BnSP-7, a Lys49 myotoxic
phospholipase A(2) homologue from Bothrops neuwiedi pauloensis
venom, was structurally and functionally characterized. Several
biological activities were assayed and compared with those of the chemically modified toxin involving specific
amino acid residues. The
cDNA produced from the total
RNA by RT-PCR contained approximately 400 bp which codified its 121
amino acid residues with a calculated pI and molecular weight of 8.9 and 13,727, respectively. Its amino acid sequence showed strong similarities with several Lys49
phospholipase A(2) homologues from other Bothrops sp.
venoms. By affinity chromatography and gel diffusion, it was demonstrated that
heparin formed a complex with BnSP-7, held at least in part by electrostatic interactions. BnSP-7 displayed bactericidal activity and promoted the blockage of the neuromuscular contraction of the chick biventer cervicis muscle. In addition to its in vivo myotoxic and
edema-inducing activity, it disrupted
artificial membranes. Both BnSP-7 and the crude
venom released
creatine kinase from the mouse gastrocnemius muscle and induced the development of a dose-dependent
edema. His, Tyr, and Lys residues of the toxin were chemically modified by
4-bromophenacyl bromide (BPB),
2-nitrobenzenesulfonyl fluoride (NBSF), and
acetic anhydride (AA), respectively. Cleavage of its N-terminal octapeptide was achieved with
cyanogen bromide (CNBr). The bactericidal action of BnSP-7 on Escherichia coli was almost completely abolished by acetylation or cleavage of the N-terminal octapeptide. The
neuromuscular effect induced by BnSP-7 was completely inhibited by
heparin, BPB, acetylation, and CNBr treatment. The
creatine kinase releasing and
edema-inducing effects were partially inhibited by
heparin or modification by BPB and almost completely abolished by acetylation or cleavage of the N-terminal octapeptide. The
rupture of
liposomes by BnSP-7 and crude
venom was dose and temperature dependent. Incubation of BnSP-7 with
EDTA did not change this effect, suggesting a Ca(2+)-independent membrane lytic activity. BnSP-7 cross-reacted with
antibodies raised against B. moojeni (MjTX-II), B. jararacussu (
BthTX-I), and B. asper (Basp-II)
myotoxins as well as against the C-terminal
peptide (residues 115-129) from Basp-II.