Cadenas / Packer Hydrogen Sulfide in Redox Biology Part A

1. Auflage 2015
ISBN: 978-0-12-801623-7
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 554, 344 Seiten

Reihe: Methods in Enzymology

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

Autoren/Hrsg.

Cadenas, Enrique

Packer, Lester

Inhaltsverzeichnis

1;Front Cover;1
2;Hydrogen Sulfide in Redox Biology, Part A;4
3;Copyright;5
4;Contents;6
5;Contributors;12
6;Preface;16
7;Section I: Hydrogen Sulfide Detection Methods;18
7.1;Chapter 1: Mechanistic Chemical Perspective of Hydrogen Sulfide Signaling;20
7.1.1;1. Introduction;22
7.1.2;2. Bioavailability of Sulfide-The Signal;22
7.1.2.1;2.1. Endogenous sulfide production;23
7.1.2.2;2.2. Sulfide catabolism;25
7.1.2.3;2.3. Endogenous sulfide buffers;26
7.1.3;3. Inorganic Polysulfides;26
7.1.3.1;3.1. Biological relevance;26
7.1.3.2;3.2. Speciation and redox capacity of polysulfides;27
7.1.3.3;3.3. Polysulfide formation by sulfide oxidation;28
7.1.3.4;3.4. Stability of polysulfides;29
7.1.4;4. Sulfide Signaling Via Protein Sulfhydration;30
7.1.4.1;4.1. Mechanisms of persulfide formation;31
7.1.4.1.1;4.1.1. Persulfide formation via disulfide reduction;32
7.1.4.1.2;4.1.2. Persulfide formation via the reactions of Cys sulfenic acid species with sulfide;33
7.1.4.1.3;4.1.3. Persulfide formation via the reactions of oxidized sulfide species with Cys thiols;34
7.1.4.1.4;4.1.4. Persulfide formation via radical pathways;35
7.1.5;5. Sulfide Signaling via Sulfide-Hemeprotein Interactions;35
7.1.5.1;5.1. Sulfide mediates heme protein functions;36
7.1.5.2;5.2. Heme proteins generate sulfide oxidation products;38
7.1.5.3;5.3. Antioxidant properties of sulfide via reduction of metal centers with higher oxidation states;39
7.1.6;6. Conclusions;40
7.1.7;Acknowledgments;41
7.1.8;References;41
7.2;Chapter 2: Measurement of H2S In Vivo and In Vitro by the Monobromobimane Method;48
7.2.1;1. Introduction;49
7.2.1.1;1.1. Properties of hydrogen sulfide;49
7.2.1.2;1.2. Hydrogen sulfide pools;49
7.2.1.3;1.3. Physiological and pathophysiological roles of hydrogen sulfide;50
7.2.1.4;1.4. Measurement of hydrogen sulfide bioavailability;51
7.2.2;2. Experimental Methods;52
7.2.2.1;2.1. Derivatization reaction of H2S with monobromobimane;52
7.2.2.1.1;2.1.1. Procedure;52
7.2.2.1.2;2.1.2. Comment and limitations;53
7.2.2.2;2.2. H2S detection in biological samples: Effects of sample preparation;53
7.2.2.3;2.3. RP-HPLC with fluorescence detection;54
7.2.2.3.1;2.3.1. Procedure;55
7.2.2.3.2;2.3.2. Preparation of SDB standard;55
7.2.2.3.3;2.3.3. Comment and limitations;56
7.2.2.4;2.4. H2S and sulfide pool detection in biological samples;56
7.2.2.4.1;2.4.1. Procedure;56
7.2.2.4.2;2.4.2. Comment and limitations;58
7.2.2.5;2.5. Confirmation of HPLC and SDB by mass spectrometer;58
7.2.2.5.1;2.5.1. Procedure;58
7.2.2.5.2;2.5.2. Comment and limitations;60
7.2.3;3. Summary;60
7.2.4;Acknowledgment;60
7.2.5;References;60
