Abstract
The two most basic β-bungarotoxins (β3- and β4-toxins) and another, less neurotoxic β-bungarotoxin (β5 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 β3 and β4-toxins were 0.066 μg and 0.072 μg/g of mouse, respectively, and their phospholipase A activities were 43.2 and 36.5 units/mg of toxin, respectively. β5-Toxin was weaker in neurotoxicity (LD50, 0.13 μg/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 β1-bungarotoxin (β1-toxin). The amino acid sequences of the A chains of the β3- and β4-toxins were identical but differed from those of the A chains of the β1- and β2-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 β3 and β4-toxins differed from each other, but they were identical with those of the B chains of the β1- and β1-toxins, respectively. The amino acid sequence of the A chain of β5 toxin differed from that of the A chain of β1-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 β5-toxin was identical with those of the B chains of β1 and β3 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 β-toxins.
