Novel neurotoxins from Taiwan banded krait (Bungarus multicinctus) venom: purification, characterization and gene...

Two novel neurotoxins BM10-1 and BM10-2 were isolated from Bungarus multicinctus (Taiwan banded krait) venom using the combinations of chromatography on a SP-Sephadex C-25 column and a reverse phase HPLC column. BM10-1 contained 66 amino acid residues including 10 Cys residues, while BM10-2 consisted of 65 amino acid residues with 8 Cys residues. The secondary structure of both BM10-1 and BM10-2 was dominated with beta-sheet, but their gross conformation differed as evidenced by CD spectra and acrylamide quenching studies. BM10-1 inhibited carbachol-induced muscle contraction in a reversible manner and the dose for achieving 50% inhibition was approximately fourfold that of alpha-bungarotoxin. BM10-2 exhibited an irreversible but weak inhibition on carbachol-induced muscle contraction. Sequence alignment of neurotoxins with BM10-1 and BM10-2 suggested that the manner in the manifestation of their activity could be partly elucidated by the residues at toxin second loop. The genomic DNAs encoding BM10-1 and BM10-1-like protein (BM10-1L) were amplified by PCR. The two genes shared virtually identical structural organization and high degree of sequence identity with B. multicinctus neurotoxin genes. Compared to intron sequences of these genes, the protein-coding regions were highly variable. The difference between BM10-1 gene and BM10-1L gene notably arose from the third exon. These results suggest the evolution of B. multicinctus neurotoxins via the path of gene duplication.