Purification and Characterization of a New Weak Hemorrhagic Metalloproteinase BmHF-1 from Bothrops marajoensis Snake Venom

Abstract

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.

Insights into the Mechanisms Involved in Strong Hemorrhage and Dermonecrosis Induced by Atroxlysin-Ia, a PI-Class Snake Venom Metalloproteinase

“…As described here, hemorrhage caused by Atroxlysin-Ia was comparable to hemorrhage induced by PIII-class SVMPs. Atroxlysin-Ia MHD was 2.2 μg, which is very similar to the values detected for the PIII-class SVMPs Batroxrhagin (1.4 µg/mice) [ 24 ] and Jararhagin (1.5 µg/mice) [ 36 ] and much higher than the MHD observed for most of the other PI-class SVMP described in the literature ranging from 10 to 50 µg/mice [ 20 , 21 , 22 , 23 ], besides many others that do not cause hemorrhage at all [ 17 , 18 , 19 ].…”

“…For example, Neuwiedase from B. neuwiedi [ 16 ], BjussuMP-II from B. jararacussu [ 17 ], BmooMP-α1 from B. moojeni [ 18 ] and Leucurolysin-A from B. leucurus [ 19 ] do not induce any hemorrhage. On the other hand, BpirMP from B. pirajai [ 20 ], BmHF-1 from B. marajoensis [ 21 ], BaP1 from B. asper [ 22 ], and Batroxase from B. atrox [ 23 ] are able to induce certain levels of hemorrhage exhibiting minimal hemorrhagic dose (MHD) of 10–50 µg/mice, which is very high compared to the MHD of PII-class and PIII-class SVMPs that is frequently below 1 µg/mice. However, many issues about the mechanism of hemorrhage induction by PI-class SVMPs still need to be resolved.…”

Snake Venom Extracellular vesicles (SVEVs) reveal wide molecular and functional proteome diversity

“…Before each proteolytic assay, the sample was submitted to probe tip sonication to release the SVEVs proteins. Every enzymatic assay performed was positive (Table 2 ), with high activity detected with azocasein substrate (566.53 ± 14.15 U/mg) when compared to others SVMPs 94 , 95 . The activity towards the BApNA substrate (0.17 ± 0,012 nmol/min) was lower compared to the Rhombeobin serine proteinase 96 .…”

Caracterização dos efeitos locais e sistémicos da metaloprotease BtaHF purificada a partir do veneno total de Bothriopsis taeniata

“…O efeito edematogênico é descrito na literatura para SVMP e acompanhado de hemorragia, dependendo da potência hemorrágica da enzima. As SVMP que possuem atividade edematogênica pertencem principalmente às classes P-I e P-III (Rodriguez et al, 2001;Gay et al, 2005;Mazzi et al, 2004;Gomez et al, 2009;Torres-Huaco et al, 2010).…”

“…A formação de edema local está relacionada com a atividade proteolítica, especialmente para SVMP da classe P-I (Rodriguez et al, 2001;Texeira et al, 2005;Torres-Huaco et al, 2010). A formação de edema local pelas SVMP é um processo tempo-dependente apresentandose na primeira hora após injeção e o efeito máximo é observado entre 1 e 3 horas.…”