our investigations on several proteases isolated from the venom of Crotalus atror (Western diamondback rattlesnake) which are probably involved in crotalid venom caused hypotension and hemorrhage. We have isolated two proteases (EI and EII) from C. atrox venom which have molecular weights of 27,500 and 29,200 and isoelectric points of 4.7 and 4.3 respectively. Both enzymes did not demonstrate any proteolytic activity on the oxidized chains of insulin and glucagon. Neither enzyme had any plasmin or fibrinolytic activity but both could release bradykinin from a kininogen analog. This proteolytic activity was inhibited by aprotinin and PMSF (phenylmethylsulfonyl fluoride) but not by EDTA (ethylenediamine tetraacetate). The enzymes were demonstrated to be structurally similar but not identical from spectroscopic, peptide mapping, and sequence analysis. The in vivo role of these kalli-krein-like proteases is uncertain; however, they may be involved in the initial symptoms of hypotension and shock. The other protease examined was hemorrhagic toxin e (Ht-e), a zinc protease from C. atrox venom. The proteolytic specificity of this protease was examined using oxidized A and B chains of insulin. The cleavage of the insulin chains was inhibited by EDTA but not aprotinin. Ht-e containing cobalt II rather than zinc cleaved the insulin chains at the same sites and at similar rates as the zinc enzyme. No structural differences between the native and the cobalt enzyme were observed using spectroscopic probes. Furthermore the cobalt protease was both hemorrhagic and proteolytic to a similar extent as the native toxin.