Isolation and characterization of the thrombin-like enzyme from Cryptelytrops purpureomaculatus venom.
A thrombin-like enzyme, purpurase, was purified from the Cryptelytrops purpureomaculatus (mangrove pit viper) venom using high performance ion-exchange and gel filtration chromatography. The purified sample (termed purpurase) yielded a homogeneous band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 35,000. The N-terminal sequence of purpurase was determined to be VVGGDECNINDHRSLVRIF and is homologous to many other venom thrombin-like enzymes. Purpurase exhibits both arginine ester hydrolase and amidase activities. Kinetic studies using tripeptide chromogenic anilide substrates showed that purpurase is not fastidious towards its substrate. The clotting times of fibrinogen by purpurase were concentration dependent, with optimum clotting activity at 3mg fibronogen/mL. The clotting activity by purpurase was in the following decreasing order: cat fibrinogen>human fibrinogen>dog fibrinogen>goat fibrinogen>>rabbit fibrinogen. Reversed-phase HPLC analysis of the products of action of purpurase on bovine fibrinogen showed that only fibrinopeptide A was released. Indirect ELISA studies showed that anti-purpurase cross-reacted strongly with venoms of most crotalid venoms, indicating the snake venom thrombin-like enzymes generally possess similar epitopes. In the more specific double-sandwich ELISA, however, anti-purpurase cross-reacted only with venoms of certain species of the Trimeresurus complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.
Tan
Nget Hong
NH
Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. tanngethong@yahoo.com.sg
D000581:Amidohydrolases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D002415:Cats; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D056148:Chromatography, Reverse-Phase; D003429:Cross Reactions; D003435:Crotalid Venoms / Q000737:chemistry / Q000201:enzymology* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005340:Fibrinogen / Q000378:metabolism; D005344:Fibrinopeptide A / Q000378:metabolism; D005345:Fibrinopeptide B / Q000378:metabolism; D006041:Goats; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D011817:Rabbits; D017385:Sequence Homology; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D017819:Viperidae / Q000378:metabolism*
D003435:Crotalid Venoms; D000939:Epitopes; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; D005340:Fibrinogen; C019724:arginine esterase; D002265:Carboxylic Ester Hydrolases; D010450:Endopeptidases; C547197:purpurase protein, Cryptelytrops purpureomaculatus; D013917:Thrombin; D000581:Amidohydrolases