Cleavage of one specific disulfide bond in papain.

Conditions leading to cleavage of all of the disulfide bridges in ribonuclease and several other proteins (0.32 M Z-mercaptoethanol in 8 M urea) caused in papain only partial reduction of the disulfide bonds. Electrophoretic studies indicated that alkylation of the partially reduced derivative yielded a unique molecular species (3-RCM papain) in which one specific disulfide bond had been split, rather than a mixture of unreduced and open chain papain. On the other hand, reduction of papain in 6 M guanidine hydrochloride, followed by alkylation, yielded a completely open polypeptide chain (7-RCM papain). Alkylation of native papain after reduction in aqueous solution resulted in the sole blocking of the active sulfhydryl group (I-RCM papain). Immunological interactions with antipapain serum indicated identity between l-RCM papain and native papain, whereas 7-RCM papain was completely unreactive. 3-RCM papain cross-reacted with 60% of the antibodies. The single cleaved disulfide bond in 3-RCM papain was identified by isolation and analysis of the peptides containing carboxymethylcysteine, obtained from a tryptic digest. It was shown to be the bond connecting the cysteine residues in positions 43 and 152 in the amino acid sequence of papain. It was also shown that during the reduction of native papain in 8 M urea autodigestion takes place. This phenomenon was avoided when I-RCM papain was reduced instead of the native enzyme; in both cases the product of reduction and alkylation was 3-RCM papain.