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Reactive sulfur species (RSS) such as H2S, HS•, H2Sn, (n = 2-7) and HS2•- are chemically similar to H2O and the reactive oxygen species (ROS) HO•, H2O2, O2•- and act on common biological effectors. RSS were present in evolution long before ROS, and because both are metabolized by catalase it has been suggested that "antioxidant" enzymes originally evolved to regulate RSS and may continue to do so today. Here we examined RSS metabolism by Cu/Zn superoxide dismutase (SOD) using amperometric electrodes for dissolved H2S, a polysulfide-specific fluorescent probe (SSP4), and mass spectrometry to identify specific polysulfides (H2S2-H2S5). H2S was concentration- and oxygen-dependently oxidized by 1μM SOD to polysulfides (mainly H2S2, and to a lesser extent H2S3 and H2S5) with an EC50 of approximately 380μM H2S. H2S concentrations > 750μM inhibited SOD oxidation (IC50 = 1.25mM) with complete inhibition when H2S > 1.75mM. Polysulfides were not metabolized by SOD. SOD oxidation preferred dissolved H2S over hydrosulfide anion (HS-), whereas HS- inhibited polysulfide production. In hypoxia, other possible electron donors such as nitrate, nitrite, sulfite, sulfate, thiosulfate and metabisulfite were ineffective. Manganese SOD also catalyzed H2S oxidation to form polysulfides, but did not metabolize polysulfides indicating common attributes of these SODs. These experiments suggest that, unlike the well-known SOD-mediated dismutation of two O2•- to form H2O2 and O2, SOD catalyzes a reaction using H2S and O2 to form persulfide. These can then combine in various ways to form polysulfides and sulfur oxides. It is also possible that H2S (or polysulfides) interact/react with SOD cysteines to affect catalytic activity or to directly contribute to sulfide metabolism. Our studies suggest that H2S metabolism by SOD may have been an ancient mechanism to detoxify sulfide or to regulate RSS and along with catalase may continue to do so in contemporary organisms.

Fluorescence spectroscopy and microscopy have been used extensively to monitor biomolecules, especially reactive oxygen species (ROS) and, more recently, reactive sulfide (RSS) species. Nearly all fluorophores are either excited by or emit light between 450 and 550 nm, which is similar to the absorbance of heme proteins and metal-centered porphyrins. Here we examined the effects of catalase (Cat), reduced and oxidized hemoglobin (Hb and metHb), albumin (alb), manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), iron protoporphyrin IX (hemin), and copper protoporphyrin IX (CuPPIX) on the fluorescence properties of fluorescein. We also examined the effects of catalase and MnTBAP on fluorophores for ROS (dichlorofluorescein, DCF), polysulfides (3′,6′-di( O-thiosalicyl)fluorescein, SSP4), and H2S (7-azido-4-methylcoumarin, AzMC) previously activated by H2O2, a mixed polysulfide (H2Sn, n = 1–7) and H2S, respectively. All except albumin concentration dependently inhibited fluorophore fluorescence and absorbed light between 450 and 550 nm, suggesting that the inhibitory effect was physical not catalytic. Catalase inhibition of fluorescein fluorescence was unaffected by sodium azide, dithiothreitol, diamide, tris(2-carboxyethyl)phosphine (TCEP), or iodoacetate, supporting a physical inhibitory mechanism. Catalase and TBAP augmented, then inhibited DCF fluorescence, but only inhibited SSP4 and AzMC fluorescence indicative of a substrate-specific catalytic oxidation of DCF and nonspecific fluorescence inhibition of all three fluorophores. These results suggest caution must be exercised when using any fluorescent tracers in the vicinity of metal-centered porphyrins.

Numerous studies have shown that HHere, we compare the effects of extended exposure (24-48 hours) to varying OAll cells continuously produced HThese results show that cellular H

