Purification and characterization of a coagulant enzyme, okinaxobin I, from the venom of Trimeresurus okinavensis (Himehabu snake) which releases fibrinopeptide B.
A coagulant enzyme, named okinaxobin I, has been purified to homogeneity from the venom of Trimeresurus okinavensis (Himehabu) by chromatographies on Sephadex G-100 and CM-Toyopearl 650M columns. The enzyme was a monomer with a molecular weight of 37,000 and its isoelectric point was 5.4. The enzyme acted on fibrinogen to form fibrin clots with a specific activity of 77 NIH units/mg. Fibrinopeptide B was released at a rate much faster than fibrinopeptide A. The enzyme exhibited 2 to 3 times higher activity toward tosyl-L-arginine methyl ester and benzoyl-L-arginine p-nitroanilide than bovine thrombin. The esterase activity was strongly inhibited by diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and to a lesser extent by tosyl-L-lysine chloromethyl ketone, indicating that the enzyme is a serine protease like thrombin. The N-terminal sequence was highly homologous to those of coagulant enzymes from T. flavoviridis and Bothrops atrox, moojeni venoms which preferentially release fibrinopeptide A. In order to remove most, if not all, of the bonded carbohydrates, the enzyme was treated with anhydrous hydrogen fluoride (HF), thereby reducing the molecular weight to 30,000. The protein contained approximately 260 amino acid residues when computation was based on this value. The HF-treated enzyme retained about 50% of the clotting and esterolytic (TAME) activities and preferentially released fibrinopeptide B from fibrinogen. The carbohydrate moiety is not crucial for enzyme activity but might be necessary for eliciting full activity.
Iwasaki
A
A
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka.
Shieh
T C
TC
Shimohigashi
Y
Y
Waki
M
M
Kihara
H
H
Ohno
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001586:Benzoylarginine Nitroanilide; D003435:Crotalid Venoms / Q000737:chemistry*; D004950:Esterases / Q000037:antagonists & inhibitors; D005344:Fibrinopeptide A / Q000378:metabolism; D005345:Fibrinopeptide B / Q000378:metabolism*; D006858:Hydrofluoric Acid / Q000494:pharmacology; D007526:Isoelectric Point; D007531:Isoflurophate; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D012911:Snakes; D013379:Substrate Specificity / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology; D014106:Tosylarginine Methyl Ester / Q000494:pharmacology; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology
D003435:Crotalid Venoms; D007531:Isoflurophate; D014107:Tosyllysine Chloromethyl Ketone; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; D010664:Phenylmethylsulfonyl Fluoride; D014106:Tosylarginine Methyl Ester; D001586:Benzoylarginine Nitroanilide; D001120:Arginine; D004950:Esterases; D012697:Serine Endopeptidases; C068144:okinaxobin I; D013917:Thrombin; D006858:Hydrofluoric Acid