Hydrogen Sulfide in Redox Biology Part A (eBook)

Enrique Cadenas, Lester Packer (Herausgeber)

eBook Download: PDF | EPUB

2015 | 1. Auflage
344 Seiten
Elsevier Science (Verlag)
978-0-12-801623-7 (ISBN)

Infos

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 the role of hydrogen sulfide in the cardiovascular and nervous systems and in inflammation

Inhalt

Front Cover 1
Hydrogen Sulfide in Redox Biology, Part A 4
Copyright 5
Contents 6
Contributors 12
Preface 16
Section I: Hydrogen Sulfide Detection Methods 18
Chapter 1: Mechanistic Chemical Perspective of Hydrogen Sulfide Signaling 20
1. Introduction 22
2. Bioavailability of Sulfide-The Signal 22
2.1. Endogenous sulfide production 23
2.2. Sulfide catabolism 25
2.3. Endogenous sulfide buffers 26
3. Inorganic Polysulfides 26
3.1. Biological relevance 26
3.2. Speciation and redox capacity of polysulfides 27
3.3. Polysulfide formation by sulfide oxidation 28
3.4. Stability of polysulfides 29
4. Sulfide Signaling Via Protein Sulfhydration 30
4.1. Mechanisms of persulfide formation 31
4.1.1. Persulfide formation via disulfide reduction 32
4.1.2. Persulfide formation via the reactions of Cys sulfenic acid species with sulfide 33
4.1.3. Persulfide formation via the reactions of oxidized sulfide species with Cys thiols 34
4.1.4. Persulfide formation via radical pathways 35
5. Sulfide Signaling via Sulfide-Hemeprotein Interactions 35
5.1. Sulfide mediates heme protein functions 36
5.2. Heme proteins generate sulfide oxidation products 38
5.3. Antioxidant properties of sulfide via reduction of metal centers with higher oxidation states 39
6. Conclusions 40
Acknowledgments 41
References 41
Chapter 2: Measurement of H2S In Vivo and In Vitro by the Monobromobimane Method 48
1. Introduction 49
1.1. Properties of hydrogen sulfide 49
1.2. Hydrogen sulfide pools 49
1.3. Physiological and pathophysiological roles of hydrogen sulfide 50
1.4. Measurement of hydrogen sulfide bioavailability 51
2. Experimental Methods 52
2.1. Derivatization reaction of H2S with monobromobimane 52
2.1.1. Procedure 52
2.1.2. Comment and limitations 53
2.2. H2S detection in biological samples: Effects of sample preparation 53
2.3. RP-HPLC with fluorescence detection 54
2.3.1. Procedure 55
2.3.2. Preparation of SDB standard 55
2.3.3. Comment and limitations 56
2.4. H2S and sulfide pool detection in biological samples 56
2.4.1. Procedure 56
2.4.2. Comment and limitations 58
2.5. Confirmation of HPLC and SDB by mass spectrometer 58
2.5.1. Procedure 58
2.5.2. Comment and limitations 60
3. Summary 60
Acknowledgment 60
References 60
Chapter 3: Hydrogen Sulfide Detection Using Nucleophilic Substitution-Cyclization-Based Fluorescent Probes 64
1. Introduction 65
2. Design and Synthesis of the Probes 66
3. Chemistry and Properties of the Probes 67
3.1. Materials 68
3.2. Test the reaction between the probes and H2S 69
3.3. Fluorescence turn-on properties by H2S 70
3.4. Test the probes selectivity for H2S 71
4. Applications of the Probes in H2S Imaging in Cell-Based Experiments 72
4.1. Materials 72
4.2. Fluorescence imaging of exogenous H2S in HeLa cells 74
4.3. Fluorescence imaging of H2S generated by persulfide-based H2S donors 74
4.4. Fluorescence imaging of H2S generated from photo-sensitive H2S donors 76
5. Conclusions 77
Acknowledgments 78
References 78
Chapter 4: Azide-Based Fluorescent Probes: Imaging Hydrogen Sulfide in Living Systems 80
1. Introduction 81
2. Fluorescent Azide-Based H2S Probes 83
2.1. Probe design 83
2.2. Reactivity 83
2.3. Use and storage of probes 85
3. In Vitro Characterization of Probes 86
3.1. Safety precautions 86
3.2. Instrumentation and materials 86
3.3. Time-course assays 87
3.4. Selectivity experiments 87
