Purification and enzymatic characterization of a novel metalloprotease from Lachesis muta rhombeata snake venom

• Published in: Journal of Venomous Animals and Toxins including Tropical Diseases
• Main Authors: Francielle Almeida Cordeiro, Bárbara Marques Coutinho, Gisele Adriano Wiezel, Karla de Castro Figueiredo Bordon, Cristiane Bregge-Silva, Nathalia Gonsales Rosa-Garzon, Hamilton Cabral, Beatrix Ueberheide, Eliane Candiani Arantes
• Format: Article in Journal/Newspaper
• Language: English
• Published: SciELO 2018
• Subjects: Lachesis muta rhombeata; Metalloprotease; Proteases; Snake venom; Arctic medicine. Tropical medicine; RC955-962; Toxicology. Poisons; RA1190-1270; Zoology; QL1-991; Arctic
• Online Access: https://doi.org/10.1186/s40409-018-0171-x; https://doaj.org/article/c438c263bc0847a789ee88fdad8f5a5a

Abstract Background Lachesis muta rhombeata (Lmr) is the largest venomous snake in Latin America and its venom contains mainly enzymatic components, such as serine and metalloproteases, L-amino acid oxidase and phospholipases A2. Metalloproteases comprise a large group of zinc-dependent proteases that cleave basement membrane components such as fibronectin, laminin and collagen type IV. These enzymes are responsible for local and systemic changes, including haemorrhage, myonecrosis and inflammation. This study aimed the isolation and enzymatic characterization of the first metalloprotease (Lmr-MP) from Lmr venom (LmrV). Methods and results Lmr-MP was purified through two chromatographic steps and submitted to enzymatic characterization. It showed proteolytic activity on azocasein with maximum activity at pH 7.0–9.0. It was inhibited by EDTA (a metal chelator that removes zinc, which is essential for enzymatic activity) and no effect was observed with PMSF, iodoacetic acid or pepstatin (inhibitors of serine, cysteine and aspartyl proteases, respectively). Ca2+, Mg2+ and Ba2+ ions increased its activity, while Al3+, Cu2+, Ni2+ and Zn2+ inhibited it. Additionally, ZnCl2 showed a dose dependent inhibition of the enzyme. Lmr-MP activity was also evaluated upon chromogenic substrates for plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) showing the highest activity on S-2302. The activity in different solutions (5 mM or 50 mM ammonium bicarbonate, pH 7.8; 0.1% trifluoroacetic acid + 50% acetonitrile; phosphate buffer saline, pH 7.4; 50 mM sodium acetate, pH 4.0 or ammonium acetate pH 4.5) was also evaluated and the results showed that its activity was abolished at acidic pHs. Its molecular mass (22,858 Da) was determined by MALDI-TOF and about 90% of its primary structure was verified by high-resolution mass spectrometry using HCD and ETD fragmentations and database search against the sequence of closely related species. It is a novel enzyme which shared high ...