Biochemistry


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1 Q&A 6048 Views Jun 5, 2019
Thiol-based redox regulation is a posttranslational protein modification that plays a key role in many biological aspects. To understand its regulatory functions, we need a method to directly assess protein redox state in vivo. Here we present a simple procedure to determine protein redox state in a model plant Arabidopsis thaliana. Our method consists of three key steps: (i) redox fixation by rapidly freezing plant tissues in the liquid nitrogen, (ii) labeling of thiol groups with the maleimide reagent, and (iii) protein detection by Western blotting. The redox state of a specific or given protein can be discriminated by the mobility change on SDS-PAGE with high sensitivity. This method provides a novel strategy to dissect the working dynamics of the redox-regulatory system in plants.
0 Q&A 5338 Views Mar 5, 2019
Thiol-disulfide exchange is a key posttranslational modification, determining the folding process of intra- and inter-protein structures. Thiols can be detected by colorimetric reagents, which are stoichiometrically reduced by free thiols, and by fluorescent adducts, showing fluorescence only after thioester formation. We adapted a simple three-step method for detection of disulfide bonds in proteins. After irreversible blocking of protein thiols, disulfide bonds are reduced, followed by the detection of thiols. The approach presented here provides an economical procedure that can be used to obtain a global overview of the thiol-disulfide status of proteins in plants. This method allows the detection of modifications in samples on a gel and can be used for semi-quantitative analysis.
0 Q&A 6008 Views May 20, 2018
Bacteria release cysteine to moderate the size of their intracellular pools. They can also evolve hydrogen sulfide, either through dissimilatory reduction of oxidized forms of sulfur or through the deliberate or inadvertent degradation of intracellular cysteine. These processes can have important consequences upon microbial communities, because excreted cysteine autoxidizes to generate hydrogen peroxide, and hydrogen sulfide is a potentially toxic species that can block aerobic respiration by inhibiting cytochrome oxidases. Lead acetate strips can be used to obtain semiquantitative data of sulfide evolution (Oguri et al., 2012). Here we describe methods that allow more-quantitative and discriminatory measures of cysteine and hydrogen sulfide release from bacterial cells. An illustrative example is provided in which Escherichia coli rapidly evolves both cysteine and sulfide upon exposure to exogenous cystine (Chonoles Imlay et al., 2015; Korshunov et al., 2016).
0 Q&A 7134 Views Jan 5, 2016
Low-molecular-weight (LMW) thiols are a class of highly reactive compounds due to their thiol moiety. They play important roles in the maintenance of cellular redox homeostasis, detoxification, and development. Monobromobimane (mBBr) is weakly fluorescent but selectively reacts with thiols to yield highly fluorescent thioethers (mBSR) products, which is especially useful for the quantification of LMW thiols. The stable mBSR products can be separated by high-performance liquid chromatography (HPLC) equipped with a fluorescent detector. The main cellular LMW thiols are L-cysteine, gamma-glutamylcysteine, and glutathione (GSH). The following protocol describes the extraction and quantification of L-cysteine, gamma-glutamylcysteine, and glutathione from Arabidopsis tissues as reported (Xiang and Oliver, 1998; Zhao et al., 2014; Wang et al., 2015) with minor revision. Modifications may be required if the HPLC system or the C18 column is different.



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