Hydrogen Sulfide in Redox Biology Part B

 
 
Academic Press
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  • erschienen am 3. März 2015
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  • 370 Seiten
 
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978-0-12-801622-0 (ISBN)
 
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
0076-6879
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  • USA
Elsevier Science
  • 10,99 MB
978-0-12-801622-0 (9780128016220)
0128016221 (0128016221)
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  • Front Cover
  • Hydrogen Sulfide in Redox Biology, Part B
  • Copyright
  • Contents
  • Contributors
  • Preface
  • Section I: The Redox Biochemistry of Hydrogen Sulfide
  • Chapter 1: Investigating the Role of H2S in 4-HNE Scavenging
  • 1. Introduction
  • 2. Experimental Compounds and Considerations
  • 2.1. H2S generation
  • 2.2. Preparation of 4-HNE solutions
  • 2.3. Preparation of 4-HNA solutions
  • 2.4. Reaction of 4-HNE with H2S
  • 2.5. Approaches to monitor the effect of H2S on protein modification by 4-HNE
  • 2.5.1. Electrophoretic mobility shift
  • 2.5.2. Immunoblotting
  • 2.6. Approaches to monitor the effect of H2S on cytotoxicity of 4-HNE
  • 2.7. Detection of 4-HNE-modified cellular proteins
  • 3. Conclusions and Perspectives
  • Acknowledgment
  • References
  • Chapter 2: Inhalation Exposure Model of Hydrogen Sulfide (H2S)-Induced Hypometabolism in the Male Sprague-Dawley Rat
  • 1. Introduction
  • 2. Exposure Protocol for H2S-Induced Hypometabolism in Rats
  • 2.1. Special gases and equipment
  • 2.2. Animals
  • 2.3. H2S exposure protocol
  • 2.4. Physiological measurements
  • 2.5. Lung histopathology assessment
  • 2.6. Tissue and blood collection after H2S exposure for experimental measurements
  • 2.7. Comments on measuring H2S and H2S-derived metabolites in blood and tissues from H2S-exposed rats
  • 3. Other Considerations for H2S Exposure Studies
  • 4. Summary
  • Acknowledgments
  • References
  • Section II: Mechanisms of H2S Cell Signaling and Transcriptional Pathways
  • Chapter 3: Use of the "Tag-Switch" Method for the Detection of Protein S-Sulfhydration
  • 1. Introduction
  • 2. The Design of "Tag-Switch" Method
  • 3. Chemistry Validation Using Small-Molecule Substrates
  • 3.1. Materials
  • 3.2. Model reactions of MSBT with thiol and persulfide substrates
  • 3.3. Model reaction of cyanoacetate with R-S-S-BT
  • 4. "Tag-Switch" Assay on Bovine Serum Albumin and GAPDH as Model Proteins
  • 4.1. Materials
  • 4.2. Preparation of BSA-SOH, BSA-SSG, and BSA-SSH
  • 4.3. "Tag-switch" labeling of model proteins for MS and dot blot
  • 4.4. Studying the mechanism of protein S-sulfhydration by "tag-switch" assay
  • 5. "Tag-Switch" Assay for the Detection on Intracellular Protein Persulfides
  • 5.1. Materials
  • 5.2. Tag-switch labeling of Jurkat cell extracts
  • 5.3. Tag-switch labeling during immunoprecipitation
  • 6. "Tag-Switch" Assay for the Detection of Intracellular S-Sulfhydration by Fluorescence Microscopy
  • 6.1. Materials
  • 6.2. Protocol
  • 6.3. Colocalization of intracellular S-sulfhydration with different organelles
  • 7. Conclusions
  • Acknowledgments
  • References
  • Chapter 4: Real-Time Assays for Monitoring the Influence of Sulfide and Sulfane Sulfur Species on Protein Thiol Redox States
  • 1. Introduction
  • 2. PTEN Activity Assay
  • 2.1. Principle
  • 2.2. Reagents and equipment
