Amino Acid Sequences of Three β-Bungarotoxins (β3-, β4-, and β5-Bungarotoxins) from Bungarus multicinctus Venom. Amino Acid Substitutions in the A Chains

Abstract

The two most basic beta-bungarotoxins (beta 3- and beta 4-toxins) and another, less neurotoxic beta-bungarotoxin (beta 5-toxin) were purified from Bungarus multicinctus venom, by a combination of CM-Sephadex C-25 column chromatography and Sephadex G-75 gel filtration. The three toxins consisted of two dissimilar polypeptides (A chain, 120 amino acid residues; B chain, 60 residues). The LD50 values of the beta 3- and beta 4-toxins were 0.066 micrograms and 0.072 micrograms/g of mouse, respectively, and their phospholipase A activities were 43.2 and 36.5 units/mg of toxin, respectively. beta 5-Toxin was weaker in neurotoxicity (LD50, 0.13 micrograms/g of mouse) than the others, and its phospholipase activity was 47.6 units/mg of toxin. Each toxin was separated into RCM-A and RCM-B chains after reduction and S-carboxymethylation. The RCM-polypeptides were maleylated and digested with TPCK-trypsin. The tryptic peptides were sequenced with manual Edman degradation or the dansyl-Edman method. The final alignment of the tryptic peptides from the respective RCM-polypeptides was deduced on the basis of the amino acid sequences of the A and B chains of beta 1-bungarotoxin (beta 1-toxin). The amino acid sequences of the A chains of the beta 3- and beta 4-toxins were identical but differed from those of the A chains of the beta 1- and beta 2-toxins by 4 amino acid substitutions in the COOH-terminal portions (residues 109-120) and substitution at position 87. The amino acid sequences of the B chains of the beta 3- and beta 4-toxins differed from each other, but they were identical with those of the B chains of the beta 1- and beta 2-toxins, respectively. The amino acid sequence of the A chain of beta 5-toxin differed from that of the A chain of beta 1-toxin by consecutive substitutions in residues 55-60 and substitutions at positions 23, 87, and 89. The amino acid sequence of the B chain of beta 5-toxin was identical with those of the B chains of beta 1- and beta 3-toxin. From our results on the effects of the amino acid displacements found in the A chains on the neurotoxicity, it was concluded that the COOH-terminal portion in the A chains was not essential to their neurotoxicity, whereas the region of residues 55-60 in the A chains appeared to participate in the constitution of the neurotoxically active site of the beta-toxins.

Recognition of Bungarus multicinctus Venom by a DNA Aptamer against β-Bungarotoxin

“…Although α-bungarotoxin (α-BuTx) is the major component (61%) of the B. multicinctus venom [23], β-bungarotoxin (β-BuTx) is more important and lethal than α-BuTx from a toxicological or clinical perspective [24]. β-BuTx constitutes >20% of the protein content of the crude venom [25]. The minimum lethal dose of β-BuTx by intraperitoneal injection of mice is 0.01 µg/g [26], while that of α-BuTx is 0.139 µg/g [27].…”

Protein complexes in snake venom

“…So far, as many as 16 isoforms have been reported from Bungarus species [5], and 8 of them, namely b1-b5 [6] and SPI-SPIII [7], have been studied in detail. Structurally, they are covalently linked heterodimers (Fig.…”

Sequence analysis and phylogenetic relationship of genes encoding heterodimeric phospholipases A2 from the venom of the scorpion Anuroctonus phaiodactylus

“…β-bungarotoxin was isolated from the snake Bungarus multicinctus and was classified in group II (Kondo et al, 1982;Delot and Bon, 1973); conodipin-m was purified from the cone snail Conus magus, classified into group IX (McIntosh et al, 1995); IpTxI and Phospholipin were isolated from the venom of the African scorpion Pandinus imperator (Zamudio et al, 1997;Conde et al, 1999) and Phaiodactylipin from the scorpion Anuroctonus phaiodactylus (Valdez-Cruz et al, 2004). The last three were placed into group III.…”