Abstract
Glutathione is an important molecule involved in the primary and secondary metabolism of all organisms. The Glutathione redox status is an indicator of the cellular redox state. Therefore, it is important to have precise methods on hand to determine the glutathione redox status in the cell. In this protocol, we describe an improved spectrophotometric method to estimate the content of reduced (GSH) and oxidized (GSSG) forms of glutathione in the extremophilic microalga Galdieria phlegrea.
Keywords: Microalgae, Galdieria phlegrea, Glutathione, Glutathione reductase, Redox state
Background
Glutathione (γ-L-glutamyl-L-cysteinyl-glycine) is an essential tripeptide existing in all known organism, ranging from bacteria to humans (Frendo et al., 2013). It is involved in several different cell protective roles (Figure 1). Glutathione is considered an excellent antioxidant molecule having redox signalling function. It is also involved in response to abiotic and biotic stress, and implicated in the primary metabolism (C, N, S metabolism) of the cell (Noctor et al., 2012; Hernández et al., 2015; Salbitani et al., 2015).In plant cells, the ratio between the reduced and oxidized forms of glutathione (GSH/GSSG) plays a significant part in signalling and in the activation of numerous defence mechanisms (Foyer and Noctor, 2012; Salbitani et al., 2015). The GSH/GSSG redox state, which is normally tightly controlled, may transiently shift towards a slightly more oxidized value during a stress condition (Tausz et al., 2004).In the past, some researchers have developed and modified spectrophotometric methods to determine the GSH and GSSG in several organism (Anderson, 1985; Bashir et al., 2013). Here we describe a protocol for a simple glutathione determination, optimized on the extremophilic microalga Galdieria phlegrea. The method illustrated is based on the reaction of GSH with the thiol reagent DTNB (5,5-dithiobis(2-nitrobenzoic acid)) to form GSSG and TNB (5-thionitrobenzoic acid), which is detected spectrophotometrically at 412 nm (Giustarini et al., 2013).Figure 1. Glutathione redox cycle. Reduced glutathione (GSH) is a tripeptide composed of cysteine, glutamic acid and glycine, which plays a key role in the control of signalling processes, detoxification and various other cell processes. Glutathione disulfide (GSSG) is the oxidized form of glutathione. It is reduced to GSH in presence of NADPH by the glutathione reductase (GR). The glutathione peroxidase (GP) converts hydrogen peroxide to water.
Materials and Reagents
Equipment
Procedure
Data analysis
Recipes
Acknowledgments
This protocol was adapted from previously published studies (Anderson, 1985; Bashir et al., 2013). This study was financed by Regione Campania (PON-Smart Generation and LR 5/2002).
References
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