3.5. Data processing and analysis 88
4. Detection of H2S in Live Cells Using Fluorescent Probes 89
4.1. Imaging exogenous H2S using confocal microscopy 89
4.1.1. Materials and instrumentation 89
4.1.2. Cell culture and dye loading 90
4.1.3. Imaging and results 90
4.2. Imaging endogenous H2S production in HUVECs 91
4.2.1. Materials and instrumentation 91
4.2.2. Cell culture and dye loading 91
4.2.3. Imaging and results 92
4.3. Interrogating pathways involved in H2S production using confocal microscopy 92
5. Conclusions 94
Acknowledgments 95
References 95
Chapter 5: Chemiluminescent Detection of Enzymatically Produced H2S 98
1. Introduction 99
2. Chemiluminescent Probes for the Determination of Sulfide 102
2.1. Probe design 102
2.2. Reactivity 103
2.3. Probe usage and storage 106
3. Examples of Routine Probe Usage 106
3.1. Instrumentation and materials 106
3.1.1. Buffer 106
3.1.2. Reactive species 107
3.1.3. Instrumentation 107
3.2. Preparation of CLSS-1 and CLSS-2 107
3.2.1. CLSS-1 107
3.2.2. CLSS-2 108
3.3. Sensing method 108
3.4. Data processing and analysis 109
4. Detection of Enzymatically Produced H2S 109
4.1. Instrumentation and materials 109
4.1.1. Instrumentation 109
4.1.2. Media 110
4.1.3. Probe 110
4.1.4. Materials 110
4.1.5. Reactive species 110
4.2. Cell culture and lysing 110
4.3. Assay for enzymatically produced H2S 111
4.4. Results and controls 112
5. Conclusions 112
Acknowledgments 113
References 113
Chapter 6: Quantification of Hydrogen Sulfide Concentration Using Methylene Blue and 5,5-Dithiobis(2-Nitrobenzoic Acid) M... 118
1. Theory 119
2. Equipment 120
3. Materials 121
3.1. Solution and buffer 121
4. Protocol 1 122
4.1. Duration 122
4.2. Preparation 122
5. Step 1: Quantification of H2S Concentration Using MB Method 123
5.1. Overview 123
5.2. Duration 123
2h 123
5.3. Tip 123
5.4. Tip 124
5.5. Tip 124
5.6. Tip 124
5.7. Tip 124
6. Protocol 2 124
6.1. Duration 124
6.2. Preparation 125
7. Step 1: Quantification of H2S concentration using 5,5-dithiobis (2-nitrobenzoicacid) method 125
7.1. Overview 125
7.2. Duration 125
2h 125
7.3. Tip 126
7.4. Tip 126
7.5. Tip 126
7.6. Tip 127
7.7. Tip 127
Acknowledgment 127
References 127
Chapter 7: H2S Analysis in Biological Samples Using Gas Chromatography with Sulfur Chemiluminescence Detection 128
1. Introduction 129
2. Principle of the GC-Coupled Sulfur Chemiluminescence Method 129
2.1. Limitations of the GC method 130
3. Protocol for GC-Coupled Sulfur Chemiluminescence Detection of H2S 131
3.1. Materials 131
3.2. Calibration standards 132
3.3. Sample manipulation 132
3.4. Chromatography conditions 132
4. Analysis of Biological Samples 133
4.1. Monitoring H2S production in tissue homogenate 133
4.2. Monitoring H2S degradation in tissue homogenates 135
4.3. Estimation of H2S production and degradation rates 136
5. Additional Technical Details 139
5.1. Column conditioning 139
5.2. Additional gas purification 139
Acknowledgment 139
References 139
Section II: Hydrogen Sulfide Donors and Their Pharmacological Activity 142
Chapter 8: Use of Phosphorodithioate-Based Compounds as Hydrogen Sulfide Donors 144
1. Introduction 145
2. Synthesis of Phosphorodithioate-Based Donors 146
2.1. Materials 146
2.2. Synthetic route 147
2.3. Protocols and data 147
3. Measurements of H2S Generation from the Donors Using Fluorescence Methods 150
3.1. Materials and instrument 150
3.2. Calibration curve for fluorescence measurements 151
3.3. Determination of H2S release from donors 152
4. H2S Release from the Donors in Cultured Cells 152
4.1. Materials 153
4.2. Cell viability test protocol and results 153
4.3. Images of H2S release in cells 154
5. Donor´s Activity Against H2O2-Induced Cell Damage 155
