Purification and properties of a coagulant thrombin-like enzyme from the venom of Bothrops leucurus.
A thrombin-like enzyme from Bothrops leucurus venom, named leucurobin (leuc), was purified by gel filtration, affinity and ion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 35 kDa monomeric glycoprotein on SDS-PAGE under reducing conditions, which decreased to 29 kDa after deglycosylation with N-glycosidase F (PNGase F). The amino acid sequence of leuc was determined by automated sequencing of the intact native protein and peptides produced by digestion of the S-pyridyl-ethylated protein with trypsin. The protein sequence exhibits significant similarities with other serine proteases reported from snake venoms, and contains two potential sites of N-linked glycosylation. The proteinase split off fibrinopeptide A (FPA) rapidly from human fibrinogen; however, only negligible traces of fibrinopeptide B (FPB) were observed. In addition, the enzyme released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bbeta-chain. Leuc could neither activate factor XIII nor release kinins from heat-treated bovine plasma. Its specific clotting activity was equivalent to 198 NIH thrombin U/mg on human fibrinogen. Kinetic properties of leuc were determined using representative chromogenic substrates. The enzyme evoked the gyroxin syndrome when injected into the tail veins of mice at levels of 0.143 microg/g mouse. The inhibitory effects of PMSF and benzamidine on the amidolytic activity suggest that leuc is a serine proteinase, and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Antibothropic serum, SBTI and EDTA had little or no effect on its amidolytic activity. However, the clotting effect of the enzyme was strongly inhibited by antibothropic serum. A Dixon plot showed that the hydrolysis of Bz-L-Arg-pNA by leuc was competitively inhibited by benzamidine (Ki=1.61+/-0.25 mM).
Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, MG 30510-010, Brazil.
Arinos
A
Magalhães
Henrique P B
HP
Magalhães
Michael
M
Richardson
Silea
S
Gontijo
Rodrigo N
RN
Ferreira
Alvair P
AP
Almeida
Eladio F
EF
Sanchez
D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation; D017837:Bothrops; D002417:Cattle; D003029:Coagulants / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D003435:Crotalid Venoms / Q000201:enzymology* / Q000302:isolation & purification / Q000494:pharmacology / Q000633:toxicity; D005176:Factor XIII / Q000187:drug effects / Q000378:metabolism; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology*
D003029:Coagulants; D003435:Crotalid Venoms; D005338:Fibrin Fibrinogen Degradation Products; D011480:Protease Inhibitors; C017356:gyroxin; D005176:Factor XIII; D012697:Serine Endopeptidases; D013917:Thrombin; C520717:leucurobin, Bothrops leucurus