Fluorescein-labeled glutathione to study protein S-glutathionylation

Lisa M. Landino, William & Mary
Carolyn M. Brown, William & Mary
Carolyn A. Edson, William & Mary
Laura J. Gilbert, William & Mary
Nathan Grega-Larson, William & Mary
Anna Jean Wirth, William & Mary
Kelly C. Lane, William & Mary

Abstract

Numerous studies of S-glutathionylation of cysteine thiols indicate that this protein modification plays a key role in redox regulation of proteins. To facilitate the study of protein S-glutathionylation, we developed a synthesis and purification to produce milligram quantities of fluorescein-labeled glutathione. The amino terminus of the glutathione tripeptide reacted with fluorescein isothiocyanate readily in ammonium bicarbonate. Purification by solid phase extraction on C8 and C18 columns separated excess reactants from desired products. Both oxidized and reduced fluorescein-labeled glutathione reacted with a variety of thiol-containing proteins to yield fluorescent proteins. (C) 2010 Elsevier Inc. All rights reserved.