The author proposes to isolate and characterize the coagulant enzyme from the venom of the red diamond rattlesnake (Crotalus ruber). The coagulant fraction will be isolated in homogeneous milligram quantities to provide samples for detailed structure and mode-of-action studies. Molecular weights of the intact enzyme and any subunits will be determined. Other physical characteristics to be determined include isoelectric point, carbohydrate content, amino acid composition and N-terminal sequence. Enzymatic parameters will be investigated for the synthetic substrates of arginine specific proteolytic enzymes including thrombin-, kallikrein-, antithrombin III-, and plasmin- like enzymes. Enzyme active site classification will be made with amino acid residue and cofactor specific inhibitors. The activity of this enzyme toward the natural substrate fibrinogen will be investigated by SDS polyacrylamide gel electrophoresis and fibrinopeptide sequence analysis. The prothrombin time coagulant activity of the venom enzyme will be characterized and coagulation factor dependencies will be investigated with the respective factor deficient plasmas. The availability of this coagulant enzyme provides an opportunity to gain a better understanding of the coagulopathy effects of venom from the red diamond rattlesnake. Detailed structural and enzymatic studies will permit the correlation of the C. ruber coagulant enzyme with other C. ruber proteins and enzymes from other species as well. The availability of well characterized pure venom components will permit long term studies into the synergistic activity of selected venom components and possibly the development of new plasma protein specific enzymes for physiological investigations.
