Abstract
A new fibrinogenolytic metalloproteinase (Bmoo FIBMP-I) was purified from Bothrops moojeni snake venom. This enzyme was isolated through a combination of three chromatographic steps (ion-exchange, molecular exclusion, and affinity chromatography). Analyses by reverse phase chromatography, followed by mass spectrometry, showed the presence of enzyme isoforms with average molecular mass of 22.8 kDa. The SDS-PAGE analyses showed a single chain of 27.6 kDa, in the presence and absence of reducing agent. The protein has a blocked N-terminal. One of the peptides obtained by enzymatic digestion of a reduced and S-alkylated isoform was completely sequenced by mass spectrometry (MS/MS). Bmoo FIBMP-I showed similarity with hemorrhagic factor and several metalloproteinases (MP). This enzyme degraded Aα-chain faster than the Bβ-chain and did not affect the γ-chain of bovine fibrinogen. The absence of proteolytic activity after treatment with EDTA, together with the observed molecular mass, led us to suggest that Bmoo FIBMP-I is a member of the P-I class of the snake venom MP family. Bmoo FIBMP-I showed pH-dependent proteolytic activity on azocasein, but was devoid of coagulant, defibrinating, or hemorrhagic activities. The kinetic parameters of proteolytic activity in azocasein were determined (V max = 0.4596 Uh−1nmol−1 ± 0.1031 and K m = 14.59 mg/mL ± 4.610).
Highlights
Snake venoms are complex mixtures of proteins and peptides with diverse pharmacological activities
This paper reports the isolation and partial characterization of a fibrinogenolytic metalloproteinase, Bmoo FIBMP-I, from B. moojeni snake venom
The high proteolytic activity of Bothrops snake venoms is primarily responsible for most of the local and systemic effects observed during envenomation by these snakes [1]
Summary
Snake venoms are complex mixtures of proteins and peptides with diverse pharmacological activities. While neurotoxicity is the most pronounced effect of envenomation by snakes from the Elapidae family, envenomation by snakes from the Viperidae family is usually characterized by local and, in severe cases, systemic effects [3, 4] These snake venoms are rich sources of metalloproteinases (MPs) which, along with the transmembrane ADAMS (desintegrin-like and metalloproteinase-containing proteins), are members of the reprolysins subfamily M12-family of metalloproteinases. (Snake venom metalloproteinases) SVMPs have been classified into three basic structural classes, P-I to P-III [5,6,7] These metalloproteinases are synthesized in the venom gland as large multidomain proteins, including a proenzyme domain and a highly conserved zinc-proteinase domain (HEBXHXBGBXH) [6, 8, 9]. Class P-II (P-IIa, P-IIb, P-IIc, P-IId, and PIIe) comprises proteins with molecular masses of 30–60 kDa that contain metalloproteinase and disintegrin-like domains. This paper reports the isolation and partial characterization of a fibrinogenolytic metalloproteinase, Bmoo FIBMP-I, from B. moojeni snake venom
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