Purification and Properties of a Thrombin-like Enzyme from the Venom of Crotalus adamanteus (Eastern Diamondback Rattlesnake)

Abstract A thrombin-like enzyme has been purified to homogeneity from the venom of Crotalus adamanteus (Eastern diamond-back rattlesnake). The enzyme acts directly on fibrinogen in vivo (and in vitro) apparently without affecting any of the other proteins involved in blood coagulation. The enzyme has esterase activity on basic amino acid esters and p-nitrophenyl esters of various N-carbobenzoxy amino acids. It exhibits no activity with a variety of N-benzoyl or N-methyl amino acid amides. Physicochemical studies indicated that the enzyme is a glycoprotein with a molecular weight of 32,700. Additionally, sedimentation equilibrium in guanidine β-mercaptoethanol showed that the enzyme contains a single polypeptide chain. The protein contains approximately 267 residues with a relatively high content of cystine. The enzyme is optimally active near pH 8 and is stable to neutral and alkaline pH; however, it loses activity upon exposure to acid pH. Both clotting and esterase activities are inhibited by diisopropyl phosphofluoridate, showing that the enzyme, like thrombin, is a serine esterase. Furthermore, the chloromethyl ketone of tosyl-l-lysine, a specific inhibitor of trypsin and thrombin, inhibits the venom enzyme indicating that a histidine residue is necessary for the activity of this enzyme. The chloromethyl ketone of tosyl-l-phenylalanine is not an inhibitor of the venom enzyme. Nitration of the enzyme with tetranitromethane causes loss of clotting activity with little effect on esterase activity suggesting that 1 or more tyrosine residues may be involved in the binding site for large substrates. Finally, the importance of disulfide bridges to the structural integrity of the venom enzyme was indicated by the rapid loss of activity in the presence of β-mercaptoethanol.