Isolation of a crotalase-like protease with alpha-fibrinogenase activity from the western diamondback rattlesnake, Crotalus atrox.
Venom toxins were isolated from rattlesnake (Crotalus atrox) venom by cation-exchange chromatography. Seven major fractions could be obtained by single-step ion-exchange chromatography with two fractions showing essentially apparent homogeneity by SDS-gel electrophoresis. All fractions showed various extents of specific proteolytic activity against alpha- or beta-chains of fibrinogen molecules. Further characterization of one of the purified fractions with alpha-fribrinogenase activity indicated that it is a single-chain thrombin-like protease with a molecular mass of about 30 kDa. It is relatively heat stable, inhibited by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone but not by soybean trypsin inhibitor and beta-mercaptoethanol. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to thrombin and crotalase characterized before from the closely related snake venoms. N-Terminal sequence analysis of the enzyme corroborated the close similarity between this enzyme and those sequences of crotalase and kallikrein-like enzymes characterized from the same Crotalidae snake family. This study is in contrast to the previous reports which indicated a lack of thrombin- and crotalase-like enzyme in the venom of Western diamondback rattlesnake.
Chiou
S H
SH
Institute of Biochemical Sciences, National Taiwan University, Taipei.
Hung
C C
CC
Lin
C W
CW
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002852:Chromatography, Ion Exchange; D003435:Crotalid Venoms / Q000201:enzymology*; D015180:Electrophoresis, Gel, Two-Dimensional; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013917:Thrombin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D003435:Crotalid Venoms; D011480:Protease Inhibitors; D008623:Mercaptoethanol; D005340:Fibrinogen; D010450:Endopeptidases; D012697:Serine Endopeptidases; D013917:Thrombin; C019948:crotalase