7.3;Chapter 3: Hydrogen Sulfide Detection Using Nucleophilic Substitution-Cyclization-Based Fluorescent Probes;64
7.3.1;1. Introduction;65
7.3.2;2. Design and Synthesis of the Probes;66
7.3.3;3. Chemistry and Properties of the Probes;67
7.3.3.1;3.1. Materials;68
7.3.3.2;3.2. Test the reaction between the probes and H2S;69
7.3.3.3;3.3. Fluorescence turn-on properties by H2S;70
7.3.3.4;3.4. Test the probes selectivity for H2S;71
7.3.4;4. Applications of the Probes in H2S Imaging in Cell-Based Experiments;72
7.3.4.1;4.1. Materials;72
7.3.4.2;4.2. Fluorescence imaging of exogenous H2S in HeLa cells;74
7.3.4.3;4.3. Fluorescence imaging of H2S generated by persulfide-based H2S donors;74
7.3.4.4;4.4. Fluorescence imaging of H2S generated from photo-sensitive H2S donors;76
7.3.5;5. Conclusions;77
7.3.6;Acknowledgments;78
7.3.7;References;78
7.4;Chapter 4: Azide-Based Fluorescent Probes: Imaging Hydrogen Sulfide in Living Systems;80
7.4.1;1. Introduction;81
7.4.2;2. Fluorescent Azide-Based H2S Probes;83
7.4.2.1;2.1. Probe design;83
7.4.2.2;2.2. Reactivity;83
7.4.2.3;2.3. Use and storage of probes;85
7.4.3;3. In Vitro Characterization of Probes;86
7.4.3.1;3.1. Safety precautions;86
7.4.3.2;3.2. Instrumentation and materials;86
7.4.3.3;3.3. Time-course assays;87
7.4.3.4;3.4. Selectivity experiments;87
7.4.3.5;3.5. Data processing and analysis;88
7.4.4;4. Detection of H2S in Live Cells Using Fluorescent Probes;89
7.4.4.1;4.1. Imaging exogenous H2S using confocal microscopy;89
7.4.4.1.1;4.1.1. Materials and instrumentation;89
7.4.4.1.2;4.1.2. Cell culture and dye loading;90
7.4.4.1.3;4.1.3. Imaging and results;90
7.4.4.2;4.2. Imaging endogenous H2S production in HUVECs;91
7.4.4.2.1;4.2.1. Materials and instrumentation;91
7.4.4.2.2;4.2.2. Cell culture and dye loading;91
7.4.4.2.3;4.2.3. Imaging and results;92
7.4.4.3;4.3. Interrogating pathways involved in H2S production using confocal microscopy;92
7.4.5;5. Conclusions;94
7.4.6;Acknowledgments;95
7.4.7;References;95
7.5;Chapter 5: Chemiluminescent Detection of Enzymatically Produced H2S;98
7.5.1;1. Introduction;99
7.5.2;2. Chemiluminescent Probes for the Determination of Sulfide;102
7.5.2.1;2.1. Probe design;102
7.5.2.2;2.2. Reactivity;103
7.5.2.3;2.3. Probe usage and storage;106
7.5.3;3. Examples of Routine Probe Usage;106
7.5.3.1;3.1. Instrumentation and materials;106
7.5.3.1.1;3.1.1. Buffer;106
7.5.3.1.2;3.1.2. Reactive species;107
7.5.3.1.3;3.1.3. Instrumentation;107
7.5.3.2;3.2. Preparation of CLSS-1 and CLSS-2;107
7.5.3.2.1;3.2.1. CLSS-1;107
7.5.3.2.2;3.2.2. CLSS-2;108
7.5.3.3;3.3. Sensing method;108
7.5.3.4;3.4. Data processing and analysis;109
7.5.4;4. Detection of Enzymatically Produced H2S;109
7.5.4.1;4.1. Instrumentation and materials;109
7.5.4.1.1;4.1.1. Instrumentation;109
7.5.4.1.2;4.1.2. Media;110
7.5.4.1.3;4.1.3. Probe;110
7.5.4.1.4;4.1.4. Materials;110
7.5.4.1.5;4.1.5. Reactive species;110
7.5.4.2;4.2. Cell culture and lysing;110
