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Abstract
Abstract Conditions leading to cleavage of all of the disulfide bridges in ribonuclease and several other proteins (0.32 m 2-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 (1-RCM papain). Immunological interactions with antipapain serum indicated identity between 1-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 1-RCM papain was reduced instead of the native enzyme; in both cases the product of reduction and alkylation was 3-RCM papain.
Highlights
The results presented in this report show that only partial reduction of native papain is attained in 8 M urea, involving the opening of only one specific disulfide bond
The results are given in Table I. -4s shown, after reduction of papain in aqueous solution only one free sulfhydryl group was det,ected, presumably the one which is involved in the active site
When the reduction was performed in the presence of 6 M guanidine hydrochloride, all three disulfide bonds were disrupted
Summary
Reduction of Papain-The reduction of papain was carried out either in aqueous solution, 8 M urea, or 6 M guanidine hydrochloride. The pH in all cases was adjusted to pH 8.2 with Tris-HCl buffer. In the case of Hz0 or 8 M urea addition of 0.5 M Tris-HCl buffer to a final concentration of 0.05 M was applied, whereas in the case of 6 M guanidine hydrochloride solid Tris powder was used. The papain concentration varied in different experiments within the region of 10 to 30 pg per ml. Nitrogen was bubbled through the papain solutions prior to the addition of the reducing agent. In most experiments the reduction was carried out by the addition of 2-mercaptoethanol to a final concentration of 0.32
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