In lung epithelial cells, hypoxia decreases the expression and activity of sodium-transporting molecules, thereby reducing the rate of transepithelial sodium absorption. The mechanisms underlying the sensing of hypoxia and subsequent coupling to sodium-transporting molecules remain unclear. Hydrogen sulfide (H2S) has recently been recognized as a cellular signaling molecule whose intracellular concentrations critically depend on oxygen levels. Therefore, it was questioned whether endogenously produced H2S contributes to hypoxic inhibition of sodium transport. In electrophysiological Ussing chamber experiments, hypoxia was established by decreasing oxygen concentrations in the chambers. Hypoxia concentration dependently and reversibly decreased amiloride-sensitive sodium absorption by cultured H441 monolayers and freshly dissected porcine tracheal epithelia due to inhibition of basolateral Na+/K+-ATPase. Exogenous application of H2S by the sulfur salt Na2S mimicked the effect of hypoxia and inhibited amiloride-sensitive sodium absorption by both tissues in an oxygen-dependent manner. Hypoxia increased intracellular concentrations of H2S and decreased the concentration of polysulfides. Pretreatment with the cystathionine-γ-lyase inhibitor d/l-propargylglycine (PAG) decreased hypoxic inhibition of sodium transport by H441 monolayers, whereas inhibition of cystathionine-β-synthase (with aminooxy-acetic acid; AOAA) or 3-mercaptopyruvate sulfurtransferase (with aspartate) had no effect. Inhibition of all of these H2S-generating enzymes with a combination of AOAA, PAG, and aspartate decreased the hypoxic inhibition of sodium transport by H441 cells and pig tracheae and decreased H2S production by tracheae. These data suggest that airway epithelial cells endogenously produce H2S during hypoxia, and this contributes to hypoxic inhibition of transepithelial sodium absorption.

The health benefits of garlic and other organosulfur-containing foods are well recognized and have been attributed to both prooxidant and antioxidant activities. The effects of garlic are surprisingly similar to those of hydrogen sulfide (H2S), which is also known to be released from garlic under certain conditions. However, recent evidence suggests that polysulfides, not H2S, may be the actual mediator of physiological signaling. In this study, we monitored formation of H2S and polysulfides from garlic oil in buffer and in human embryonic kidney (HEK) 293 cells with fluorescent dyes, 7-azido-4-methylcoumarin and SSP4, respectively and redox activity with two redox indicators redox-sensitive green fluorescent protein (roGFP) and DCF. Our results show that H2S release from garlic oil in buffer requires other low-molecular-weight thiols, such as cysteine (Cys) or glutathione (GSH), whereas polysulfides are readily detected in garlic oil alone. Administration of garlic oil to cells rapidly increases intracellular polysulfide but has minimal effects on H2S unless Cys or GSH are also present in the extracellular medium. We also observed that garlic oil and diallyltrisulfide (DATS) potently oxidized roGFP in buffer but did not affect DCF. This appears to be a direct polysulfide-mediated oxidation that does not require a reactive oxygen species intermediate. Conversely, when applied to cells, garlic oil became a significant intracellular reductant independent of extracellular Cys or GSH. This suggests that intracellular metabolism and further processing of the sulfur moieties are necessary to confer antioxidant properties to garlic oil in vivo.

Reactive sulfur species (RSS) such as H2S, HS•, H2Sn, (n = 2–7) and HS2•- are chemically similar to H2O and the reactive oxygen species (ROS) HO•, H2O2, O2•- and act on common biological effectors. RSS were present in evolution long before ROS, and because both are metabolized by catalase it has been suggested that “antioxidant” enzymes originally evolved to regulate RSS and may continue to do so today. Here we examined RSS metabolism by Cu/Zn superoxide dismutase (SOD) using amperometric electrodes for dissolved H2S, a polysulfide-specific fluorescent probe (SSP4), and mass spectrometry to identify specific polysulfides (H2S2-H2S5). H2S was concentration- and oxygen-dependently oxidized by 1 μM SOD to polysulfides (mainly H2S2, and to a lesser extent H2S3 and H2S5) with an EC50 of approximately 380 μM H2S. H2S concentrations > 750 μM inhibited SOD oxidation (IC50 = 1.25 mM) with complete inhibition when H2S > 1.75 mM. Polysulfides were not metabolized by SOD. SOD oxidation preferred dissolved H2S over hydrosulfide anion (HS-), whereas HS- inhibited polysulfide production. In hypoxia, other possible electron donors such as nitrate, nitrite, sulfite, sulfate, thiosulfate and metabisulfite were ineffective. Manganese SOD also catalyzed H2S oxidation to form polysulfides, but did not metabolize polysulfides indicating common attributes of these SODs. These experiments suggest that, unlike the well-known SOD-mediated dismutation of two O2•- to form H2O2 and O2, SOD catalyzes a reaction using H2S and O2 to form persulfide. These can then combine in various ways to form polysulfides and sulfur oxides. It is also possible that H2S (or polysulfides) interact/react with SOD cysteines to affect catalytic activity or to directly contribute to sulfide metabolism. Our studies suggest that H2S metabolism by SOD may have been an ancient mechanism to detoxify sulfide or to regulate RSS and along with catalase may continue to do so in contemporary organisms., Highlights • Polysulfides are reactive sulfide species (RSS) and are similar to reactive oxygen species (ROS). • RSS may be the antecedent of redox regulatory and stress-related modalities. • RSS likely persist in modern-day organisms and are regulated by SOD.

