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Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?

Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?
Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?

BACKGROUND AND PURPOSE: Posttranslational modifications of cysteine (Cys) residues represent a major aspect of redox biology, and their reliable detection is key in providing mechanistic insights. The metastable character of these modifications and cell lysis-induced artifactual oxidation render current state-of-the-art protocols to rely on alkylation-based stabilization of labile Cys derivatives before cell/tissue rupture. An untested assumption in these procedures is that for all Cys derivatives alkylation rates are faster than their dynamic interchange. However, when the interconversion of Cys derivatives is not rate-limiting, then electrophilic labeling is under Curtin-Hammett control and hence the final alkylated mixture may not represent the speciation that prevailed before alkylation.

KEY RESULTS: We here present evidence that in the majority of cases, the speciation of alkylated polysulfide/thiol derivatives indeed depends on the experimental conditions. Our results reveal that alkylation perturbs sulfur speciation in both a concentration- and time-dependent manner, and that strong alkylating agents can cleave polysulfur chains. Moreover, we show that labeling of sulfenic acids with dimedone also affects Cys speciation, suggesting that part of the endogenous pool of products previously believed to represent sulfenic acid species may in fact represent polysulfides.

EXPERIMENTAL APPROACH: These observations were obtained using buffered aqueous solutions of inorganic-, organic-, cysteine-, glutathione- and GAPDH-polysulfide species. Additional experiments in human plasma and serum revealed that monobromobimane can extract sulfide from the endogenous sulfur pool by shifting speciation equilibria, suggesting caution should be exercised when interpreting experimental results using this tool.

CONCLUSION AND IMPLICATION: We highlight methodological caveats potentially arising from these pitfalls and conclude that current derivatization strategies often fail to adequately capture physiologic speciation of sulfur species.

Journal Article
0007-1188
Bogdándi, Virág
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Ida, Tomoaki
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Sutton, Thomas R.
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Bianco, Christopher
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Ditrói, Tamás
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Koster, Grielof
e404c38a-6f48-430a-adf0-5208228cb9e7
Henthorn, Hillary A
69c5b64e-07d6-4094-b058-45eb3b1678ab
Minnion, Magda
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Toscano, John P
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van der Vliet, Albert
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Pluth, Michael D
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Feelisch, Martin
8c1b9965-8614-4e85-b2c6-458a2e17eafd
Fukuto, Jon M.
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Akaike, Takaaki
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Nagy, Péter
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Bogdándi, Virág
8938002e-9f5b-46d8-8ccf-6660b0fe507f
Ida, Tomoaki
ebbb63cc-ecd5-4ac7-b3c0-04c6df0963f9
Sutton, Thomas R.
dbc4cb4f-cab0-46ee-b911-8945f50563cd
Bianco, Christopher
094ce550-2655-4575-8bd6-5fa43b4ef38e
Ditrói, Tamás
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Koster, Grielof
e404c38a-6f48-430a-adf0-5208228cb9e7
Henthorn, Hillary A
69c5b64e-07d6-4094-b058-45eb3b1678ab
Minnion, Magda
4e765870-10de-4f9a-87d9-0f35e6592e87
Toscano, John P
05e067be-b931-4abd-bbad-f7f349ae3ec2
van der Vliet, Albert
3f483143-10f9-4e89-9573-29e6f0c90809
Pluth, Michael D
dbaa6de6-3a2c-4bbc-a11f-ab49da892609
Feelisch, Martin
8c1b9965-8614-4e85-b2c6-458a2e17eafd
Fukuto, Jon M.
222058bd-beef-4b4e-856c-54b1fc7eb4a9
Akaike, Takaaki
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Nagy, Péter
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Bogdándi, Virág, Ida, Tomoaki, Sutton, Thomas R., Bianco, Christopher, Ditrói, Tamás, Koster, Grielof, Henthorn, Hillary A, Minnion, Magda, Toscano, John P, van der Vliet, Albert, Pluth, Michael D, Feelisch, Martin, Fukuto, Jon M., Akaike, Takaaki and Nagy, Péter (2018) Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell? British Journal of Pharmacology. (doi:10.1111/bph.14394).

Record type: Article

Abstract

BACKGROUND AND PURPOSE: Posttranslational modifications of cysteine (Cys) residues represent a major aspect of redox biology, and their reliable detection is key in providing mechanistic insights. The metastable character of these modifications and cell lysis-induced artifactual oxidation render current state-of-the-art protocols to rely on alkylation-based stabilization of labile Cys derivatives before cell/tissue rupture. An untested assumption in these procedures is that for all Cys derivatives alkylation rates are faster than their dynamic interchange. However, when the interconversion of Cys derivatives is not rate-limiting, then electrophilic labeling is under Curtin-Hammett control and hence the final alkylated mixture may not represent the speciation that prevailed before alkylation.

KEY RESULTS: We here present evidence that in the majority of cases, the speciation of alkylated polysulfide/thiol derivatives indeed depends on the experimental conditions. Our results reveal that alkylation perturbs sulfur speciation in both a concentration- and time-dependent manner, and that strong alkylating agents can cleave polysulfur chains. Moreover, we show that labeling of sulfenic acids with dimedone also affects Cys speciation, suggesting that part of the endogenous pool of products previously believed to represent sulfenic acid species may in fact represent polysulfides.

EXPERIMENTAL APPROACH: These observations were obtained using buffered aqueous solutions of inorganic-, organic-, cysteine-, glutathione- and GAPDH-polysulfide species. Additional experiments in human plasma and serum revealed that monobromobimane can extract sulfide from the endogenous sulfur pool by shifting speciation equilibria, suggesting caution should be exercised when interpreting experimental results using this tool.

CONCLUSION AND IMPLICATION: We highlight methodological caveats potentially arising from these pitfalls and conclude that current derivatization strategies often fail to adequately capture physiologic speciation of sulfur species.

Text
Bogd-ndi_et_al-2018-British_Journal_of_Pharmacology - Accepted Manuscript
Restricted to Repository staff only until 17 June 2019.
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Accepted/In Press date: 5 June 2018
e-pub ahead of print date: 17 June 2018
Additional Information: This article is protected by copyright. All rights reserved.
Keywords: Journal Article

Identifiers

Local EPrints ID: 422437
URI: https://eprints.soton.ac.uk/id/eprint/422437
ISSN: 0007-1188
PURE UUID: aedc05ca-40a8-4209-aedd-179c38f07960
ORCID for Martin Feelisch: ORCID iD orcid.org/0000-0003-2320-1158

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Date deposited: 24 Jul 2018 16:30
Last modified: 14 Mar 2019 01:36

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