Proteolytic, edematogenic and myotoxic activities of a hemorrhagic metalloproteinase isolated from Bothrops alternatus venom.
A hemorrhagic metalloproteinase has been isolated from Bothrops alternatus venom from specimens that inhabit the north-east region of Argentina. The present study aimed at evaluating the proteolytic, hemorrhagic, edematogenic and myotoxic activities of the purified metalloproteinase, in order to consider its participation on the phatophysiology of the intoxication by Bothrops alternatus venom. The hemorrhagic metalloproteinase was isolated by a combination of DEAE-Cellulose chromatography and gel filtration on Sephadex G-75. The enzyme showed a molecular mass around 55k Da, it exhibited a hemorrhagic activity with a minimal hemorrhagic dose of 1.9 microg, almost two fold minor than the whole venom (3.6 microg). The enzyme showed a weak proteolytic activity on casein (18.72 U/mg enzyme), similar to the one exhibited by the whole venom (20 U/mg venom). Besides, the ability to degrade casein could be detected by SDS-PAGE; beta-casein was the fraction that showed the higher degradation, followed by alphas(1)-casein and kappa-casein degradation. The hemorrhagic metalloproteinase rapidly hydrolysed the A alpha-chain of fibrinogen, followed by B beta-chain degradation and leaving the gamma-chain unaffected. Proteolytic activities were inhibited by EDTA whereas they were not inhibited by benzamidine and PMSF. The metalloproteinase showed several polypeptides chains after autocatalytic processing, including a chain of 28k Da, it could be the processed disintegrin-like and cysteine-rich domains. The isolated enzyme exhibited myotoxic activity with high CK levels at 6h, due to local ischemia resulting of its hemorrhagic activity, and a significant edema-inducing effect (MED=1.3 microg), corroborated both results by the histological observations of samples of gastrocnemius muscle. These findings showed that this hemorrhagic metalloproteinase, possesses high edematogenic and myotoxic activities and, in despite of exhibiting a weak proteolytic activity, it is able to degrade fibrinogen. So, this enzyme would contribute markedly to the phatophysiology of the bothropic envenomation.
Gay
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Facultad de Ciencias Exactas y Naturales y Agrimensura, Universidad Nacional del Nordeste (UNNE). Av. Libertad 5470, Corrientes 3400, Argentina.
Leiva
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Maruñak
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Teibler
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Acosta de Pérez
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D000818:Animals; D017837:Bothrops / Q000502:physiology*; D002364:Caseins / Q000737:chemistry; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D003435:Crotalid Venoms / Q000201:enzymology / Q000633:toxicity*; D003636:DEAE-Cellulose; D004487:Edema / Q000139:chemically induced* / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000737:chemistry; D006470:Hemorrhage / Q000097:blood / Q000139:chemically induced*; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D051379:Mice; D018482:Muscle, Skeletal / Q000473:pathology; D009135:Muscular Diseases / Q000139:chemically induced* / Q000473:pathology; D009336:Necrosis
D002364:Caseins; D003435:Crotalid Venoms; D005340:Fibrinogen; D003636:DEAE-Cellulose; D045726:Metalloproteases