Hydrogen sulfide (H2S) is a novel signaling molecule most recently found to be of fundamental importance in cellular function as a regulator of apoptosis, inflammation, and perfusion. Mechanisms of endogenous H2S signaling are poorly understood; however, signal transmission is thought to occur via persulfidation at reactive cysteine residues on proteins. Although much has been discovered about how H2S is synthesized in the body, less is known about how it is metabolized. Recent studies have discovered a multitude of different targets for H2S therapy, including those related to protein modification, intracellular signaling, and ion channel depolarization. The most difficult part of studying hydrogen sulfide has been finding a way to accurately and reproducibly measure it. The purpose of this review is to: elaborate on the biosynthesis and catabolism of H2S in the human body, review current knowledge of the mechanisms of action of this gas in relation to ischemic injury, define strategies for physiological measurement of H2S in biological systems, and review potential novel therapies that use H2S for treatment.

Catalase is well-known as an antioxidant dismutating H2O2 to O2 and H2O. However, catalases evolved when metabolism was largely sulfur-based, long before O2 and reactive oxygen species (ROS) became abundant, suggesting catalase metabolizes reactive sulfide species (RSS). Here we examine catalase metabolism of H2Sn, the sulfur analog of H2O2, hydrogen sulfide (H2S) and other sulfur-bearing molecules using H2S-specific amperometric electrodes and fluorophores to measure polysulfides (H2Sn; SSP4) and ROS (dichlorofluorescein, DCF). Catalase eliminated H2Sn, but did not anaerobically generate H2S, the expected product of dismutation. Instead, catalase concentration- and oxygen-dependently metabolized H2S and in so doing acted as a sulfide oxidase with a P50 of 20 mmHg. H2O2 had little effect on catalase-mediated H2S metabolism but in the presence of the catalase inhibitor, sodium azide (Az), H2O2 rapidly and efficiently expedited H2S metabolism in both normoxia and hypoxia suggesting H2O2 is an effective electron acceptor in this reaction. Unexpectedly, catalase concentration-dependently generated H2S from dithiothreitol (DTT) in both normoxia and hypoxia, concomitantly oxidizing H2S in the presence of O2. H2S production from DTT was inhibited by carbon monoxide and augmented by NADPH suggesting that catalase heme-iron is the catalytic site and that NADPH provides reducing equivalents. Catalase also generated H2S from garlic oil, diallyltrisulfide, thioredoxin and sulfur dioxide, but not from sulfite, metabisulfite, carbonyl sulfide, cysteine, cystine, glutathione or oxidized glutathione. Oxidase activity was also present in catalase from Aspergillus niger. These results show that catalase can act as either a sulfide oxidase or sulfur reductase and they suggest that these activities likely played a prominent role in sulfur metabolism during evolution and may continue do so in modern cells as well. This also appears to be the first observation of catalase reductase activity independent of peroxide dismutation. Keywords: Hydrogen sulfide, Polysulfide, Garlic, Reactive oxygen species, Thioredoxin, Evolution, Aspergillus niger

The mechanism(s) by which cells detect hypoxia and transduce this into appropriate homeostatic responses is a contentious issue. Vascular oxygen sensing is generally attributed to hypoxic generatio...

Hydrogen sulfide (H(2)S) is a volatile gas of considerable interest as a physiologically relevant signaling molecule, but this volatility has typically been overlooked in the context of biological experiments. We examined volatility of 10 and 100 μM H(2)S (Na(2)S·9H(2)O) in real time with polarographic electrodes in three commonly employed experimental apparatuses: 24-well tissue culture plates (WP), muscle myograph baths (MB), and the Langendorff perfused heart apparatus (LPH). H(2)S loss from all apparatuses was rapid and exponential, with half-times (t(1/2)) of 5 min (WP), less than 4 min (MB), and less than 0.5 min (LPH). The t(1/2) for H(2)S loss from MB bubbled with 100% oxygen was slightly longer than that for MB bubbled with 100% nitrogen; both were significantly shorter than stirred but unbubbled MB (9 min). Therefore, even without tissue, H(2)S rapidly disappears from buffer under a variety of experimental conditions, and this is due to volatilization, not oxidation. The inability to maintain H(2)S concentration, even briefly, questions the accuracy of dose-response studies and the relevance of long-term (10 min) exposure to a single treatment of H(2)S. These results also help to explain the discrepancy between low H(2)S concentrations in blood and tissues versus high concentrations of exogenous H(2)S required to produce physiological responses.

