Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2′-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiols

Abstract

1. The u.v.-spectral characteristics of 5,5′-dithiobis-(2-nitrobenzoic acid) (Nbs2), 2,2′-dipyridyl disulphide (2-Py–S–S–2-Py), 4,4′-dipyridyl disulphide (4-Py–S–S–4-Py), 5-mercapto-2-nitrobenzoic acid (Nbs), 2-thiopyridone (Py–2-SH) and 4-thiopyridone (Py–4-SH) were determined over a wide range of pH and used to calculate their acid dissociation constants. 2. The reactions of l-cysteine, 2-mercaptoethanol and papain with the above-mentioned disulphides were investigated spectrophotometrically in the pH range 2.5–8.5. 3. Under the conditions of concentration used in this study the reactions of both low-molecular-weight thiols with all three disulphides resulted in the stoicheiometric release of the thiol or thione fragments Nbs, Py–2-SH and Py–4-SH at all pH values. The rates of these reactions are considerably faster at pH8 than at pH4, which suggests that the predominant reaction pathway in approximately neutral media is nucleophilic attack of the thiolate ion on the unprotonated disulphide. 4. The reaction of papain with Nbs2 is markedly reversible in the acid region, and the pH-dependence of the equilibrium constant for this system in the pH range 5–8 at 25°C and I=0.1 is described by: [Formula: see text] 5. Papain reacts with both 2-Py–S–S–2-Py and 4-Py–S–S–4-Py in the pH range 2.5–8.5 to provide release of the thione fragments, stoicheiometric with the thiol content of the enzyme. 6. Whereas the ratios of the second-order rate constant for the reaction at pH4 to that at pH8 for the cysteine–2-Py–S–S–2-Py reaction (kpH4/kpH8=0.015) and for the papain–4-Py–S–S–4-Py reaction (kpH4/kpH8=0.06) are less than 1, that for the papain–2-Py–S–S–2-Py reaction is greater than 1 (kpH4/kpH8=15). 7. This high reactivity of papain has been shown to involve reaction of the thiol group of cysteine-25, the enzyme's only cysteine residue, which is part of its catalytic site. 8. That this rapid and stoicheiometric reaction of the thiol group of native papain is not shown either by low-molecular-weight thiols or by the thiol group of papain after its active conformation has been destroyed by acid or heat denaturation, strongly commends 2-Py–S–S–2-Py as one of the most useful papain active-site titrants discovered to date. This reagent has been shown to allow accurate titration of papain active sites in the presence of up to 10-fold molar excess of l-cysteine and up to 100-fold molar excess of 2-mercaptoethanol.