Isolation of two basic phospholipases A2 from Bothrops diporus snake venom: Comparative characterization and synergism between Asp49 and Lys49 variants

Abstract Bothrops diporus , previously considered a subspecies of the B. neuwiedi complex, is a medically relevant viperid in Northeastern Argentina. The venom of this species causes local tissue damage characterized by myonecrosis, hemorrhage, blistering, and edema. In the present study, two basic phospholipases A 2 (PLA 2 -I and PLA 2 -II) were isolated from this venom, and their pathological effects upon murine skeletal muscle and myogenic cells in culture were analyzed. Partial amino acid sequencing showed that PLA 2 -I and PLA 2 -II are Asp49 and Lys49 PLA 2 s, respectively. In agreement with this, PLA 2 -I showed PLA 2 activity, whereas PLA 2 -II did not. Functional assays revealed differences in their myotoxicity, cytotoxicity, and anti-adhesion activity, and in the ability to inhibit cell migration, all of which were greater for the Lys49 variant. Native electrophoresis showed that PLA 2 -I was less basic than PLA 2 -II. The two proteins act synergistically to affect the integrity of C 2 C 12 myogenic cells, providing a further example of the concerted action of coexisting snake venom components. PLA 2 -I and PLA 2 -II, together with additional basic PLA 2 s revealed by RP-HPLC, probably play an important role in myonecrosis after envenomation by B. diporus .