Isolation of multiple isoforms of alpha-fibrinogenase from the Western diamondback rattlesnake, Crotalus atrox: N-terminal sequence homology with ancrod, an antithrombotic agent from Malayan viper.
Three fibrinogenolytic proteases from the rattlesnake (Crotalus atrox) venom were isolated and purified from multiple-step chromatographies including ion-exchange chromatography, gel permeation and reversed-phase HPLC. The fractions were shown to be homogeneous as judged by SDS-gel electrophoresis. In vitro, they also showed a high and specific proteolytic activity against alpha-chains of fibrinogen molecules. Further characterization of these purified fractions with fibrinogenase activity indicated that they are all single-chain proteins with similar molecular weights of about 33,000-35,000. Their stability at high temperatures was examined and the cleavage specificity of various proteases was studied using oxidized insulin B-chains as substrates. The reactivity toward fibrinogen molecules in the presence of varied protease inhibitors including EDTA, beta-mercaptoethanol, soybean trypsin inhibitor, aprotinin and phenylmethanesulfonyl fluoride was also investigated in order to classify these proteases based on their reaction mechanisms. Amino acid analysis indicates that these purified venom toxins possess closely-similar compositions with regard to most amino acids including cysteine, methionine and tyrosine. N-Terminal sequence analysis of these proteases revealed that they are similar to ancrod, an antithrombotic agent isolated from the Malayan pit viper. This study points to the existence of a family of novel ancrod-like fibrinogenases in crotalid rattlesnakes, which may be useful as effective venom-based anticoagulants.
Hung
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CC
Institute of Biochemical Sciences, National Taiwan University, Taipei.
Chiou
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SH
D000595:Amino Acid Sequence; D000721:Ancrod / Q000737:chemistry; D000818:Animals; D003435:Crotalid Venoms / Q000201:enzymology*; D017839:Crotalus; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis; D006358:Hot Temperature; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry; D013379:Substrate Specificity
D003435:Crotalid Venoms; D011480:Protease Inhibitors; D005340:Fibrinogen; D000721:Ancrod; D012697:Serine Endopeptidases; C019948:crotalase; D008666:Metalloendopeptidases; C076549:catroxase