7.5.4.3;4.3. Assay for enzymatically produced H2S;111
7.5.4.4;4.4. Results and controls;112
7.5.5;5. Conclusions;112
7.5.6;Acknowledgments;113
7.5.7;References;113
7.6;Chapter 6: Quantification of Hydrogen Sulfide Concentration Using Methylene Blue and 5,5-Dithiobis(2-Nitrobenzoic Acid) M...;118
7.6.1;1. Theory;119
7.6.2;2. Equipment;120
7.6.3;3. Materials;121
7.6.3.1;3.1. Solution and buffer;121
7.6.4;4. Protocol 1;122
7.6.4.1;4.1. Duration;122
7.6.4.2;4.2. Preparation;122
7.6.5;5. Step 1: Quantification of H2S Concentration Using MB Method;123
7.6.5.1;5.1. Overview;123
7.6.5.2;5.2. Duration;123
7.6.6;2h;123
7.6.6.1;5.3. Tip;123
7.6.6.2;5.4. Tip;124
7.6.6.3;5.5. Tip;124
7.6.6.4;5.6. Tip;124
7.6.6.5;5.7. Tip;124
7.6.7;6. Protocol 2;124
7.6.7.1;6.1. Duration;124
7.6.7.2;6.2. Preparation;125
7.6.8;7. Step 1: Quantification of H2S concentration using 5,5-dithiobis (2-nitrobenzoicacid) method;125
7.6.8.1;7.1. Overview;125
7.6.8.2;7.2. Duration;125
7.6.9;2h;125
7.6.9.1;7.3. Tip;126
7.6.9.2;7.4. Tip;126
7.6.9.3;7.5. Tip;126
7.6.9.4;7.6. Tip;127
7.6.9.5;7.7. Tip;127
7.6.10;Acknowledgment;127
7.6.11;References;127
7.7;Chapter 7: H2S Analysis in Biological Samples Using Gas Chromatography with Sulfur Chemiluminescence Detection;128
7.7.1;1. Introduction;129
7.7.2;2. Principle of the GC-Coupled Sulfur Chemiluminescence Method;129
7.7.2.1;2.1. Limitations of the GC method;130
7.7.3;3. Protocol for GC-Coupled Sulfur Chemiluminescence Detection of H2S;131
7.7.3.1;3.1. Materials;131
7.7.3.2;3.2. Calibration standards;132
7.7.3.3;3.3. Sample manipulation;132
7.7.3.4;3.4. Chromatography conditions;132
7.7.4;4. Analysis of Biological Samples;133
7.7.4.1;4.1. Monitoring H2S production in tissue homogenate;133
7.7.4.2;4.2. Monitoring H2S degradation in tissue homogenates;135
7.7.4.3;4.3. Estimation of H2S production and degradation rates;136
7.7.5;5. Additional Technical Details;139
7.7.5.1;5.1. Column conditioning;139
7.7.5.2;5.2. Additional gas purification;139
7.7.6;Acknowledgment;139
7.7.7;References;139
8;Section II: Hydrogen Sulfide Donors and Their Pharmacological Activity;142
8.1;Chapter 8: Use of Phosphorodithioate-Based Compounds as Hydrogen Sulfide Donors;144
8.1.1;1. Introduction;145
8.1.2;2. Synthesis of Phosphorodithioate-Based Donors;146
8.1.2.1;2.1. Materials;146
8.1.2.2;2.2. Synthetic route;147
8.1.2.3;2.3. Protocols and data;147
8.1.3;3. Measurements of H2S Generation from the Donors Using Fluorescence Methods;150
8.1.3.1;3.1. Materials and instrument;150
8.1.3.2;3.2. Calibration curve for fluorescence measurements;151
8.1.3.3;3.3. Determination of H2S release from donors;152
8.1.4;4. H2S Release from the Donors in Cultured Cells;152
8.1.4.1;4.1. Materials;153
8.1.4.2;4.2. Cell viability test protocol and results;153
8.1.4.3;4.3. Images of H2S release in cells;154
8.1.5;5. Donor´s Activity Against H2O2-Induced Cell Damage;155
8.1.5.1;5.1. Materials;155
8.1.5.2;5.2. The optimal concentration of H2O2 for cell damage experiments;155
