Separation and characterization of the A chain and B chain in beta 1-bungarotoxin from Bungarus multicinctus (Taiwan banded krait) venom.

The interchain disulfide bond between A chain and B chain of beta 1-bungarotoxin (beta 1-Bgt) was selectively cleaved by dithiothreitol, and the A and B chains were separated by HPLC. The separated A and B chains did not show detectable enzymatic activity and lethal toxicity, but exhibited an immunoreactivity with anti-beta 1-Bgt antibody. Analytical isoelectrofocusing revealed that the A chain is a neutral subunit with pI = 7.4, and the B chain is a basic one with pI = 9.6. The A chain exhibited a Ca(2+)-binding ability as revealed by fluorescence measurement. Moreover, fluorescence studies showed that the intact interchain disulfide bond is essential for maintaining the hydrophobic character of substrate binding site in beta 1-Bgt and stabilizing the architectural environment of Trp-19 in the A chain. However, combination of the A chain and B chain failed to restore the biological activities and physiochemical properties which the intact beta 1-Bgt possessed. These, together with our previous result that the Trp-19 of the A chain is involved in substrate binding, suggest that the integrity of the interchain disulfide bond favors the maintenance of the active conformation of beta 1-Bgt.