5.1. Materials 155
5.2. The optimal concentration of H2O2 for cell damage experiments 155
5.3. Evaluation of donor´s protective effects against H2O2 damage 156
6. Summary 157
Acknowledgments 157
References 157
Chapter 9: GYY4137, a Novel Water-Soluble, H2S-Releasing Molecule 160
1. Introduction 161
2. Why Slow Releasing H2S Donors? 163
3. The Development and Characterization of GYY4137 164
4. Facile Synthesis and Chemical Characterization of GYY4137 165
5. Biological Effects of GYY4137: An Overview and Potential Role in Disease? 166
5.1. Cardiovascular system: Vascular smooth muscle and platelet function 169
5.2. Effect of GYY4137 on nonvascular smooth muscle 170
5.3. Inflammation: Is GYY4137 pro- or anti-inflammatory? 170
5.4. Effect of GYY4137 in the reproductive system 172
5.5. GYY4137: Apoptosis and cell cycle progression 173
5.6. GYY4137 and aging 175
6. The Effect of GYY4137 in Nonmammalian Systems 176
7. Conclusion 178
References 179
Chapter 10: Neuroprotective Effects of Hydrogen Sulfide in Parkinson´s Disease Animal Models: Methods and Protocols 186
1. Introduction 187
2. PD Animal Models 187
2.1. 6-OHDA-induced PD rat model 189
2.2. Rotenone-induced PD rat model 191
2.3. MPTP-induced subacute PD mice model 192
2.3.1. Postoperative care 193
3. H2S and Its Releasing Compound Treatment 193
3.1. NaHS 193
3.2. ACS84 194
4. Behavior Tests 194
4.1. Rotational behavior test 194
4.2. Locomotor activity test 195
4.3. Rearing activity test 195
5. Immunohistochemical Assay 195
5.1. Tyrosine-hydroxylase positive neurons 196
5.2. Glia activation 197
6. Brain H2S Activity Tests 197
6.1. CSE, CBS, and 3-MST expression 197
6.2. Brain H2S generating enzyme activity test 198
6.2.1. Brain tissue H2S-producing capacity 198
7. Prospects of H2S Therapy on PD and Conclusions 199
Acknowledgment 201
References 201
Section III: Hydrogen Sulfide Metabolism in Mammalian Tissues 204
Chapter 11: Assay Methods for H2S Biogenesis and Catabolism Enzymes 206
1. Introduction 206
2. Assays for H2S Biogenesis 208
2.1. Assays for CBS and CSE 209
2.1.1. H2S formation from cysteine or cysteine+homocysteine 209
Reagents 209
2.1.2. Methanethiol formation from methylcysteine 209
Reagents 209
2.1.3. Assessing H2S production by CBS versus CSE in tissue samples 210
Reagents 210
2.2. Assays for MST 211
2.2.1. MST assay using small molecule acceptors 211
Reagents 211
2.2.2. MST assay using thioredoxin 211
Reagents 211
3. Assays for Enzymes Involved in H2S Catabolism 212
3.1. Assay for SQR 213
Reagents 213
3.2. Assay for sulfur dioxygenase (or persulfide dioxygenase or ETHE1) 213
3.2.1. Preparation of GSSH 213
Reagents 213
3.2.2. Oxygen consumption assay 214
Reagents 214
3.3. Assays for rhodanese 214
3.3.1. Assay for thiocyanate formation by rhodanese 214
Reagents 214
3.3.2. Assay for thiosulfate production by rhodanese 215
Reagents 215
3.3.3. Assay for H2S production by rhodanese 215
Reagents 215
Acknowledgments 216
References 216
Chapter 12: Oxidation of H2S in Mammalian Cells and Mitochondria 218
1. Introduction 219
1.1. Sulfide gasotransmitters and mitochondria 219
1.2. Issues treated in and audience of this chapter 220
2. Sulfide in the Context of Mitochondrial Bioenergetics 220
2.1. Cellular bioenergetics and mitochondria 220
2.2. Multiple hydrogen donors to the mitochondrial coenzyme Q 222
2.3. Sulfide and gaseous transmitters are toxic to mitochondria 223
2.4. Positive feedback loops for sulfide oxidation/inhibition 224
3. Practical Issues 225
3.1. Oxygen consumption 225
3.2. Use of inhibitors of mitochondrial respiration 226
3.3. Other measurements 226
3.4. Sulfide solutions 227
3.5. Cellular and mitochondrial models 228
4. Sulfide Oxidation Experiments 231
