The reaction of papain with Ellman's reagent (5,5'-dithiobis- (2-nitrobenzoate) dianion).
1. The report by Robyt et al. (1971) that the 2-nitro-5-mercaptobenzoate dianion (Nbs(2-)) produced by reaction of papain with the 5,5'-dithiobis-(2-nitrobenzoate) dianion (Nbs(2) (2-); Ellman's 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 Nbs(2) (2-) at pH8, Nbs(2) (2-) reacts with the protein in an amount stoicheiometric with the cysteinyl thiol group of papain to produce Nbs(2-) 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 Nbs(2) (2-) at pH10.5 probably by nucleophilic catalysis involving the enzyme's 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.
D001565:Benzoates; D002384:Catalysis; D003545:Cysteine; D003553:Cystine; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007202:Indicators and Reagents; D010206:Papain; D011506:Proteins; D013440:Sulfides
D001565:Benzoates; D007202:Indicators and Reagents; D011506:Proteins; D013440:Sulfides; D003553:Cystine; D010206:Papain; D003545:Cysteine