  • 2.3. Procedure
  • 2.4. Comments
  • 2.5. Expected results
  • 3. roGFP2 Redox Assay
  • 3.1. Principle
  • 3.2. Reagents and equipment
  • 3.3. Procedure
  • 3.4. Comments
  • 3.5. Expected results
  • 4. Application of "H2S Donors" and Polysulfides
  • 4.1. General considerations
  • 4.2. Expected results
  • 5. Quantitation of Sulfane Sulfur by Cold Cyanolysis
  • 5.1. Principle
  • 5.2. Reagents and equipment
  • 5.3. Procedure
  • 5.4. Comments
  • 5.5. Expected results
  • 6. Elimination of Sulfane Sulfur by Cold Cyanolysis
  • 6.1. Principle
  • 6.2. Elimination of pre-existing sulfane sulfur
  • 6.2.1. Reagents and equipment
  • 6.2.2. Procedure
  • 6.2.3. Expected results
  • 6.3. Prevention of the de novo formation of sulfane sulfur
  • 6.3.1. Reagents and equipment
  • 6.3.2. Procedure
  • 6.3.3. Expected results
  • Acknowledgments
  • References
  • Chapter 5: Protein Sulfhydration
  • 1. Introduction
  • 1.1. Protein S-sulfhydration
  • 1.2. Reciprocity of sulfhydration and nitrosylation
  • 2. Detection of Sulfhydration Using the Modified Biotin Switch Assay
  • 2.1. Treatment of cell cultures to assess sulfhydration
  • 2.2. Reagents for the modified biotin switch assay
  • 2.3. Solutions and buffers for the modified biotin switch assay
  • 2.4. Protocol
  • 3. Detection of Sulfhydration Using the Maleimide Assay
  • 3.1. Solutions and buffers for the maleimide assay
  • 3.2. Protocol
  • 3.3. Alternate protocol
  • 3.4. Considerations
  • 4. Summary
  • Acknowledgments
  • References
  • Section III: H2S in Cell Signaling in the Cardiovascular and Nervous System and Inflammatory Processes
  • Chapter 6: Intravital Microscopic Methods to Evaluate Anti-inflammatory Effects and Signaling Mechanisms Evoked by Hydrog...
  • 1. Introduction
  • 2. Molecular Determinants of Neutrophil/Endothelial Cell Adhesive Interactions
  • 3. Intravital Microscopic Approaches to Study Leukocyte/Endothelial Cell Adhesive Interactions
  • 4. Assessing Leukocyte Rolling, Adhesion, and Emigration in the Intact Microcirculation
  • 5. Detection of Chemokine and Adhesion Molecule Expression using Intravital Microscopy
  • 6. Intravital Microscopic Methods to Assess Changes in Microvascular Permeability
  • 7. Assessment of Reactive Oxygen Species Generation Using Intravital Microscopy
  • 8. Fluorescence Detection of Cell Injury using Intravital Microscopy
  • 9. Perfused Capillary Density Assessment with Intravital Microscopy
  • 10. Acute and Preconditioning-Induced Anti-inflammatory Actions of Hydrogen Sulfide: Assessment Using Intravital Microscopy
  • 11. Conclusion and Perspectives
  • Acknowledgment
  • References
  • Chapter 7: Attenuation of Inflammatory Responses by Hydrogen Sulfide (H2S) in Ischemia/Reperfusion Injury
  • 1. Introduction
  • 2. Ischemia-Reperfusion Injury
  • 3. Central Nervous System
  • 4. Respiratory System
  • 5. Cardiovascular System
  • 6. Gastrointestinal System
  • 6.1. Stomach
  • 6.2. Small intestine
  • 7. Hepatobiliary System
  • 8. Renal System
  • 9. Musculoskeletal
  • 10. Summary
  • References
  • Chapter 8: CD47-Dependent Regulation of H2S Biosynthesis and Signaling in T Cells
  • 1. Introduction
  • 2. Regulation of H2S Biosynthesis in T Cells
  • 2.1. Biosynthesis of H2S in normal T cells
  • 2.2. Biosynthesis of H2S by lymphoid malignancies
  • 3. Catabolism of H2S
  • 4. Regulation of T cell Activation by H2S Signaling
