Purification and characterization of a novel metalloproteinase, acurhagin, from Agkistrodon acutus venom.
Acurhagin, a high-molecular mass hemorrhagic metalloproteinase, was purified from the crude venom of Agkistrodon acutus using anion-exchange and hydrophobic interaction chromatography. Acurhagin is a monomer with a molecular mass of 51.4 kDa under non-reducing conditions on SDS-PAGE and 48,133 Da by mass spectrometry. Partial amino acid sequence of its metalloproteinase domain is homologous to other high-molecular mass metalloproteinases from snake venoms. It preferentially cleaved Aalpa chain of fibrinogen, followed by Bbeta chain, while gamma chains was minimally affected. Monitored by RP-HPLC, it extensively degraded fibrinogen into various peptide fragments. In aqueous solution, acurhagin autoproteolyzed to a 30 kDa fragment at 37 degrees C. The N-terminal sequence of the 30 kDa fragment of acurhagin showed a high homology to those proteins consisting of disintegrin-like and cysteine-rich domains. Caseinolytic assay showed that the proteinase activity of acurhagin was slightly enhanced by Ca2+ and Mg2+, but completely inhibited by Zn2+. When treated with metal chelators, acurhagin was completely inactivated. Furthermore, acurhagin exerts an inhibitory effect on ADP-induced platelet aggregation of platelet-rich plasma in an incubation-time dependent manner. It also impairs collagen- and ristocetin-induced platelet aggregation by cleaving collagen and vWF, respectively.
Wang
Wen-Jeng
WJ
Department of Pharmacology, College of Medicine, National Taiwan University, Taipei.
Huang
Tur-Fu
TF
D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors / Q000201:enzymology* / Q000302:isolation & purification* / Q000493:pharmacokinetics / Q000633:toxicity; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010974:Platelet Aggregation / Q000187:drug effects; D011480:Protease Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology
C031442:Agkistrodon venoms; C115499:BBeta fibrinogen; D002413:Cations, Divalent; D002614:Chelating Agents; D003435:Crotalid Venoms; D011480:Protease Inhibitors; C469546:acurhagin protein, Agkistrodon acutus; C464794:fibrinogen Aalpha; D005340:Fibrinogen; D015032:Zinc