CHARACTERIZATION OF A PROTEASE WITH ALPHA-FIBRINOGENASE AND BETA-FIBRINOGENASE ACTIVITY FROM THE WESTERN DIAMONDBACK RATTLESNAKE, CROTALUS-ATROX

A metalloprotease from the rattlesnake Crotalus atrox venom was isolated and purified from multiple-step chromatographies including anion-exchange chromatography, gel permeation and reversed-phase HPLC. The fraction was shown to be homogeneous as judged by SDS-gel electrophoresis. It also showed a high proteolytic activity against α- and β-chains of fibrinogen molecules. Further characterization of the purified fraction with fribrinogenase activity indicated that it is a single-chain protease with a molecular mass of about 24 kDa and an acidic isoelectric point. It is relatively heat stable up to about 65°C, inhibited by EDTA, β-mercaptoethanol, but not by phenylmethanesulfonyl fluoride, Nα-p-tosyl-l-phenylalanine chloromethyl ketone and Nα-p-tosyl-l-lysine chloromethyl ketone, soybean trypsin inhibitor and aprotinin. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to some metalloproteases characterized before from the closely related rattlesnake venoms. n-Terminal sequence analysis of the enzyme corroborated some similarity between this enzyme and the reported sequences of these enzymes characterized from the Crotalidae snake family. This study indicated the presence of a novel fibrinogenase (termed Catroxase) with n-terminal sequence different from the metalloprotease with hemorrhagic activity isolated from the same Western diamondback rattlesnake. © 1992 Academic Press, Inc.