SUMMARY Hydrogen sulfide (H 2 S) has been shown to affect gastrointestinal (GI) motility and signaling in mammals and O 2 -dependent H 2 S metabolism has been proposed to serve as an O 2 ‘sensor’ that couples hypoxic stimuli to effector responses in a variety of other O 2 -sensing tissues. The low P O2 values and high H 2 S concentrations routinely encountered in the GI tract suggest that H 2 S might also be involved in hypoxic responses in these tissues. In the present study we examined the effect of H 2 S on stomach, esophagus, gallbladder and intestinal motility in the rainbow trout ( Oncorhynchus mykiss ) and coho salmon ( Oncorhynchus kisutch ) and we evaluated the potential for H 2 S in oxygen sensing by examining GI responses to hypoxia in the presence of known inhibitors of H 2 S biosynthesis and by adding the sulfide donor cysteine (Cys). We also measured H 2 S production by intestinal tissue in real time and in the presence and absence of oxygen. In tissues exhibiting spontaneous contractions, H 2 S inhibited contraction magnitude (area under the curve and amplitude) and frequency, and in all tissues it reduced baseline tension in a concentration-dependent relationship. Longitudinal intestinal smooth muscle was significantly more sensitive to H 2 S than other tissues, exhibiting significant inhibitory responses at 1–10 μmol l –1 H 2 S. The effects of hypoxia were essentially identical to those of H 2 S in longitudinal and circular intestinal smooth muscle; of special note was a unique transient stimulatory effect upon application of both hypoxia and H 2 S. Inhibitors of enzymes implicated in H 2 S biosynthesis (cystathionine β-synthase and cystathionine γ-lyase) partially inhibited the effects of hypoxia whereas the hypoxic effects were augmented by the sulfide donor Cys. Furthermore, tissue production of H 2 S was inversely related to O 2 ; addition of Cys to intestinal tissue homogenate stimulated H 2 S production when the tissue was gassed with 100% nitrogen (∼0% O 2 ), whereas addition of oxygen (∼10% O 2 ) reversed this to net H 2 S consumption. This study shows that the inhibitory effects of H 2 S on the GI tract of a non-mammalian vertebrate are identical to those reported in mammals and they provide further evidence that H 2 S is a key mediator of the hypoxic response in a variety of O 2 -sensitive tissues.

Stepwise one-electron reduction of oxygen to water produces reactive oxygen species (ROS) that are chemically and biochemically similar to reactive sulfide species (RSS) derived from one-electron oxidations of hydrogen sulfide to elemental sulfur. Both ROS and RSS are endogenously generated and signal via protein thiols. Given the similarities between ROS and RSS, we wondered whether extant methods for measuring the former would also detect the latter. Here, we compared ROS to RSS sensitivity of five common ROS methods: redox-sensitive green fluorescent protein (roGFP), 2′, 7′-dihydrodichlorofluorescein, MitoSox Red, Amplex Red, and amperometric electrodes. All methods detected RSS and were as, or more, sensitive to RSS than to ROS. roGFP, arguably the “gold standard” for ROS measurement, was more than 200-fold more sensitive to the mixed polysulfide H2Sn ( n = 1–8) than to H2O2. These findings suggest that RSS may be far more prevalent in intracellular signaling than previously appreciated and that the contribution of ROS may be overestimated. This conclusion is further supported by the observation that estimated daily sulfur metabolism and ROS production are approximately equal and the fact that both RSS and antioxidant mechanisms have been present since the origin of life, nearly 4 billion years ago, long before the rise in environmental oxygen 600 million years ago. Although ROS are assumed to be the most biologically relevant oxidants, our results question this paradigm. We also anticipate our findings will direct attention toward development of novel and clinically relevant anti-(RSS)-oxidants.

