title: Kinetic and thermodynamic studies on the disulfide-bond reducing potential of hydrogen sulfide.
abstract: The significance of persulfide species in hydrogen sulfide biology is increasingly recognized. However, the molecular mechanisms of their formation remain largely elusive. The obvious pathway of the reduction of biologically abundant disulfide moieties by sulfide was challenged on both thermodynamic and kinetic grounds. Using DTNB (5,5'-dithiobis-(2-nitrobenzoic acid), also known as Ellman's reagent) as a model disulfide we conducted a comprehensive kinetic study for its reaction with sulfide. The bimolecular reaction is relatively fast with a second-order rate constant of 889 ± 12 M(-1)s(-1) at pH = 7.4. pH dependence of the rate law revealed that the reaction proceeds via the bisulfide anion species with an initial nucleophilic thiol-disulfide exchange reaction to give 5-thio-2-nitrobenzoic acid (TNB) and TNB-persulfide with a pH independent second-order rate constant of 1090 ± 12 M(-1)s(-1). However, kinetic studies and stoichiometric analyses in a wide range of reactant ratios together with kinetic simulations revealed that it is a multistep process that proceeds via kinetically driven, practically irreversible reactions along the disulfide → persulfide → inorganic polysulfides axis. The kinetic model postulated here, which is fully consistent with the experimental data, suggests that the TNB-persulfide is further reduced by sulfide with a second-order rate constant in the range of 5 × 10(3) - 5 × 10(4) M(-1)s(-1) at pH 7.4 and eventually yields inorganic polysulfides and TNB. The reactions of cystine and GSSG with sulfide were found to be significantly slower and to occur via more complicated reaction schemes. (1)H NMR studies suggest that these reactions also generate Cys-persulfide and inorganic polysulfide species, but in contrast with DTNB, in consecutive equilibrium processes that are sensitive to changes in the reactant and product ratios. Collectively, our results demonstrate that the reaction of disulfides with sulfide is a highly system specific process from both thermodynamic and kinetic aspects, which together with the considerable steady-state concentrations of the reactants in biological systems signifies physiological relevance.
authors.affiliation: Department of Molecular Immunology and Toxicology, National Institute of Oncology, Ráth György utca 7-9, Budapest 1122, Hungary; Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem tér 1, Debrecen 4010, Hungary.
authors.forename: Anita
authors.initials: A
authors.lastname: Vasas
authors.affiliation: Department of Molecular Immunology and Toxicology, National Institute of Oncology, Ráth György utca 7-9, Budapest 1122, Hungary; Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem tér 1, Debrecen 4010, Hungary.
authors.forename: Éva
authors.initials: É
authors.lastname: Dóka
authors.affiliation: Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem tér 1, Debrecen 4010, Hungary.
authors.forename: István
authors.initials: I
authors.lastname: Fábián
authors.affiliation: Department of Molecular Immunology and Toxicology, National Institute of Oncology, Ráth György utca 7-9, Budapest 1122, Hungary. Electronic address: peter.nagy@oncol.hu.
authors.forename: Péter
authors.initials: P
authors.lastname: Nagy
mesh_terms: D004220:Disulfides / Q000737:chemistry*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D006862:Hydrogen Sulfide / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D013816:Thermodynamics
chemical_list: D004220:Disulfides; D004228:Dithionitrobenzoic Acid; D006862:Hydrogen Sulfide
keywords: Disulfide reduction; Hydrogen sulfide; Kinetics and mechanisms; Persulfide; Speciation