4.1. Addition of defined concentrations of sulfide 231
4.2. Safe and toxic range for free sulfide concentration 232
4.3. Concentration dependence of SOU activity 235
4.4. Establishment of steady states by infusion 236
4.5. Seahorse 237
5. Treatment, Expression, and Interpretation of Results 239
5.1. Steady-state experiments 239
5.2. Injection experiments 241
6. Originality and Interest with Regard to Bioenergetics 242
6.1. Stoichiometric calculations 242
6.2. Reduction of coenzyme Q and competition between electron donors 243
6.3. Reverse flux in complex I 243
Acknowledgments 244
References 244
Chapter 13: Redox Regulation of Mammalian 3-Mercaptopyruvate Sulfurtransferase 246
1. Introduction 247
1.1. History and molecular properties 247
1.2. Catalytic properties 249
1.3. Distribution 251
1.4. Biological function 251
2. Redox Regulation of Cysteine Metabolism and MST 252
3. Regulation of MST Activity via Redox-Sensing Molecular Switches 252
3.1. Redox-sensing molecular switches 252
3.2. Catalytic site cysteine as a redox-sensing molecular switch 254
3.3. Cysteine(s) residue(s) on the surface of MST as a redox-sensing molecular switch 256
4. MST Knockout Mouse 262
5. Other Investigation 268
References 269
Chapter 14: Role of Human Sulfide: Quinone Oxidoreductase in H2S Metabolism 272
1. Introduction 273
2. Expression of Human SQOR in E. coli 274
3. Purification of Recombinant Human SQOR 274
4. Catalytic Assays 276
5. Spectral Properties of Recombinant Human SQOR 277
6. Survey of Potential Sulfane Sulfur Acceptors for Human SQOR 278
7. Spectral Course of SQOR Catalytic Assays with Sulfite, Cyanide, or Sulfide as Sulfane Sulfur Acceptor 280
8. Steady-State Kinetic Parameters for H2S Oxidation by SQOR with Sulfite, Cyanide, or Sulfide as Sulfane Sulfur Acceptor 282
9. Role of Human SQOR in H2S Metabolism 284
Acknowledgment 285
References 286
Chapter 15: H2S Regulation of Nitric Oxide Metabolism 288
1. Introduction 289
1.1. Nitric oxide and hydrogen sulfide: Key gaseous signaling molecules and their interactions 289
1.2. How does H2S influence NOS and production of NO and its metabolites? 289
1.3. Novel adducts formation from H2S-NO interactions 291
2. Techniques Determining Enzymatic Activity and Expression of NOS 292
2.1. High-sensitive radiolabeled detection of NOS 293
2.1.1. Key points and limitations 293
2.2. Western blotting for detection of NOS expression 294
2.3. Determination of mRNA expression of NOSs by qRT-PCR 294
3. Detection of NO and Its Metabolites 295
3.1. Griess assay: Classic biochemical assay for nitrite/nitrate/nitrosothiol detection 296
3.1.1. Protocol 296
3.1.2. Key points and limitations 297
3.2. Chemiluminescent detection of NO metabolites 297
3.2.1. Protocol 298
3.2.2. Preparation of samples for analysis 299
3.2.3. Process for analyses of nitrate, nitrite, S-nitrosothiols, and XNO 299
3.2.4. Experimental protocol to identify interference of H2S on NO detection 300
3.2.5. Key points and limitations 300
3.3. Real-time detection of NO by electrode probe 302
3.3.1. Calibration 302
3.3.2. Key points and limitations 303
3.4. ESR detection of NO 303
3.5. Fluorescent detection of NO 304
4. Novel Adducts from H2S-NO Interactions 304
4.1. Peroxynitrite (ONOOH/ONOO-) 305
4.2. H2S interactions with NO donors 305
4.3. S-nitrosothiols 306
4.4. Experimental procedures 307
4.4.1. UV-vis and stopped-flow spectroscopy 307
4.4.1.1. Protocol 307
4.4.1.2. Key points 308
4.4.2. Mass spectrometry 308
4.4.2.1. Protocol 308
4.4.2.2. Key comments 308
4.4.3. HNO/NO- imaging with CuBOT1 309
4.4.3.1. Protocol 309
4.4.3.2. Key points 309
5. Conclusion 309
Acknowledgments 310
References 310
Author Index 316
Subject Index 334
Color Plate 346

