Purification and characterization of two high molecular mass snake venom metalloproteinases (P-III SVMPs), named SV-PAD-2 and HR-Ele-1, from the venom of Protobothrops elegans (Sakishima-habu)

We herein identified two high molecular mass metalloproteinases, named SV-PAD-2 and HR-Ele-1, in the venom of Protobothrops elegans. HR-Ele-1 appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) regard under reducing and non-reducing conditions, and the molecular mass of this protease was approximately 60 kDa under reducing conditions. On the other hand, the molecular masses of SV-PAD-2 on SDS-PAGE were 110 kDa under the non-reducing condition and 52 kDa under the reducing condition. These SVMPs exhibited fibrinogenolytic and enzymatic activities against synthetic substrates for matrix metalloproteinases (MMPs) and the insulin B-chain, and were both inhibited by EDTA. SV-PAD-2 inhibited ADP- and collagen-induced platelet aggregation, with IC50 values of 240 nM and 185 nM, respectively. HR-Ele-1 exhibited hemorrhagic activity, and its minimum hemorrhagic dose (MHD) was 0.05 μg in the guinea pig.