Protein-bound glutathione measurement in cultured cells by CZE with LIF detection.
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 cmx75 microm 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.
Zinellu
Angelo
A
Department of Biomedical Sciences, University of Sassari, Sassari, Italy. angelozinellu@libero.it
Sotgia
Salvatore
S
Posadino
Anna Maria
AM
Pasciu
Valeria
V
Zinellu
Elisabetta
E
Usai
Maria Franca
MF
Scanu
Bastianina
B
Chessa
Roberto
R
Gaspa
Leonardo
L
Tadolini
Bruna
B
Deiana
Luca
L
Carru
Ciriaco
C
D002138:Calibration; D002478:Cells, Cultured; D019075:Electrophoresis, Capillary / Q000379:methods*; D005978:Glutathione / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*
D011506:Proteins; D005978:Glutathione