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Bothropic envenomation is characterised by severe local damage caused by the toxic action of venom components and aggravated by induced inflammation. In this comparative study, the local inflammatory effects caused by the venoms of Bothrops alternatus and Bothrops moojeni, two snakes of epidemiological importance in Brazil, were investigated. The toxic action of venom components induced by bothropic venom was also characterised. Herein, the oedema, hyperalgesia and myotoxicity induced by bothropic venom were monitored for various lengths of time after venom injection in experimental animals. The intensity of the local effects caused by B. moojeni venom is considerably more potent than B. alternatus venom. Our results also indicate that metalloproteases and phospholipases A2 have a central role in the local damage induced by bothropic venoms, but serine proteases also contribute to the effects of these venoms. Furthermore, we observed that specific anti-inflammatory drugs were able to considerably reduce the oedema, the pain and the muscle damage caused by both venoms. The inflammatory reaction induced by B. moojeni venom is mediated by eicosanoid action, histamine and nitric oxide, with significant participation of bradykinin on the hyperalgesic and myotoxic effects of this venom. These mediators also participate to inflammation caused by B. alternatus venom. However, the inefficient anti-inflammatory effects of some local modulation suggest that histamine, leukotrienes and nitric oxide have little role in the oedema or myotoxicity caused by B. alternatus venom.

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A fibrinogenolytic metalloproteinase from Bothrops moojeni venom, named moojenin, was purified by a combination of ion-exchange chromatography on DEAE-Sephacel and gel filtration on Sephacryl S-300. SDS-PAGE analysis indicated that moojenin consists of a single polypeptide chain and has a molecular mass about 45 kDa. Sequencing of moojenin by Edman degradation revealed the amino acid sequence LGPDIVSPPVCGNELLEVGEECDCGTPENCQNE, which showed strong identity with many other snake venom metalloproteinases (SVMPs). The enzyme cleaves the Aα-chain of fibrinogen first, followed by the Bβ-chain, and shows no effects on the γ-chain. Moojenin showed a coagulant activity on bovine plasma about 3.1 fold lower than crude venom. The fibrinogenolytic and coagulant activities of the moojenin were abolished by preincubation with EDTA, 1,10-phenanthroline and β-mercaptoethanol. Moojenin showed maximum activity at temperatures ranging from 30 to 40 °C and its optimal pH was 4.0. Its activity was completely lost at temperatures above 50 °C. Moojenin induced necrosis in liver and muscle, evidenced by morphological alterations, but did not cause histological alterations in mouse lungs, kidney or heart. Moojenin rendered the blood uncoagulatable when it was intraperitoneally administered into mice. This metalloproteinase may be of medical interest because of its anticoagulant activity.

In this work, we describe the purification and characterization of a new serine protease enzyme from Bothrops moojeni snake venom (BmooSP). On SDS-PAGE, BmooSP was found to be a single-chain protein with an apparent molecular mass of 36,000 and 32,000 under reduced and non-reduced conditions, respectively. Mass spectrometry analysis showed that the BmooSP is composed by two isoforms with molecular mass of 30,363 and 30,070, respectively. The purified enzyme consists of 277 amino acid residues, disregarding the cysteine and tryptophan residues that have been degraded by acid hydrolysis, and its N-terminal sequence showed similarity with other serine protease enzymes. BmooSP induced blood-clotting in vitro, defibrination in vivo, caseinolytic and fibrin(ogen)olytic activities. The enzyme is stable at high temperatures (up to 100 °C) and shows maximum activity at pH around 7.0. Preliminary results show that BmooSP can induce the formation of a stable fibrin clot for more than 10 days. BmooSP presents medical interest because it can be used as biodegradable fibrin glue and for the treatment and prevention of cardiovascular disorders because of its ability to promote the defibrination in vivo, decreasing blood viscosity and improving blood circulation.