Textauszug

Chapter Two

Measurement of H2S In Vivo and In Vitro by the Monobromobimane Method

Xinggui Shen; Gopi K. Kolluru; Shuai Yuan; Christopher G. Kevil1    Department of Pathology, Louisiana State University Health Sciences Center–Shreveport, Shreveport, Louisiana, USA
1 Corresponding author: email address: [e-mail]

Abstract

The gasotransmitter hydrogen sulfide (H2S) is known as an important regulator in several physiological and pathological responses. Among the challenges facing the field is the accurate and reliable measurement of hydrogen sulfide bioavailability. We have reported an approach to discretely measure sulfide and sulfide pools using the monobromobimane (MBB) method coupled with reversed phase high-performance liquid chromatography (RP-HPLC). The method involves the derivatization of sulfide with excess MBB under precise reaction conditions at room temperature to form sulfide dibimane (SDB). The resultant fluorescent SDB is analyzed by RP-HPLC using fluorescence detection with the limit of detection for SDB (2 nM). Care must be taken to avoid conditions that may confound H2S measurement with this method. Overall, RP-HPLC with fluorescence detection of SDB is a useful and powerful tool to measure biological sulfide levels.

Keywords

Fluorescence

High-performance liquid chromatography

Mass spectrometry

Oxygen

pH

Sulfide

1 Introduction

1.1 Properties of hydrogen sulfide

Hydrogen sulfide (H2S) is a colorless gas with the odor of rotten eggs and can be oxidized to form sulfur dioxide, sulfates, sulfite, and elemental sulfur. Based on its lipophilic property, hydrogen sulfide easily penetrates the lipid bilayer of cell membranes (Wang, 2012); however, it is less membrane permeable than nitric oxide (NO) and carbon monoxide (CO). The difference in membrane permeability between NO, CO, and hydrogen sulfide is also reflected by their dipole moments, which have values of 0.16, 0.13, and 0.97, respectively. Hydrogen sulfide is slightly soluble in water and acts as a weak acid with an acid dissociation constant (pKa1) of 7.04 and pKa2 of 19 at 37 °C (Hughes, Centelles, & Moore, 2009). It can dissociate into H+ and hydrosulfide anion (HS−), which in turn may dissociate into H+ and sulfide anion (S2 −) in the following reaction:

2S↔H++HS−↔2H++S2−

At physiological pH and 37 °C, ~ 20% of sulfide is present as H2S, whereas at physiological pH and 25 °C, ~ 40% of sulfide is present as H2S conversely at pH 9.5, hydrogen sulfide mainly exists as HS− (Hughes et al., 2009; Shen et al., 2011). In vivo, pH favors sulfide existence primarily as H2S and its highly reactive anion, HS−.

1.2 Hydrogen sulfide pools

Hydrogen sulfide is produced from a variety of sources, including chemical reactions (e.g., hydrogen gas and elemental sulfur, ferrous sulfide and HCl, aluminum sulfide and water), sulfate-reducing bacteria, and in mammalian tissues. During hydrogen sulfide production in mammalian tissues, there are three tissue-specific enzymes involved, viz., cystathionine-β-synthase, cystathionine-γ-lyase, and 3-mercaptosulfurtransferase (Moore, Bhatia, & Moochhala, 2003).

Acid-labile sulfide and bound sulfane sulfur are two main forms of hydrogen sulfide stored in mammalian cells. They can release hydrogen sulfide under acidic and on reducing conditions, respectively (Shen, Peter, Bir, Wang, & Kevil, 2012). Examples of bound sulfane sulfur include thiosulfate, persulfide, thiosulfonate, polysulfides, polythionates, and elemental sulfur. Overall, these different biochemical forms are important for regulating the amount of bioavailable hydrogen sulfide (Ishigami et al., 2009; Wintner et al., 2010).

1.3 Physiological and pathophysiological roles of hydrogen sulfide

Hydrogen sulfide is best known for its toxicity. Indeed, H2S at high concentrations irreversibly inhibits the respiratory chain by binding to the ferric heme a3 center and the CuB center of cytochrome c oxidase. Similarly, H2S reacts with oxygenated ferrous hemoglobin and myoglobin and converts them to sulfhemoglobin or sulfmyoglobin, which are unable to carry O2. However, mounting evidence implicates H2S as an endogenous signaling molecule that plays important roles in physiological and pathological processes (Kolluru, Shen, Bir, & Kevil, 2013; Kolluru, Shen, & Kevil, 2013; Wang, 2011).

