Cyanogen Bromide Cleavage of the Two Constituent Polypeptide Chains of Ricin D

Two constituent polypeptide chains of ricin D, a highly toxic protein from castor bean seeds, were cleaved with cyanogen bromide (CNBr). Four CNBr fragments, α-CB I, α-CB II, α-CB III, and α-CB IV, were isolated from the Ile (α) chain by gel filtration through Bio-Gel P–30 and characterized. N-Terminal sequence analyses revealed that the alignment of these fragments was (α-CB I)-(α-CB IV)-(α-CB II)-(α-CB III). CNBr cleavage of Ala (ß) chain, followed by reduction and S-carboxymethylation, yielded four fragments, ß–CB I, ß–CB II, ß–CB III, and ß–CB IV. Methionine residue located at 5th position from N-terminus of Ala chain was found to be resistant to CNBr cleavage. Sequence analyses of these fragments revealed that fragment ß-CB IV was originated from fragment ß-CB III by acid cleavage, not by CNBr cleavage of aspartyl-prolyl bond during CNBr treatment, and the alignment of CNBr fragments was established to be (ß-CB II)-(ß-CB I)-(ß-CB III).