Three three-finger proteins (BM8, BM11, BM14) were isolated from Bungarus multicinctus (Taiwan banded krait) venom using successive chromatography on a ion-exchanger column and reverse phase HPLC column. BM8 and BM14 contain 82 amino acid residues including 10 cysteine residues, and BM11 contain 68 amino acid residues including 10 cysteine residues. Unlike snake venom cardiotoxins and a-neurotoxins, the three proteins contain two additional cysteine residues in their N-terminal region. Noticeably, only two amino acid substitutions occurred at positions 37 and 38 were observed between BM8 and BM14. CD measurement revealed that their secondary structures were dominant with b-sheet structure as noted with cardiotoxins and a-neurotoxins. However, the gross conformations of BM8, BM11 and BM14 were not the same as evidenced by CD spectra and acrylamide quenching studies. In contract to BM8, BM11 and BM14 exhibit an activity on blocking [3H]QNB binding to muscarinic acetylcholine receptor (mAChR). Modification of Lys residues in BM14 resulted in a decreased binding activity, indicating that the Lys residues may be involved in the mAChR binding. The genomic DNA with the size of approximate 2.3 kb and 2.4 kb, respectively, encoded the precursors of BM14 and BM11 was amplified by PCR. The genes shared virtually identical overall organization with snake venom cardiotoxin and£\-neurotoxin genes, composed of three exons and two introns. Comparison of BM11 and BM14 genes with cardiotoxins and a-neurotoxins genes showed that the exon regions were more diversified than intron regions. This implicated that the accelerated evolution may be involved in the evolution of these genes in order to acquire newly arising functions. Nevertheless, several transcriptional factor binding sites including SP-1, CACCC-binding factor and NF-KB were highly conserved in the promoter regions. It reflected a common regulation mechanism in controlling the transcription of these genes. These observations indicate that the three proteins are evolutionarily related to cardiotoxins and a-neurotoxins.

beta-Bungarotoxin (beta-Bgt), the presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of the A chain and the B chain, cross-linked by an interchain disulfide bond. In this study, two novel beta-Bgt isotoxins were purified from Bungarus multicinctus venom by the combinations of ion-exchange chromatography and reverse phase HPLC. Amino acid sequencing, peptide mapping and mass analyses revealed that they probably contained the same A chain, but their B chain differed. Consequently, the discrepancies in their biological activity and fine structure reflected the role of B chain in intact of beta-Bgt. In Yeast-Two-Hybrid system, a potassium channel binding protein was identified to interact with the B chain of beta-Bgt. Although the recombinant potassium channel binding protein functionally bound with Ca2+, but it could not prove to bind with BM12 and BM13 as revealed by in vitro cross-linking assay. The A chain genes including A1 chain, A2 chain and A8 chain genes were amplified by PCR reaction. Their nucleotide sequences shared up to 97.5% identity. Alignment of the determined A chain genes with A chain cDNAs revealed that the A1 chain gene was organized with four exon and three intron, while A2 chain gene comprised three exons and two introns. When A2 chain is expressed, the region corresponds to the first exon of A1 chain gene is skipped instead of inclusion of intronic sequence adjacent to the second intron. The resulting A2 chain mRNA encoded a 25 residues signal peptide, which different from A1 chain mRNA with a 27 residues signal peptide. Comparative analyses on phospholipase A2 genes and cDNAs suggest that this is the first report on skipping of exon changes the signal peptide sequence of snake venom proteins.