8.1.5.3;5.3. Evaluation of donor´s protective effects against H2O2 damage;156
8.1.6;6. Summary;157
8.1.7;Acknowledgments;157
8.1.8;References;157
8.2;Chapter 9: GYY4137, a Novel Water-Soluble, H2S-Releasing Molecule;160
8.2.1;1. Introduction;161
8.2.2;2. Why Slow Releasing H2S Donors?;163
8.2.3;3. The Development and Characterization of GYY4137;164
8.2.4;4. Facile Synthesis and Chemical Characterization of GYY4137;165
8.2.5;5. Biological Effects of GYY4137: An Overview and Potential Role in Disease?;166
8.2.5.1;5.1. Cardiovascular system: Vascular smooth muscle and platelet function;169
8.2.5.2;5.2. Effect of GYY4137 on nonvascular smooth muscle;170
8.2.5.3;5.3. Inflammation: Is GYY4137 pro- or anti-inflammatory?;170
8.2.5.4;5.4. Effect of GYY4137 in the reproductive system;172
8.2.5.5;5.5. GYY4137: Apoptosis and cell cycle progression;173
8.2.5.6;5.6. GYY4137 and aging;175
8.2.6;6. The Effect of GYY4137 in Nonmammalian Systems;176
8.2.7;7. Conclusion;178
8.2.8;References;179
8.3;Chapter 10: Neuroprotective Effects of Hydrogen Sulfide in Parkinson´s Disease Animal Models: Methods and Protocols;186
8.3.1;1. Introduction;187
8.3.2;2. PD Animal Models;187
8.3.2.1;2.1. 6-OHDA-induced PD rat model;189
8.3.2.2;2.2. Rotenone-induced PD rat model;191
8.3.2.3;2.3. MPTP-induced subacute PD mice model;192
8.3.2.3.1;2.3.1. Postoperative care;193
8.3.3;3. H2S and Its Releasing Compound Treatment;193
8.3.3.1;3.1. NaHS;193
8.3.3.2;3.2. ACS84;194
8.3.4;4. Behavior Tests;194
8.3.4.1;4.1. Rotational behavior test;194
8.3.4.2;4.2. Locomotor activity test;195
8.3.4.3;4.3. Rearing activity test;195
8.3.5;5. Immunohistochemical Assay;195
8.3.5.1;5.1. Tyrosine-hydroxylase positive neurons;196
8.3.5.2;5.2. Glia activation;197
8.3.6;6. Brain H2S Activity Tests;197
8.3.6.1;6.1. CSE, CBS, and 3-MST expression;197
8.3.6.2;6.2. Brain H2S generating enzyme activity test;198
8.3.6.2.1;6.2.1. Brain tissue H2S-producing capacity;198
8.3.7;7. Prospects of H2S Therapy on PD and Conclusions;199
8.3.8;Acknowledgment;201
8.3.9;References;201
9;Section III: Hydrogen Sulfide Metabolism in Mammalian Tissues;204
9.1;Chapter 11: Assay Methods for H2S Biogenesis and Catabolism Enzymes;206
9.1.1;1. Introduction;206
9.1.2;2. Assays for H2S Biogenesis;208
9.1.2.1;2.1. Assays for CBS and CSE;209
9.1.2.1.1;2.1.1. H2S formation from cysteine or cysteine+homocysteine;209
9.1.3;Reagents;209
9.1.3.1;2.1.2. Methanethiol formation from methylcysteine;209
9.1.4;Reagents;209
9.1.4.1;2.1.3. Assessing H2S production by CBS versus CSE in tissue samples;210
9.1.5;Reagents;210
9.1.5.1;2.2. Assays for MST;211
9.1.5.1.1;2.2.1. MST assay using small molecule acceptors;211
9.1.6;Reagents;211
9.1.6.1;2.2.2. MST assay using thioredoxin;211
9.1.7;Reagents;211
9.1.8;3. Assays for Enzymes Involved in H2S Catabolism;212
9.1.8.1;3.1. Assay for SQR;213
9.1.9;Reagents;213
9.1.9.1;3.2. Assay for sulfur dioxygenase (or persulfide dioxygenase or ETHE1);213