Hydrogen sulfide (H2S) signaling has been implicated in physiological processes in practically all organ systems studied to date. At times the excitement of this new field has outpaced the technical expertise or practical knowledge with which to accurately assess these advancements. Recently, the myriad of proposed H2S actions has spawned interest in using indicators of H2S metabolism, especially plasma H2S concentrations, as a means of identifying a variety of pathophysiological conditions or to predict clinical outcomes. While this is a noteworthy endeavor, there are a number of contraindications to this practice at this time. First, there is little consensus regarding normal, i.e., "physiological" concentrations of H2S in either plasma or tissue. In fact, it has been shown that the methods most often employed for these measurements are associated with substantial artifact. Second, interactions, or presumed lack thereof, of H2S with other biomolecules (e.g., O2, H2O2, pH, etc.) or analytical reagents (e.g., reducing reagents, N-ethylmaleimide, phenylarsine, etc.) are often assumed but not evaluated. Third, the experimental design and/or statistical analyses may not be sufficient to justify using H2S concentration in tissue or blood as a predictive biomarker of pathophysiology. In this study, we first briefly review the problems associated with plasma and tissue H2S measurements and the associated errors and we provide some simple methods to evaluate whether the data obtained is physiologically relevant. Second we provide a brief analysis of H2S interactions with the above biomolecules. Third, we provide a statistical tool with which to determine the clinical applicability of H2S measurements. It is hoped that these points will provide a rational background for future work.

H2S derived from organic thiol metabolism has been proposed serve as an oxygen sensor in a variety of systems because of its susceptibility to oxidation and its ability to mimic hypoxic responses in numerous oxygen-sensing tissues. Thiosulfate, an intermediate in oxidative H2S metabolism can alternatively be reduced and regenerate H2S. We propose that this contributes to the H2S-mediated oxygen-sensing mechanism. H2S formation from thiosulfate in buffers and in a variety of mammalian tissues and in lamprey dorsal aorta was examined in real time using a polarographic H2S sensor. Inferences of intracellular H2S production were made by examining hypoxic pulmonary vasoconstriction (HPV) in bovine pulmonary arteries under conditions in which increased H2S production would be expected and in mouse and rat aortas, where reducing conditions should mediate vasorelaxation. In Krebs-Henseleit (mammalian) and Cortland (lamprey) buffers, H2S was generated from thiosulfate in the presence of the exogenous reducing agent, DTT, or the endogenous reductant dihydrolipoic acid (DHLA). Both the magnitude and rate of H2S production were greatly increased by these reductants in the presence of tissue, with the most notable effects occurring in the liver. H2S production was only observed when tissues were hypoxic; exposure to room air, or injecting oxygen inhibited H2S production and resulted in net H2S consumption. Both DTT and DHLA augmented HPV, and DHLA dose-dependently relaxed precontracted mouse and rat aortas. These results indicate that thiosulfate can contribute to H2S signaling under hypoxic conditions and that this is not only a ready source of H2S production but also serves as a means of recycling sulfur and thereby conserving biologically relevant thiols.

Multiphoton Microscopy and Phosphorescence Lifetime Microscopy are combined to produce full-frame lifetime resolved 3D images in vivo. Parallel excitation and acquisition of two-photon emission is achieved via a frequency multiplexed multi-foci excitation beam.

We present work looking at the two-photon optical characterization of water soluble chemical probes based on surfactant nanomicelles encapsulation and ormosil PEBBLEs as well as in-vitro two-photon imaging of oxygen and hydrogen sulfide.

“Sulfhydration” has been proposed to be a means of hydrogen sulfide (H 2 S) signaling in which the H 2 S reacts with the sulfur of a cysteine residue in proteins, thereby forming a polysulfide and changing the function of the latter, as described by the equation RSH + H 2 S → RSSH. However, this requires oxidation of the cysteine RSH, which cannot be accomplished by H 2 S, as the sulfur is in its most reduced form (oxidation state of −2). Hydropersulfides (H 2 S n , n = 2–8, oxidation number = 0), can oxidize RSH and have been suggested to be a contaminant of salts commonly used to produce H 2 S (NaSH, Na 2 S) and account for much of the activity originally attributed to H 2 S. Here, we use the green fluorescent protein, roGFP2, that contains two reactive cysteine residues (Cys147, Cys204) and is a specific reporter of redox status, being fully oxidized by 30 mM tert-butyl hydroperoxide (t-BuOOH), and fully reduced by dithiothreitol (DTT, 30 mM), to determine if H 2 S can spontaneously form hydro-persulfides in normoxic (21% O 2 ) laboratory conditions. We show that all concentrations ranging from 1 μ M-300uM H 2 S have very little reductive effect on roGFP, but that H 2 S > 300 uM slowly reoxidizes roGFP in a closed system given an hour to react. We attribute this to spontaneous oxidation of H 2 S to hydropersulfides. Addition of mixed persulfides (K 2 S n , n = 1–8) oxidizes roGFP at low concentrations (1uM-300uM), supporting our hypothesis. These results show that H 2 S n can be formed from H 2 S and thereby signal via sulfhydration. Support, NSF IOS-1051627 and NSF DGE-1313583.