Purification and characterization of a new weak hemorrhagic metalloproteinase BmHF-1 from Bothrops marajoensis snake venom.
BmHF-1, from the venom of Bothrops marajoensis, was purified by Sephadex G-75 and HPLC-RP on micro-Bondapak C-18 column chromatography. It presented a molecular mass of 27162.36 Da determined by MALDI-TOF MS. BmHF-1 had a sequence of 238 residues of amino acids. The multiple alignment of its amino acid sequence and those of other snake venom metalloproteinases showed high structural similarity, mainly among P-I class. The enzyme initially cleaves the Aalpha-chain of fibrinogen, followed by the Bbeta-chain, and shows no effects on the gamma-chain. BmHF-1 had, caseinolytic and weakly hemorrhagic activities, which were inhibited by EDTA. In contrast, PMSF did not affect these activities. The caseinolytic activity of BmHF-1 had a pH optimum of 8.0 and was stable in solution up to 40 degrees C; activity was completely lost at > or = 70 degrees C. The proteolytic activity was also inhibited by sDa (opossum sera) and Da2-1, Da2-II, antihemorrhagic factors isolated from the opossum sera of Didelphis albiventris. BmHF-1 presents weak hemorrhagic activity, with a MHD of 41.14 microg and it induces dose-dependent edema. We could concluded that, despite its weak hemorrhagic activity, BmHF-1 contributes to local tissue damage by inducing edema, releasing pharmacologically active mediators from protein precursors due to its enzymatic action.
Torres-Huaco
Frank Denis
FD
Department of Biochemistry, Institute of Biology (IB), State University of Campinas (UNICAMP), P.O. Box 6109, Campinas, SP 13083-970, Brazil.
Ponce-Soto
Luis Alberto
LA
Martins-de-Souza
Daniel
D
Marangoni
Sergio
S
D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D017837:Bothrops / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D003435:Crotalid Venoms / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D006470:Hemorrhage / Q000139:chemically induced / Q000201:enzymology; D008297:Male; D045726:Metalloproteases / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013694:Temperament
D003435:Crotalid Venoms; D005340:Fibrinogen; D045726:Metalloproteases