Sulfhydryl reagent

About: Sulfhydryl reagent is a research topic. Over the lifetime, 780 publications have been published within this topic receiving 323750 citations.

Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method.

Abstract: The spectrophotometric/microplate reader assay method for glutathione (GSH) involves oxidation of GSH by the sulfhydryl reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) to form the yellow derivative 5'-thio-2-nitrobenzoic acid (TNB), measurable at 412 nm. The glutathione disulfide (GSSG) formed can be recycled to GSH by glutathione reductase in the presence of NADPH. The assay is composed of two parts: the preparation of cell cytosolic/tissue extracts and the detection of total glutathione (GSH and GSSG). The method is simple, convenient, sensitive and accurate. The lowest detection for GSH and GSSG is 0.103 nM in a 96-well plate. This method is rapid and the whole procedure takes no longer than 15 min including reagent preparation. The method can assay GSH in whole blood, plasma, serum, lung lavage fluid, cerebrospinal fluid, urine, tissues and cell extracts and can be extended for drug discovery/pharmacology and toxicology protocols to study the effects of drugs and toxic compounds on glutathione metabolism.

Autoxidation versus covalent binding of quinones as the mechanism of toxicity of dopamine, 6-hydroxydopamine, and related compounds toward C1300 neuroblastoma cells in vitro

Abstract: The mechanism of cytotoxicity of 6-hydroxydopamine, 2,4,5-trihydroxyphenylalanine, dopa, dopamine, norepinephrine, and epinephrine was explored by asking whether cytotoxicity was a reflection of the potential for autoxidation of each polyphenol or of the sulfhydryl reactivity of its quinone products. The cytotoxicity of the polyphenols, as measured by inhibition of [3H]thymidine incorporation into DNA by C1300 neuroblastoma cells in tissue culture, correlated with the rate of autoxidation, as measured spectrophotometrically or by oxygen electrode studies. Polarographic determinations of the oxidation potentials of the polyphenols were also predictive of cytotoxicity; the most cytotoxic compounds had the most negative half-wave potentials and thus were the most readily oxidized. By contrast, the sulfhydryl reactivity of the quinone oxidation products of the polyphenols, as measured by inhibition of purified calf thymus DNA polymerase α, exhibited an inverse relationship to the cytotoxicity of the polyphenols; the most toxic compounds, 6-hydroxydopamine and 2,4,5-trihydroxyphenylalanine, were oxidized to the least reactive quinone products. An alternative mechanism of toxicity was observed with N -acetyldopamine, which was oxidized to 4-(2- N -acetylaminoethyl)-1,2-benzoquinone, a potent sulfhydryl reagent. N -Acetyldopamine was more toxic than predicted by its half-wave potential or its rate of autoxidation. Furthermore, while norepinephrine completely neutralized 6-hydroxydopamine and 2,4,5-trihydroxyphenylalanine as cytotoxic agents, the toxicity of N -acetyldopamine was minimally affected. Thus we conclude that 6-hydroxydopamine and 2,4,5-trihydroxyphenylalanine kill cells through the production of H2O2, O2[unknown], and OH·, while for dopamine and dopa the reaction of quinone oxidation products with nucleophiles probably also contributes to their cytotoxicity.

[37] Reaction of protein sulfhydryl groups with Ellman's reagent.

Abstract: Publisher Summary This chapter discusses a reaction of protein sulfhydryl groups with Ellman's reagent. The reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is developed by Ellman as a sulfhydryl reagent. DTNB is found to be a sensitive tool for the assay of thiol groups in tissues, body fluids, and proteins. Ellman described a method for its synthesis. DTNB is an aromatic disulfide, and, since it has a higher standard oxidation-reduction potential than aliphatic analogs, it will react with aliphatic thiols by an exchange reaction to form a mixed disulfide of the protein and 1 mole of 2-nitro-5-thiobenzoate per mole of protein sulfhydryl group. Sulfhydryl-disulfide exchange reactions between disulfide compounds with sulfur directly attached to aromatic groups and simple alkyl mercaptans should go to completion. Protein sulfhydryl (SH) groups may behave similarly to simple alkyl mercaptans unless steric factors interfere with the course of reaction. The sulfhydryl groups in proteins exhibit variable reactivity toward DTNB owing to steric factors. Determination of total sulfhydryl content requires that the protein be denatured, preferably with sodium dodecyl sulfate.