9.1.9.1.1;3.2.1. Preparation of GSSH;213
9.1.10;Reagents;213
9.1.10.1;3.2.2. Oxygen consumption assay;214
9.1.11;Reagents;214
9.1.11.1;3.3. Assays for rhodanese;214
9.1.11.1.1;3.3.1. Assay for thiocyanate formation by rhodanese;214
9.1.12;Reagents;214
9.1.12.1;3.3.2. Assay for thiosulfate production by rhodanese;215
9.1.13;Reagents;215
9.1.13.1;3.3.3. Assay for H2S production by rhodanese;215
9.1.14;Reagents;215
9.1.15;Acknowledgments;216
9.1.16;References;216
9.2;Chapter 12: Oxidation of H2S in Mammalian Cells and Mitochondria;218
9.2.1;1. Introduction;219
9.2.1.1;1.1. Sulfide gasotransmitters and mitochondria;219
9.2.1.2;1.2. Issues treated in and audience of this chapter;220
9.2.2;2. Sulfide in the Context of Mitochondrial Bioenergetics;220
9.2.2.1;2.1. Cellular bioenergetics and mitochondria;220
9.2.2.2;2.2. Multiple hydrogen donors to the mitochondrial coenzyme Q;222
9.2.2.3;2.3. Sulfide and gaseous transmitters are toxic to mitochondria;223
9.2.2.4;2.4. Positive feedback loops for sulfide oxidation/inhibition;224
9.2.3;3. Practical Issues;225
9.2.3.1;3.1. Oxygen consumption;225
9.2.3.2;3.2. Use of inhibitors of mitochondrial respiration;226
9.2.3.3;3.3. Other measurements;226
9.2.3.4;3.4. Sulfide solutions;227
9.2.3.5;3.5. Cellular and mitochondrial models;228
9.2.4;4. Sulfide Oxidation Experiments;231
9.2.4.1;4.1. Addition of defined concentrations of sulfide;231
9.2.4.2;4.2. Safe and toxic range for free sulfide concentration;232
9.2.4.3;4.3. Concentration dependence of SOU activity;235
9.2.4.4;4.4. Establishment of steady states by infusion;236
9.2.4.5;4.5. Seahorse;237
9.2.5;5. Treatment, Expression, and Interpretation of Results;239
9.2.5.1;5.1. Steady-state experiments;239
9.2.5.2;5.2. Injection experiments;241
9.2.6;6. Originality and Interest with Regard to Bioenergetics;242
9.2.6.1;6.1. Stoichiometric calculations;242
9.2.6.2;6.2. Reduction of coenzyme Q and competition between electron donors;243
9.2.6.3;6.3. Reverse flux in complex I;243
9.2.7;Acknowledgments;244
9.2.8;References;244
9.3;Chapter 13: Redox Regulation of Mammalian 3-Mercaptopyruvate Sulfurtransferase;246
9.3.1;1. Introduction;247
9.3.1.1;1.1. History and molecular properties;247
9.3.1.2;1.2. Catalytic properties;249
9.3.1.3;1.3. Distribution;251
9.3.1.4;1.4. Biological function;251
9.3.2;2. Redox Regulation of Cysteine Metabolism and MST;252
9.3.3;3. Regulation of MST Activity via Redox-Sensing Molecular Switches;252
9.3.3.1;3.1. Redox-sensing molecular switches;252
9.3.3.2;3.2. Catalytic site cysteine as a redox-sensing molecular switch;254
9.3.3.3;3.3. Cysteine(s) residue(s) on the surface of MST as a redox-sensing molecular switch;256
9.3.4;4. MST Knockout Mouse;262
9.3.5;5. Other Investigation;268
9.3.6;References;269
9.4;Chapter 14: Role of Human Sulfide: Quinone Oxidoreductase in H2S Metabolism;272
9.4.1;1. Introduction;273
9.4.2;2. Expression of Human SQOR in E. coli;274
9.4.3;3. Purification of Recombinant Human SQOR;274
