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Protein-bound glutathione measurement in cultured cells by CZE with LIF detection

Zinellu, Angelo and Sotgia, Salvatore and Posadino, Anna Maria and Pasciu, Valeria and Zinellu, Elisabetta and Usai, Maria Franca and Scanu, Bastianina and Chessa, Roberto and Gaspa, Leonardo and Tadolini, Bruna and Deiana, Luca and Carru, Ciriaco (2007) Protein-bound glutathione measurement in cultured cells by CZE with LIF detection. Electrophoresis, Vol. 28 (18), p. 3277-3283. eISSN 1522-2683. Article.

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DOI: 10.1002/elps.200700042

Abstract

Protein modification due to S-glutathio(ny)lation, usually a reversible process in intact cells, arises interest as a possible mode of regulatory events that may potentially modify a large number of cellular processes. However, since less than 1% of the total protein is S-thiolated in resting cells, high sensitivity methods are required for its evaluation. We set up a new method by CE with LIF detection that allows to measure all forms of intracellular GSH involved in the process. For total and reduced glutathione, cell lysates were rapidly derivatized by 5-iodoacetoamidofluorescein (5-IAF), a selective reagent which traps thiol groups, thus minimizing auto-oxidation. Derivatized samples were separated in a 47 cm×75 μm id capillary by using 7 mmol/L sodium phosphate at pH 11.6. For the evaluation of S-glutathio(ny)lation, intracellular proteins from cell lysates were precipitated and washed to eliminate free GSH. After protein resuspension with NaOH and reduction treatment with tri-n-butylphosphine (TBP), the freed GSH was dried in a vacuum concentrator and directly dissolved in the derivatization mixture. GSH-IAF adduct was detected in a 6 mmol/L sodium phosphate, 3 mmol/L boric acid, and 75 mmol/L N-methylglucamine run buffer in less than 5 min. The high sensitivity ensured by 5-IAF use and sample concentration, allowed to quantify GSH at levels as low as 5 nmol/L, value suitable for the evaluation of protein S-glutathio(ny)lation. The method suitability was checked both in HUVEC and ECV304 cultured cells.

Item Type:Article
ID Code:2371
Status:Published
Refereed:Yes
Uncontrolled Keywords:CE, Glutathione, S-Glutathio(ny)lation
Subjects:Area 05 - Scienze biologiche > BIO/10 Biochimica
Area 05 - Scienze biologiche > BIO/12 Biochimica clinica e biologia molecolare clinica
Divisions:001 Università di Sassari > 01 Dipartimenti > Scienze fisiologiche, biochimiche e cellulari
001 Università di Sassari > 01 Dipartimenti > Scienze biomediche
002 Altri enti e centri di ricerca del Nord Sardegna > National laboratory of the national institute of biostructures and biosystems, Osilo
Publisher:Wiley
eISSN:1522-2683
Deposited On:18 Aug 2009 10:07

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