Reassessment of Ellman's reagent.

http://www.protocol-online.org/forums/uploads/monthly_03_2010/msg-16499-1268346199.ipb

Ellman's reagent: 5,5'-dithiobis(2-nitrobenzoic acid)--a reexamination.

The model hydrogen peroxide-myeloperoxidase-chloride system is capable of generating the powerful oxidant hypochlorous acid, which can be quantitated by trapping the generated species with the beta-amino acid, taurine. The resultant stable product, taurine chloramine, can be quantitated by its ability to oxidize the sulfhydryl compound, 5-thio-2-nitro-benzoic acid to the disulfide, 5,5'-dithiobis(2-nitroben-zoic acid) or to oxidize iodide to iodine. Using this system, purified myeloperoxidase in the presence of chloride and taurine converted stoichiometric quantities of hydrogen peroxide to taurine chloramine. Chloramine generation was absolutely dependent on hydrogen peroxide, myeloperoxidase, and chloride and could be inhibited by catalase, myeloperoxidase inhibitors, or chloride-free conditions. In the presence of taurine, intact human neutrophils stimulated with either phorbol myristate acetate or opsonized zymosan particles generated a stable species capable of oxidizing 5-thio-2-nitrobenzoic acid or iodide. Resting cells did not form this species. The oxidant formed by the stimulated neutrophils was identified as taurine chloramine by both ultraviolet spectrophotometry and electrophoresis. Taurine chloramine formation by the neutrophil was dependent on the taurine concentration, time, and cell number. Neutrophil-dependent chloramine generation was inhibited by catalase, the myeloperoxidase inhibitors, azide, cyanide, or aminotriazole and by chloride-free conditions, but not by superoxide dismutase or hydroxyl radical scavengers. Thus, it appears that stimulated human neutrophils can utilize the hydrogen peroxide-myeloperoxidase-chloride system to generate taurine chloramine. Based on the demonstrated ability of the myeloperoxidase system to generate free hypochlorous acid we conclude that neutrophils chlorinate taurine by producing this powerful oxidant. The biologic reactivity and cytotoxic potential of hypochlorous acid and its chloramine derivatives suggest that these oxidants play an important role in the inflammatory response and host defense.

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Chlorination of taurine by human neutrophils. Evidence for hypochlorous acid generation.

Sulfenic acid chemistry, detection and cellular lifetime

A model study of the radical addition of 2-mercaptoethanol onto oleic acid was performed under mild conditions (generation of radicals under UV light at room temperature without any photoinitiator). To evaluate the efficiency and the robustness of thiol−ene reaction, experimental parameters were varied, such as the irradiation intensity (ranging from 0.5 to 15.0 W/cm2), the thiol/double bond ratio (ranging from 1.2/1 to 5.0/1), the solvent/double bond ratio (ranging from 0/1 to 500/1), and the number of double bonds per chain. It was especially shown that the higher the content of polyunsaturated fatty chains, the lower the rate of 2-mercaptoethanol grafting. Best yields were reached in 1 h reaction for a thiol/ene ratio of 3/1, without solvent, using maximum UV intensity. Side reactions, identified by NMR, FT-IR, LC-MS, and iodine titration, were upon esterification between the carboxylic acid function of oleic acid and the hydroxyl function of 2-mercaptoethanol, disulfide formation, double-bond isomeriz...

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Synthesis of Biobased Polyols by Thiol−Ene Coupling from Vegetable Oils

Dithionite, bisulfite, and thiosulfate can be determined quantitatively in the presence of each other with satisfactory precision by a method involving three operationally distinct iodometric titrations. Data are presented graphically for the decomposition of aqueous solutions of sodi.um dithionite as a function of pH at initial 0.1M dithionite; initial concentrations of dithionite at pH 5.5; and initial concentrations of bisulfite, thiosulfate, and hydrosulfide at pH 5.5 and initial 0.1 M dithionite. The cumulative ratio of bisulfite formed to dithionite decomposed does not vary significantly from unity over the span of 16 to 94% decomposition in the range of pH 4.00 to 6.75, but the ratio of bisulfite formed to thiosulfate formed is consistently higher than 3:1 through much of the course of reaction, though it usually falls to a cumulative value of a little more than 2:1 terminally.

Iodometric method for the determination of dithionite, bisulfite, and thiosulfate in the presence of each other and its use in following the decomposition of aqueous solutions of sodium dithionite.

Oxidation of organic divalent sulfur by iodine. I. Alternative pathways for thiols as determined by structure

Alkaline decomposition of organic disulfides. II. Alternative pathways as determined by structure

Alkaline decomposition of organic disulfides. IV. Limitation on the use of Ellman's reagent. 2,2'-Dinitro-5,5'-dithiodibenzoic acid

Oxidation of organic divalent sulfur by iodine. II. Equilibrating thiol-iodine-disulfide-hydrogen iodide system in acetic acid and evidence for sulfenyl iodide intermediates

James P. Danehy

Papers

Iodometric method for the determination of dithionite, bisulfite, and thiosulfate in the presence of each other and its use in following the decomposition of aqueous solutions of sodium dithionite.

Oxidation of organic divalent sulfur by iodine. I. Alternative pathways for thiols as determined by structure

Alkaline decomposition of organic disulfides. II. Alternative pathways as determined by structure

Alkaline decomposition of organic disulfides. IV. Limitation on the use of Ellman's reagent. 2,2'-Dinitro-5,5'-dithiodibenzoic acid

Oxidation of organic divalent sulfur by iodine. II. Equilibrating thiol-iodine-disulfide-hydrogen iodide system in acetic acid and evidence for sulfenyl iodide intermediates