Characterization of a protease with alpha- 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 alpha- and beta-chains of fibrinogen molecules. Further characterization of the purified fraction with fibrinogenase 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 degrees C, inhibited by EDTA, beta-mercaptoethanol, but not by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-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.
Chiou
S H
SH
Institute of Biochemical Sciences, National Taiwan University, Taipei.
Hung
C C
CC
Huang
K F
KF
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000737:chemistry*; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D003435:Crotalid Venoms; D011480:Protease Inhibitors; D013917:Thrombin; D008666:Metalloendopeptidases; C076549:catroxase