To date, a large spectrum of proteins has been shown to be targeted by H2S. H2S post-translationally modifies the regulatory sulfonylurea receptor of the ATP-sensitive potassium (KATP) channel in vascular smooth muscle cells (SMCs), resulting in an increased potassium flow, resultant hyperpolarization, and vasodilation (Tang, Wu, Liang, & Wang, 2005). The wide distribution of KATP and its isoforms makes H2S important for the regulation of heart contractility and rate (cardiomyocytes), sensation (neurons), insulin secretion (β-islet cells), and mitochondrial functions (mitoKATP). Other ion channels are also shown to be the target of H2S, including intermediate and small conductance potassium channels (IKCa/SKCa), L-type calcium channels, and the transient receptor potential cation channel A1 (Avanzato et al., 2014; Mustafa et al., 2011; Streng et al., 2008; Tang, Zhang, Yang, Wu, & Wang, 2013).

Moreover, recent evidence shows vascular endothelial growth factor receptor 2 is modified by H2S to facilitate its activation after ligand binding (Tao et al., 2013). Meanwhile, SMC proliferation and survival have been shown to be inhibited by H2S involving ERK activation, which indicates critical regulation of vascular remodeling by H2S (Baskar, Sparatore, Del Soldato, & Moore, 2008). H2S also targets and inhibits PTP1B and regulates endoplasmic reticulum stress (Krishnan, Fu, Pappin, & Tonks, 2011). Additionally, persulfidation of p65 by H2S promotes its nucleus translocation and increases transcription of antiapoptotic proteins (Sen et al., 2012). Last but not least, H2S can serve as an anti-oxidant that counter balances with reactive oxygen species, including superoxide, hypochlorous acid, peroxynitrite, and lipid peroxidation. Interestingly, H2S is also thought to interact with the other gasotransmitters NO and CO on the levels of enzymatic synthesis, oxidative stress, and small adducts, such as HNO, HSNO, and GSNO (Nagy et al., 2014).

1.4 Measurement of hydrogen sulfide bioavailability

Accurate and reliable measurement of biological hydrogen sulfide can provide critical information associated with various pathophysiological functions. However, significant uncertainty exists regarding levels of H2S associated with health, disease, and therapeutics. While there are several reasons for the current uncertainties it is now clear that a wide range of values for hydrogen sulfide have been reported (Levitt, Abdel-Rehim, & Furne, 2011; Nagy et al., 2014; Shen et al., 2011, 2012; Zheng et al., 2012).

The methylene blue method is the most commonly reported method used in the literature to measure hydrogen sulfide in biological samples (Zhu et al., 2007). The method is based on spectrophotometry of methylene blue dye after the reaction of sulfide and N,N-dimethyl-p-phenylenediamine. However, this method can be highly problematic, making it inappropriate for measuring biological levels of hydrogen sulfide (Shen et al., 2011). Key problems include: (1) interference of other colored substances, (2) methylene blue dimer and trimer formation, (3) strong acid chemical pretreatment, and (4) low sensitivity.

Other analytical methods have been reported but are limited for various reasons. Gas chromatography is sensitive enough to measure physiological sulfide levels, but it potentially liberates loosely-bound sulfide because of irreversible sulfide binding or shifts in phase transition equilibria (Levitt et al., 2011; Ubuka, Abe, Kajikawa, & Morino, 2001). Sulfide-specific ion-selective electrodes have also been in use to detect H2S levels in biological samples, with a detection range of 1–10 μM but are prone to fouling and limited sensitivity detection. Lastly, fluorescent probes for intracellular measurement of hydrogen sulfide have greatly evolved in the last couple of years. Yet, a major challenge exists with the regard to interference by other thiol species (Nagy et al., 2014).

The purpose of this chapter is to provide detailed techniques to perform the MBB derivatized method for detecting hydrogen sulfide in various biological matrices. The main methodology used is RP-HPLC with fluorescence detection or in combination with mass spectrometry. Additionally, different sample treatment workflows allow for the separation and quantification of free sulfide, acid-labile sulfide, and bound sulfane sulfur (Shen et al., 2012). With a 2.0 nM limit of detection, this method is sensitive and reliable enough for use with most biological samples.

2 Experimental Methods

2.1...

Produkt-Details

• Erscheint lt. Verlag: 25.2.2015
• Sprache: englisch
• Themenwelt: Studium ► 1. Studienabschnitt (Vorklinik) ► Physiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• ISBN-10: 0-12-801623-X / 012801623X
• ISBN-13: 978-0-12-801623-7 / 9780128016237