9.4.4;4. Catalytic Assays;276
9.4.5;5. Spectral Properties of Recombinant Human SQOR;277
9.4.6;6. Survey of Potential Sulfane Sulfur Acceptors for Human SQOR;278
9.4.7;7. Spectral Course of SQOR Catalytic Assays with Sulfite, Cyanide, or Sulfide as Sulfane Sulfur Acceptor;280
9.4.8;8. Steady-State Kinetic Parameters for H2S Oxidation by SQOR with Sulfite, Cyanide, or Sulfide as Sulfane Sulfur Acceptor;282
9.4.9;9. Role of Human SQOR in H2S Metabolism;284
9.4.10;Acknowledgment;285
9.4.11;References;286
9.5;Chapter 15: H2S Regulation of Nitric Oxide Metabolism;288
9.5.1;1. Introduction;289
9.5.1.1;1.1. Nitric oxide and hydrogen sulfide: Key gaseous signaling molecules and their interactions;289
9.5.1.2;1.2. How does H2S influence NOS and production of NO and its metabolites?;289
9.5.1.3;1.3. Novel adducts formation from H2S-NO interactions;291
9.5.2;2. Techniques Determining Enzymatic Activity and Expression of NOS;292
9.5.2.1;2.1. High-sensitive radiolabeled detection of NOS;293
9.5.2.1.1;2.1.1. Key points and limitations;293
9.5.2.2;2.2. Western blotting for detection of NOS expression;294
9.5.2.3;2.3. Determination of mRNA expression of NOSs by qRT-PCR;294
9.5.3;3. Detection of NO and Its Metabolites;295
9.5.3.1;3.1. Griess assay: Classic biochemical assay for nitrite/nitrate/nitrosothiol detection;296
9.5.3.1.1;3.1.1. Protocol;296
9.5.3.1.2;3.1.2. Key points and limitations;297
9.5.3.2;3.2. Chemiluminescent detection of NO metabolites;297
9.5.3.2.1;3.2.1. Protocol;298
9.5.3.2.2;3.2.2. Preparation of samples for analysis;299
9.5.3.2.3;3.2.3. Process for analyses of nitrate, nitrite, S-nitrosothiols, and XNO;299
9.5.3.2.4;3.2.4. Experimental protocol to identify interference of H2S on NO detection;300
9.5.3.2.5;3.2.5. Key points and limitations;300
9.5.3.3;3.3. Real-time detection of NO by electrode probe;302
9.5.3.3.1;3.3.1. Calibration;302
9.5.3.3.2;3.3.2. Key points and limitations;303
9.5.3.4;3.4. ESR detection of NO;303
9.5.3.5;3.5. Fluorescent detection of NO;304
9.5.4;4. Novel Adducts from H2S-NO Interactions;304
9.5.4.1;4.1. Peroxynitrite (ONOOH/ONOO-);305
9.5.4.2;4.2. H2S interactions with NO donors;305
9.5.4.3;4.3. S-nitrosothiols;306
9.5.4.4;4.4. Experimental procedures;307
9.5.4.4.1;4.4.1. UV-vis and stopped-flow spectroscopy;307
9.5.4.4.1.1;4.4.1.1. Protocol;307
9.5.4.4.1.2;4.4.1.2. Key points;308
9.5.4.4.2;4.4.2. Mass spectrometry;308
9.5.4.4.2.1;4.4.2.1. Protocol;308
9.5.4.4.2.2;4.4.2.2. Key comments;308
9.5.4.4.3;4.4.3. HNO/NO- imaging with CuBOT1;309
9.5.4.4.3.1;4.4.3.1. Protocol;309
9.5.4.4.3.2;4.4.3.2. Key points;309
9.5.5;5. Conclusion;309
9.5.6;Acknowledgments;310
9.5.7;References;310
10;Author Index;316
11;Subject Index;334
12;Color Plate;346

Leseproben

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-ß-synthase, cystathionine-?-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 (ß-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...