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

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 (PLA-I and PLA-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-I and PLA-II are Asp49 and Lys49 PLAs, respectively. In agreement with this, PLA-I showed PLA activity, whereas PLA-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-I was less basic than PLA-II. The two proteins act synergistically to affect the integrity of CC myogenic cells, providing a further example of the concerted action of coexisting snake venom components. PLA-I and PLA-II, together with additional basic PLAs revealed by RP-HPLC, probably play an important role in myonecrosis after envenomation by B. diporus.