These new volumes of Methods in Enzymology
(554 and 555) on Hydrogen Sulfide Signaling continue the legacy established by previous volumes on another gasotransmitter, nitric oxide (Methods in Enzymology
volumes 359, 396, 440, and 441), with quality chapters authored by leaders in the field of hydrogen sulfide research. These volumes of Methods in Enzymology
were designed as a compendium for hydrogen sulfide detection methods, the pharmacological activity of hydrogen sulfide donors, the redox biochemistry of hydrogen sulfide and its metabolism in mammalian tissues, the mechanisms inherent in hydrogen sulfide cell signaling and transcriptional pathways, and cell signaling in specific systems, such as cardiovascular and nervous system as well as its function in inflammatory responses. Two chapters are also devoted to hydrogen sulfide in plants and a newcomer, molecular hydrogen, its function as a novel antioxidant.
- Continues the legacy of this premier serial with quality chapters on hydrogen sulfide research authored by leaders in the field
- Covers conventional and new hydrogen sulfide detection methods
- Covers the pharmacological activity of hydrogen sulfide donors
- Contains chapters on important topics on hydrogen sulfide modulation of cell signaling and transcriptional pathways, and the role of hydrogen sulfide in the cardiovascular and nervous systems and in inflammation
Investigating the Role of H2S in 4-HNE Scavenging
Hilde Laggner1; Bernhard M.K. Gmeiner1 Department of Medical Chemistry and Pathobiochemistry, Center of Pathobiochemistry and Genetics, Medical University of Vienna, Vienna, Austria
1 Corresponding authors: email address: email@example.com, firstname.lastname@example.org
4-HNE (4-hydroxy-2-nonenal) is a highly reactive a,ß-unsaturated aldehyde generated from oxidation of polyunsaturated fatty acids and has been suggested to play a role in the pathogenesis of several diseases. 4-HNE can bind to amino acids, proteins, polynucleotides, and lipids and exert cytotoxicity. 4-HNE forms adducts (Michael adducts) with cysteine, lysine, as well as histidine on proteins with the thiol function as the most reactive nucleophilic moiety. Thus, detoxification strategies by 4-HNE scavenging compounds might be of interest. Recently, hydrogen sulfide (H2S) has been identified as an endogenous vascular gasotransmitter and neuromodulator. Assuming that the low-molecular thiol H2S may react with 4-HNE, methods to monitor the ability of H2S to counteract the protein-modifying and cytotoxic activity of 4-HNE are described in this chapter.
4-Hydroxy-2-nonenal (4-HNE) is one of the reaction products of lipid hydroperoxide break down occurring in response to oxidative stress (Esterbauer, Schaur, & Zollner, 1991; Spiteller, Kern, Reiner, & Spiteller, 2001). The chemical reactivity of this and other a,ß-unsaturated aldehydes has been extensively studied in the past (Esterbauer, Ertl, & Scholz, 1976; Esterbauer et al., 1991; Schultz, Yarbrough, & Johnson, 2005; Spiteller et al., 2001).
4-HNE has been shown to be toxic to cells (Esterbauer et al., 1991). Beside its bare cytotoxic ability, 4-HNE-modified proteins may play a mechanism in the pathogenesis of human diseases and in addition, 4-HNE may act as signaling molecule (Petersen & Doorn, 2004). Most of the biochemical effects of 4-HNE may be due to its easy reaction of the CC bond (Michael addition) with the nucleophilic thiol and amino groups of free or protein-bound amino acids (cysteine, histidine, and lysine). Lipids (phosphatidyl-ethanol amine) and nucleic acids are also targets of this highly reactive aldehyde (Schaur, 2003). The double bond can be reduced by an NAD(P)H-dependent alkenal/one oxidoreductase forming 4-HNA (4-hydroxy-nonanal), thus detoxifying 4-HNE (Dick, Kwak, Sutter, & Kensler, 2001). Epoxidation of 4-HNE can also take place in presence of hydroperoxide (Schaur, 2003).
The CO group can undergo hemi-acetal and acetal formation with alcohols or thiols. Schiff base formation with primary amino groups (e.g., lysine) and enzymatic oxidation (aldehyde dehydrogenase/NAD) and reduction (alcohol dehydrogenase/NADH) results in 4-hydroxy-nonenoic acid and 1,4-dihydroxy-nonen formation, respectively (Schaur, 2003). Oxidation of the 4-hydroxy group results in the formation of 4-ONE (4-oxo-2-nonenal), an extremely neurotoxic derivative (Lin et al., 2005).
As 4-HNE is suggested to play a role in the pathogenesis of several diseases, molecular strategies should be developed to detoxify this highly reactive compound (Aldini, Carini, Yeum, & Vistoli, 2014; Mali & Palaniyandi, 2013). Possible approaches are (i) inhibiting 4-HNE formation, (ii) activating/upregulating detoxifying enzymes, and (iii) scavenging of 4-HNE by low-molecular-weight compounds (Aldini et al., 2014). The latter are the cysteine-mimetic, lysine-mimetic, and histidine-mimetic HNE-sequestering agents directly reacting via Michael adduct and Schiff base formation. The reaction of 4-HNE with thiol compounds has received particular attention. Glutathione (GSH) reacts readily with 4-HNE, a reaction which has been attributed to the HNE-detoxifying action of GSH (Esterbauer, Zollner, & Scholz, 1975).
