Determination of glutaredoxin enzyme activity and protein S-glutathionylation using fluorescent eosin-glutathione

Analytical Biochemistry
Lucia CoppoA Holmgren

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 activi...Continue Reading

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Citations

Jul 7, 2017·Luminescence : the Journal of Biological and Chemical Luminescence·Mani Vedamalai, Iti Gupta
Dec 16, 2017·Chemical Society Reviews·Lisa J AlcockJustin M Chalker
Jul 11, 2018·Nature Medicine·Vikas AnathyYvonne M W Janssen-Heininger
Nov 7, 2019·American Journal of Physiology. Cell Physiology·Shi B ChiaYvonne M W Janssen-Heininger

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