1. The report by Robyt et al. (1971) that the 2-nitro-5-mercaptobenzoate dianion (Nbs2−) produced by reaction of papain with the 5,5′-dithiobis-(2-nitrobenzoate) dianion (Nbs22−; Ellman9s reagent) cleaves the three disulphide bonds in papain is shown to be incorrect. 2. When partially active papain containing approx. 0.4 mol of thiol/mol of protein is incubated with excess of Nbs22− at pH8, Nbs22− reacts with the protein in an amount stoicheiometric with the cysteinyl thiol group of papain to produce Nbs2− in an amount stoicheiometric with the original papain cysteinyl thiol group, and the catalytically inactive mixed disulphide, papain–Nbs−. 3. Papain catalyses the hydrolysis of Nbs22− at pH10.5 probably by nucleophilic catalysis involving the enzyme9s thiol group. 4. These results cast very serious doubts on the claim by Robyt et al. (1971) to have established a new general method for the determination of cystinyl disulphide residues in proteins.