Recently, H2S (hydrogen sulfide) has been identified as the third gasotransmitter, beside NO and CO, in the vasculature (Lefer, 2007; Leffler, Parfenova, Jaggar, & Wang, 2006; Wang, 2002; Zhao, Zhang, Lu, & Wang, 2001). The enzymes cystathionine-ß-synthase (CBS EC 220.127.116.11), cystathionine-?-lyase (CSE EC 18.104.22.168), and 3-mercapto-pyruvate sulfurtransferase (3MST EC 22.214.171.124) are responsible for the endogenous production of H2S (Kabil & Banerjee, 2014; Shibuya et al., 2009).
In the brain (human, rat, and bovine), CBS is highly expressed and the primary physiological source of H2S (Wang, 2012). Thus, a neuromodulatory action of H2S has been proposed (Abe & Kimura, 1996; Kimura, 2000; Moore, Bhatia, & Moochhala, 2003). Perturbed H2S production in the brain has been linked to certain diseases. Decreased S-adenosylmethionine concentration has been reported for Alzheimer's disease (Morrison, Smith, & Kish, 1996) which may lead to diminished CBS activity and result in low endogenous H2S levels. An overproduction of H2S was found in Down syndrome patients, where the CBS gene located on chromosome 21 is overexpressed as reported by Kamoun, Belardinelli, Chabli, Lallouchi, and Chadefaux-Vekemans (2003). The synthesis of endogenous H2S is significantly lower but 4-HNE is markedly increased in Alzheimer's disease (Butterfield et al., 2006; Liu, Raina, Smith, Sayre, & Perry, 2003; Lovell, Ehmann, Mattson, & Markesbery, 1997). We found that the low-molecular-weight thiol H2S exerts protective activity against 4-HNE induced cytotoxicity and HNE protein-adduct formation (Schreier et al., 2010). Here we describe various approaches to monitor the interaction of 4-HNE with H2S. We refer to scavenging reactions and inhibition of protein modifying and cytoprotective properties of H2S using a neuroblastoma cell line (SH-SY5Y).
2 Experimental Compounds and Considerations
2.1 H2S generation
Sodium hydrogen sulfide (NaHS) and disodium sulfide (Na2S) can be purchased from Aldrich. NaHS or Na2S stock solutions (100 mmol/L) are prepared daily in distilled water and stored on ice in the dark (?230 nm = 7700 M- 1 cm- 1) (Nagy et al., 2014). Stock solutions are diluted to the desired concentrations in the respective buffer.
At pH 7.4, H2S concentration is taken as 30% of the NaHS or Na2S concentration (Beauchamp, Bus, Popp, Boreiko, & Andjelkovich, 1984; Reiffenstein, Hulbert, & Roth, 1992). The term H2S for the sum of the sulfur-species H2S, HS-, and S2 - is used according to Whiteman et al. (2004).
2.2 Preparation of 4-HNE solutions
4-Hydroxy-2-nonenal-dimethyl acetal (4-HNE-DMA) is supplied from Sigma-Aldrich. 4-HNE is prepared from 4-HNE-DMA by acid hydrolysis. An aliquot of hexane solution containing 4-HNE-DMA is evaporated under a gentle stream of nitrogen at room temperature. An equal volume of cold HCl (1 mmol/L) is added and the sample incubated at 4 °C for 45 min under nitrogen atmosphere and gentle agitation. At the end of incubation, the concentration of 4-HNE is determined using ? = 13,600 M- 1 cm- 1 at 222 nm. The stock solution is further diluted into buffer solution to the desired concentration.
4-HNE is also supplied in ethanol as solvent (Cayman Chemical). In this case, an aliquot of the stock solution is evaporated under a stream of nitrogen and subsequently 4-HNE dissolved in PBS. In aqueous media, 4-HNE can be dissolved at final concentrations between 42 mmol/L (6.6 mg/mL water) (Esterbauer et al., 1991) and about 6.4 mmol/L (1 mg/mL PBS) according to the supplier. 4-HNE in buffered working solutions should be prepared daily fresh and kept at 4 °C in the dark.
2.3 Preparation of 4-HNA solutions
4-HNA can be synthesised from 4-oxohexanal (Long, Smoliakova, Honzatko, & Picklo, 2008; Picklo, Amarnath, McIntyre, Graham, & Montine, 1999). 4-HNA was a generous gift of Dr. Matthew J. Picklo, Dept of Pharmacology, Physiology and Therapeutics, University of North Dakota School of Medicine, Grand Forks, USA. Present address: USDA-ARS Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota, USA.
2.4 Reaction of 4-HNE with H2S
All reactions are carried out in 50 mmol/L phosphate buffer pH 7.4 at 37 °C. The reaction is monitored as the decrease of 4-HNE (absorbance at 222 nm) according to Esterbauer et al. (1975). As NaHS in solution absorbs at 230 nm (Nagy et al., 2014), which is to close to the Amax of 4-HNE, HCl is added to the incubations (final concentration 50 mmol/L, pH < 1) destroying 94-99% of NaHS, and the absorbance of the samples is recorded after 5 min.
Figure 1 shows the time- (A) and concentration- (B) dependent reaction of H2S with 4-HNE...