Purification and characterization of a hemorrhagic metalloproteinase from Bothrops lanceolatus (Fer-de-lance) snake venom.
Bothrops snake venoms contain metalloproteinases that contribute to the local effects seen after envenoming. In this work, a hemorrhagic metalloproteinase (BlaH1) was purified from the venom of the snake Bothrops lanceolatus by a combination of gel filtration, affinity (metal chelating) and hydrophobic interaction chromatographies. The hemorrhagin was homogeneous by SDS-PAGE and had a molecular mass of 28 kDa that was unaltered by treatment with beta-mercaptoethanol. BlaH1 gave a single band in immunoelectrophoresis and immunoblotting using commercial bothropic antivenom. BlaH1 had hemorrhagic, caseinolytic, fibrinogenolytic, collagenolytic and elastinolytic activities, but no phospholipase A(2) activity. The hemorrhagic and caseinolytic activities were inhibited by EDTA, indicating that they were metal ion-dependent. In contrast, aprotinin, benzamidine and PMSF did not affect these activities. The caseinolytic activity of BlaH1 had a pH optimum of 8.0 and was stable in solution at up to 40 degrees C; activity was completely lost at > or =70 degrees C. The hemorrhagic activity was neutralized by commercial bothropic antivenom. These properties suggest that this new hemorrhagin belongs to class P-I snake venom metalloproteinases.
Stroka
Alessandra
A
Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), CP 6111, 13083-970 Campinas, SP, Brazil.
Donato
José L
JL
Bon
Cassian
C
Hyslop
Stephen
S
de Araújo
Albetiza Lôbo
AL
D000818:Animals; D007611:Aprotinin / Q000378:metabolism; D001550:Benzamidines / Q000378:metabolism; D017837:Bothrops; D002364:Caseins / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D017364:Collagenases / Q000378:metabolism; D003435:Crotalid Venoms / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004492:Edetic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004950:Esterases / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D015151:Immunoblotting; D008297:Male; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature
D001550:Benzamidines; D002364:Caseins; D003435:Crotalid Venoms; D005340:Fibrinogen; D007611:Aprotinin; D004492:Edetic Acid; D004950:Esterases; D010741:Phospholipases A; D010450:Endopeptidases; C041605:hemorrhagic proteinase IV; D017364:Collagenases; D008666:Metalloendopeptidases; C032157:benzamidine