  • 4.1. Biphasic effects of H2S on T cells
  • 5. Autocrine and Paracrine Roles of H2S in T cell Activation
  • 6. Role of H2S in the Cytoskeleton
  • 7. T Cell Regulation by TSP1/CD47 Signaling
  • 8. H2S Regulation of Leukocyte Adhesion
  • 9. Role of H2S in Diseases Associated with Altered T cell Immunity
  • 9.1. Inflammatory bowel disease
  • 9.2. Renal injury
  • 9.3. Rheumatoid arthritis
  • 9.4. Asthma and allergy
  • 9.5. Psoriasis
  • 9.6. Systemic lupus erythematosus
  • 10. Future Prospective
  • Acknowledgments
  • References
  • Chapter 9: Anti-inflammatory and Cytoprotective Properties of Hydrogen Sulfide
  • 1. Introduction
  • 2. Enzymatic Synthesis of H2S
  • 3. Healing and Resolution of Inflammation
  • 4. Mechanisms of Anti-inflammatory Effects of H2S
  • 5. Effects of H2S on Visceral Pain
  • 6. Cytoprotective Actions of H2S
  • 7. Therapeutic Applications of H2S-Releasing Drugs
  • 7.1. Inflammation and pain
  • 7.2. Cardiovascular disease
  • 7.3. Spinal cord injury and neurodegenerative diseases
  • 7.4. Inflammatory bowel disease
  • 7.5. Visceral analgesia
  • Acknowledgments
  • References
  • Chapter 10: H2S and Substance P in Inflammation
  • 1. Introduction
  • 2. Disease Models Used to Study the Role of H2S and Substance P
  • 2.1. Caerulein-induced acute pancreatitis
  • 2.2. Lipopolysaccharide-induced endotoxemia and cecal ligation and puncture-induced sepsis
  • 2.3. Burn injury
  • 2.4. Carrageenan-induced hindpaw edema
  • 3. H2S and Substance P-What Are They Doing Together?
  • 4. Summary
  • Acknowledgments
  • References
  • Chapter 11: Role of Hydrogen Sulfide in Brain Synaptic Remodeling
  • 1. Introduction
  • 2. Pharmacological and Physiological Effect of H2S
  • 3. Effect of H2S on the CNS
  • 4. Effect of H2S on Brain Cells (Astrocyte, Microglia, and Oligodendrocyte)
  • 5. Synapse
  • 6. Glia and Neurons Interactions
  • 7. Effect of H2S on Neuronal Redox Stress
  • 8. Effect of H2S on Glutamate Neurotransmission
  • 9. Effect of H2S on NMDA Receptor Regulation
  • 10. Effect of H2S on GABA-Mediated Neurotransmission
  • 11. Effect of H2S on Calmodulin Kinase
  • 12. Conclusion
  • Acknowledgment
  • References
  • Section IV: H2S in Plants
  • Chapter 12: Detection of Thiol Modifications by Hydrogen Sulfide
  • 1. Introduction
  • 2. Hydrogen Sulfide Acts as a Signal in Cells
  • 3. Modification of Thiols by Signaling Molecules
  • 4. Identification of Modified Thiols by Other Methods
  • 5. Experimental Protocols
  • 6. Caenorhabditis elegans as a Model Organism
  • 7. Growth of C. elegans
  • 8. Treatment of Samples with H2S
  • 9. Estimation of Toxicity of H2S Compounds
  • 10. Treatment of Samples with Thiol Tag
  • 11. Isolation and Analysis of Modified Proteins
  • 12. Estimation of Protein Concentrations in Samples
  • 13. Further Analysis and Identification of Modified Proteins
  • 14. Concluding Remarks
  • References
  • Chapter 13: Analysis of Some Enzymes Activities of Hydrogen Sulfide Metabolism in Plants
  • 1. Theory
  • 2. Equipment
  • 3. Materials
  • 3.1. Solution and buffer
  • 4. Protocol 1
  • 4.1. Duration
  • 4.2. Preparation
  • 5. Step 1: Analyze of l-/d-Cysteine Desulfhydrase Activity
  • 5.1. Overview
  • 5.2. Duration
  • 5.3. Tip
  • 5.4. Tip
  • 5.5. Tip
  • 5.6. Tip
  • 5.7. Tip
  • 5.8. Tip
  • 6. Protocol 2
  • 6.1. Duration
  • 6.2. Preparation
  • 7. Step 1: Analyze of Sulfite Reductase Activity
  • 7.1. Overview
