1. The u.v.-spectral characteristics of 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs(2)), 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 Nbs(2) 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 degrees 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 (k(pH4)/k(pH8)=0.015) and for the papain-4-Py-S-S-4-Py reaction (k(pH4)/k(pH8)=0.06) are less than 1, that for the papain-2-Py-S-S-2-Py reaction is greater than 1 (k(pH4)/k(pH8)=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.

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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.

Reactivities of the Various Protonic States in the Reactions of Papain and of L-Cysteine with 2,2'and with 4,4'-Dipyridyl Disulphide:EvidenceforNucleophilic Reactivity in the Un-ionized Thiol Group of the Cysteine-25 Residue of Papain Occasioned by its Interaction with the Histidine-159-Asparagine-175 Hydrogen-Bonded System

The pH‐depcndcncc ol reactions at room temperature undcrgonc by model phenolic alcohols and ethers is examined. The kinetics of the self‐condensation of 3,5‐dimethyl‐4‐hydroxyhenzyl alcohol and of its condensation with 2,6‐xylenol are determined and mechanisms of reaction deduced. The setting o f plicnol‐formaldelivde resins a t room temperature is discussed in the light of thcsc experiments.

In the accompanying paper evidence is presented for the existence of nucleophilic reactivity in the un-ionized thiol group of cysteine-25 in papain (EC 3.4.4.10) occasioned by its interaction with the imidazole group of the histidine-159-asparagine-175 hydrogen-bonded system. This evidence arises from kinetic analysis of the reactions of the papain thiol group with 2,2'and with 4,4'-dipyridyl disulphide.These results, together with the principle of microscopic reversibility, suggest that the reaction of a thiolate ion with a mixed disulphide composed of papain and another thiol should be subject to general acid catalysis by the imidazolium ion ofhistidine-159. The reactions with the above disulphides are effectively irreversible because the 'thiol' products exist very largely in the tautomeric thione forms.To investigate the reverse reaction of a thioldisulphide interchange involving papain, we carried out a kinetic analysis of the reaction of papain with 5,5'-dithiobis-(2-nitrobenzoate) dianion and its reverse, the reaction of 5-mercapto-2-nitrobenzoate dianion with the papain-5-mercapto-2-nitrobenzoate disulphide. This demonstrates a pH-rate profile for the reverse reaction characteristic of the expected general acid catalysis by histidine-159. It demonstrates also a profile for the forward reaction characteristic of nucleophilic reactivity in the un-ionized thiol group of papain towards a disulphide that, by contrast with the pyridyl disulphides, cannot effectively increase its electrophilicity by protonation. Materials and methodsActive enzyme was prepared as reported previously (Brocklehurst & Little, 1970). 5,5'-Dithiobis-(2nitrobenzoic acid) (m.p. 238-240°C), recrystallized from acetic acid, was shown to contain less than 0.1 % of 5-mercapto-2-nitrobenzoic acid by spectrophotometric analysis. Apparent dissociation constants at 25.0°C and I0.1 determined spectrophotometrically are: 5,5'-dithiobis-(2-nitrobenzoic acid), 4.4.The stoicheiometries and kinetics of the reactions of 5,5'-dithiobis-(2-nitrobenzoate) dianion with papain, L-cysteine and 2-mercaptoethanol at 25.0°C and 10.1 were determined spectrophotometrically at 412nm in deoxygenated buffers containing 1 mm-EDTA. Stoicheiometries were calculated by using E412= 1.36 x 104/(1 + [H+]/4 x 10-5)M-1 cm-1. In the papain reaction at pH values below 7 the infinity readings of preliminary experiments showed that progress curves represent reactions approaching a pH-dependent equilibrium position (eqn. 1) in which Pap-SH represents papain and Ar represents 02N\ HO2C k+2 Pap-SH + Ar-S-S-Ar^. Pap-S-S-Ar + Ar-SH k-2 (1) The analysis in terms of eqn. (1) when [Ar-S-S-Aro] > [Pap-SHo] and [Ar-SHO]=[Pap-S-S-ArO]=0 was carried out as described by Frost & Pearson (1963) with a computer program. The computer printed out values of k+2, k2 and Keq. (=k+2/k_2). Results and discussionThe reactions of 5,5'-dithiobis-(2-nitrobenzoate) dianion with L-cysteine and with 2-mercaptoethanol in the pH range 5-9 are first-order in each reactant. The pH-dependences of the second-ord...