Pharmacological and partial biochemical characterization of Bmaj-9 isolated from Bothrops marajoensis snake venom

Bmaj-9, a basic PLA₂ (13679.33 Da), was isolated from Bothrops marajoensis snake venom through only one chromatographic step in reversed phase HPLC on μ-Bondapak C-18 column. The amino acid composition showed that Bmaj-9 had a high content of Lys, His, and Arg, typical of a basic PLA₂. The sequence of Bmaj-9 contains 124 amino acid residues with a pI value of 8.55, such as DLWQWGQMIL KETGKLPFSY YTAYGCYCGW GGRGGKPKAD TDRCCFVHDC, revealing a high homology with Asp49 PLA₂ from other snake venoms. It also exhibited a pronounced phospholipase A₂ activity when compared with crude venom. In chick biventer cervicis preparations, the time for 50% and 100% neuromuscular paralysis was respectively (in minutes): 110 ± 10 (1 μg/mL); 40 ± 6 and 90 ± 2 (5 μg/mL); 30 ± 3 and 70 ± 5 (10 μg/mL); 42 ± 1 and 60 ± 2 (20 μg/mL), with no effect on the contractures elicited by either exogenous ACh (110 μM) or KCl (20 mM). Bmaj-9 (10 μg/mL) neither interfered with the muscular response to direct electrical stimulation in curarized preparations nor significantly altered the release of CK at 0, 15, 30 and 60 minutes incubations (27.4 ± 5, 74.2 ± 8, 161.0 ± 21 and 353.0 ± 47, respectively). The histological analysis showed that, even causing blockade at the maximum dosage (5 μg/mL), the toxin does not induce significant morphological alterations such as necrosis or infiltration of inflammatory cells. These results identified Bmaj-9 as a new member of the basic Asp49 PLA₂ family able to interact with the motor nerve terminal membrane, thereby inducing a presynaptic neuromuscular blockade.