Isolation of Snake Venom Metalloproteinases

The PI SVMP BaP1 was isolated from the venom of Bothrops asper, as described by Gutiérrez et al. [22 (link)] and Watanabe et al. [23 (link)], by a combination of ion-exchange chromatography on CM-Sepharose, followed by affinity chromatography on Affi-gel Blue. The PIII SVMP CsH1 was isolated from Crotalus simus venom, as described by Herrera et al. [7 (link)], by ion-exchange chromatography on DEAE-Sepharose, followed by gel filtration on a Superdex TM 200 10/300GL (GE Healthcare, LifeSciences) column (10 x 300 mm) using an ÄKTA FPLC (GE Healthcare, Life Sciences). Homogeneity of SVMP preparations was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Both toxins were isolated from the venoms of more than 40 adult specimens of each species collected in Costa Rica and maintained at the serpentarium of Instituto Clodomiro Picado, Costa Rica. After collection, venoms of each species were separately pooled, lyophilized, and stored at -20°C until used. The Minimum Hemorrhagic Dose (MHD), corresponding to the amount of enzyme that induces a hemorrhagic spot of 10 mm diameter in mice 2 h after injection, is 20 μg for BaP1 [22 (link)] and 2.2 μg for CsH1 [7 (link)].