  • 7.2. Duration
  • 7.3. Tip
  • 7.4. Tip
  • 7.5. Tip
  • 7.6. Tip
  • 7.7. Tip
  • 7.8. Tip
  • 8. Protocol 3
  • 8.1. Duration
  • 8.2. Preparation
  • 9. Step 1: Analyze of ß-Cyano-l-Alanine Synthase Activity
  • 9.1. Overview
  • 9.2. Duration
  • 9.3. Tip
  • 9.4. Tip
  • 9.5. Tip
  • 9.6. Tip
  • 9.7. Tip
  • 10. Protocol 4
  • 10.1. Duration
  • 10.2. Preparation
  • 11. Step 1: Analyze of l-Cysteine Synthase Activity
  • 11.1. Overview
  • 11.2. Duration
  • 11.3. Tip
  • 11.4. Tip
  • 11.5. Tip
  • 11.6. Tip
  • Acknowledgment
  • References
  • Chapter 14: Sulfide Detoxification in Plant Mitochondria
  • 1. Introduction
  • 1.1. Formation of sulfide in plant cells
  • 1.2. Sulfide toxicity and detoxification mechanisms in plant mitochondria
  • 2. Methods
  • 2.1. Determination of CAS activity
  • 2.2. SAT affinity purification of OAS-TL from plant tissue and determination of enzymatic activity
  • 2.2.1. Recombinant expression and immobilization of His6:AtSAT5 on a nickel-loaded chromatography column
  • 2.2.2. Large-scale extraction of soluble proteins from plant tissue
  • 2.2.3. Purification of OAS-TL proteins from soluble protein extract
  • 2.2.4. Determination of OAS-TL activity
  • 2.3. Discrimination between CAS and OAS-TL
  • 2.4. Determination of SDO activity
  • 3. Summary
  • Acknowledgments
  • References
  • Section V: Molecular Hydrogen
  • Chapter 15: Molecular Hydrogen as a Novel Antioxidant: Overview of the Advantages of Hydrogen for Medical Applications
  • 1. Introduction
  • 2. Comparison of H2 with Other Medical Gasses
  • 3. Oxidative Stress as Pathogenic Sources
  • 4. Physiological Roles of H2O2
  • 5. Measurement of H2 Gas Concentration
  • 6. Advantages of Hydrogen in Medical Applications
  • 6.1. Selective reaction of H2 with highly reactive ROS
  • 6.2. Rapid diffusion
  • 7. Methods of Ingesting Molecular Hydrogen
  • 7.1. Inhalation of hydrogen gas
  • 7.2. Oral ingestion by drinking hydrogen water
  • 7.3. Injection of hydrogen-saline
  • 7.4. Direct incorporation of molecular hydrogen by diffusion: Eye drops, bath, and cosmetics
  • 7.5. Maternal intake of H2
  • 8. Medical Effects of H2
  • 8.1. Acute oxidative stress by ischemia/reperfusion
  • 8.2. Chronic oxidative stress loading to neurodegeneration
  • 8.3. Stimulatory effects on energy metabolism
  • 8.4. Anti-inflammatory effects
  • 9. Possible Molecular Mechanisms Underlying Various Effects of Molecular Hydrogen
  • 9.1. Direct reduction of hydroxyl radicals with molecular hydrogen
  • 9.2. Direct reduction of peroxynitrite with molecular hydrogen to regulate gene expression
  • 9.3. Indirect reduction of oxidative stress by regulating gene expression
  • 10. Unresolved Questions and Closing Remarks
  • References
  • Author Index
  • Subject Index
  • Color Plate
Chapter One

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: hildegard.laggner@meduniwien.ac.at, bernhard.gmeiner@meduniwien.ac.at

Abstract


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.

Keywords

4-Hydroxy-2-nonenal

4-Hydroxy-nonanal

Hydrogen sulfide

Michael adduct

Neuroblastoma

SH-SY5Y cells

a,ß-Unsaturated aldehyde

1 Introduction


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 4.2.1.22), cystathionine-?-lyase (CSE EC 4.4.1.1), and 3-mercapto-pyruvate sulfurtransferase (3MST EC 2.8.1.2) 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...

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