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Thiol Quantification Using Colorimetric Thiol-Disulfide Exchange in Nonaqueous Solvents.

ABSTRACT: A careful analysis of two (thiol-disulfide exchange) thiol quantification chromophores' behavior (Ellman's reagent and Aldrithiol-4) in nonaqueous solvents is presented. A wide range of kinetic profiles and response factors were measured to exhibit a large variance for nonaqueous systems. We report several robust benchtop and room-temperature methods using different organic solvents compared to aqueous conditions. Validation of analytical analyses in nonaqueous systems and quantification of the cysteine content of ovalbumin are also presented. This work serves as a treatise on the utilization of thiol-disulfide exchange chromophores under nonaqueous conditions for the quantification of thiols.

SUBMITTER: Schmidt R

PROVIDER: S-EPMC10018523 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Thiol Quantification Using Colorimetric Thiol-Disulfide Exchange in Nonaqueous Solvents.

Schmidt Ryan R Logan Matthew G MG Patty Sarah S Ferracane Jack L JL Pfeifer Carmem S CS Kendall Alexander J AJ

ACS omega 20230228 10

A careful analysis of two (thiol-disulfide exchange) thiol quantification chromophores' behavior (Ellman's reagent and Aldrithiol-4) in nonaqueous solvents is presented. A wide range of kinetic profiles and response factors were measured to exhibit a large variance for nonaqueous systems. We report several robust benchtop and room-temperature methods using different organic solvents compared to aqueous conditions. Validation of analytical analyses in nonaqueous systems and quantification of the ...[more]

PMID: 36936287

Similar Datasets

Thiol-Disulfide Exchange in Human Growth Hormone.

Project description:PurposeThiol-disulfide exchange was monitored in recombinant human growth hormone (hGH) and in model tryptic peptides derived from hGH to investigate the effects of higher-order structure on the reaction.MethodsDifferent free thiol-containing peptides, varying in length and amino acid sequence, were used to initiate the reaction at pH 7.0 and 37°C in hGH. Protein samples were digested with trypsin and analyzed for native disulfides, scrambled disulfides and free thiols using LC/MS. The loss of native disulfide and disulfide exchange was compared with model peptides derived from hGH.ResultsLoss of native disulfide in cyclic (cT20-T21) and linear peptides (T20-T21pep) derived from the C-terminal hGH disulfide during the first 60 min of reaction was greater than loss of the C-terminal disulfide in hGH itself. Of the thiols tested, glutathione (GSH) was the most reactive, forming the highest percentage of mixed disulfides in intact hGH and in the model peptides. At longer reaction times (>240 min), native disulfides in both hGH and cT20-T21 were regenerated. The fastest rates of regeneration were observed for Cys and the di- or tripeptide containing an Arg residue adjacent to Cys, suggesting that they may be useful in refolding.ConclusionsThiol-disulfide exchange reactions in hGH and related model peptides were influenced by higher order structure, by the size of the thiol reactant and by an Arg residue adjacent to Cys in the thiol reactant. Reduction of disulfide bonds in hGH did not affect higher order structure as measured by CD and HDX-MS.

| S-EPMC4854756 | biostudies-literature

Thiol-Disulfide Exchange Reactions in the Mammalian Extracellular Environment.

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Thiol-disulfide exchange in peptides derived from human growth hormone.

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| S-EPMC4283463 | biostudies-literature

Kinetics and mechanisms of thiol-disulfide exchange covering direct substitution and thiol oxidation-mediated pathways.

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| S-EPMC3613173 | biostudies-literature

The atypical thiol-disulfide exchange protein α-DsbA2 from Wolbachia pipientis is a homotrimeric disulfide isomerase.

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| S-EPMC9452476 | biostudies-literature

Control of periplasmic interdomain thiol:disulfide exchange in the transmembrane oxidoreductase DsbD.

Project description:The bacterial protein DsbD transfers reductant from the cytoplasm to the otherwise oxidizing environment of the periplasm. This reducing power is required for several essential pathways, including disulfide bond formation and cytochrome c maturation. DsbD includes a transmembrane domain (tmDsbD) flanked by two globular periplasmic domains (nDsbD/cDsbD); each contains a cysteine pair involved in electron transfer via a disulfide exchange cascade. The final step in the cascade involves reduction of the Cys(103)-Cys(109) disulfide of nDsbD by Cys(461) of cDsbD. Here we show that a complex between the globular periplasmic domains is trapped in vivo only when both are linked by tmDsbD. We have found previously ( Mavridou, D. A., Stevens, J. M., Ferguson, S. J., & Redfield, C. (2007) J. Mol. Biol. 370, 643-658 ) that the attacking cysteine (Cys(461)) in isolated cDsbD has a high pK(a) value (10.5) that makes this thiol relatively unreactive toward the target disulfide in nDsbD. Here we show using NMR that active-site pK(a) values change significantly when cDsbD forms a complex with nDsbD. This modulation of pK(a) values is critical for the specificity and function of cDsbD. Uncomplexed cDsbD is a poor nucleophile, allowing it to avoid nonspecific reoxidation; however, in complex with nDsbD, the nucleophilicity of cDsbD increases permitting reductant transfer. The observation of significant changes in active-site pK(a) values upon complex formation has wider implications for understanding reactivity in thiol:disulfide oxidoreductases.

| S-EPMC2631958 | biostudies-literature

Fast-Forming Dissolvable Redox-Responsive Hydrogels: Exploiting the Orthogonality of Thiol-Maleimide and Thiol-Disulfide Exchange Chemistry.

Project description:Fast-forming yet easily dissolvable hydrogels (HGs) have potential applications in wound healing, burn incidences, and delivery of therapeutic agents. Herein, a combination of a thiol-maleimide conjugation and thiol-disulfide exchange reaction is employed to fabricate fast-forming HGs which rapidly dissolve upon exposure to dithiothreitol (DTT), a nontoxic thiol-containing hydrophilic molecule. In particular, maleimide disulfide-terminated telechelic linear poly(ethylene glycol) (PEG) polymer and PEG-based tetrathiol macromonomers are employed as gel precursors, which upon mixing yield HGs within a minute. The selectivity of the thiol-maleimide conjugation in the presence of a disulfide linkage was established through 1H NMR spectroscopy and Ellman's test. Rapid degradation of HGs in the presence of thiol-containing solution was evident from the reduction in storage modulus. HGs encapsulated with fluorescent dye-labeled dextran polymers and bovine serum albumin were fabricated, and their cargo release was investigated under passive and active conditions upon exposure to DTT. One can envision that the rapid gelation and fast on-demand dissolution under relatively benign conditions would make these polymeric materials attractive for a range of biomedical applications.

| S-EPMC9472223 | biostudies-literature

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