title: Determination of glutaredoxin enzyme activity and protein S-glutathionylation using fluorescent eosin-glutathione.
abstract: Glutaredoxins catalyze glutathione-dependent disulfide oxidoreductions, particularly reduction of glutathione (GSH)-protein mixed disulfides. Mammalian glutaredoxins are present in the cytosol/nucleus as Grx1 or in mitochondria as Grx2a. Here we describe di-eosin-glutathione disulfide (Di-E-GSSG) as a new tool to study glutaredoxin (Grx) activity. Di-E-GSSG has almost no fluorescence in its disulfide form due to self-quenching, whereas the reduced form (E-GSH) has a large fluorescence emission at 545 nm after excitation at 520 nm. Di-E-GSSG was a very poor substrate for glutathione reductase, but we discovered that the molecule was an excellent substrate for glutaredoxin in a coupled assay system with GSH, nicotinamide adenine dinucleotide phosphate (NADPH), and glutathione reductase or with lipoamide, NADH, and lipoamide dehydrogenase. In addition, Di-E-GSSG was used to glutathionylate the free SH group of bovine serum albumin (BSA), yielding eosin-glutathionylated BSA (E-GS-BSA) readily observed in ultraviolet (UV) light. E-GS-BSA also displayed a quenched fluorescence, and its Grx-catalyzed reduction could be followed by the formation of E-GSH by fluorescence emission using microtiter plates. This way of measuring Grx activity provided an ultrasensitive method that detected Grx1 and Grx2 at picomolar levels. Human Grx1 was readily quantified in 40 μl of plasma and determined to be 680 ± 208 pM in healthy controls.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm, Sweden. Electronic address: lucia.coppo@ki.se.
authors.forename: Lucia
authors.initials: L
authors.lastname: Coppo
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm, Sweden.
authors.forename: Sergio J
authors.initials: SJ
authors.lastname: Montano
authors.affiliation: Department of Biochemistry and Molecular Biology, Campus de Rabanales, University of Córdoba, 14071, Córdoba, Spain.
authors.forename: Alicia C
authors.initials: AC
authors.lastname: Padilla
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm, Sweden. Electronic address: arne.holmgren@ki.se.
authors.forename: Arne
authors.initials: A
authors.lastname: Holmgren
mesh_terms: D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004801:Eosine Yellowish-(YS) / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D054477:Glutaredoxins / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015394:Molecular Structure; D017293:Protein S / Q000737:chemistry / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D004220:Disulfides; D005456:Fluorescent Dyes; C516005:GLRX protein, human; D054477:Glutaredoxins; D017293:Protein S; D012710:Serum Albumin, Bovine; D005978:Glutathione; D004801:Eosine Yellowish-(YS)
keywords: Deglutathionylation; Di-eosin-glutathione; Fluorescence assay; Glutaredoxin activity; Redox