Nitrosation-modulating effect of ascorbate in a model dynamic system of coexisting nitric oxide and superoxide

Abstract

The coexistence of nitric oxide and superoxide leads to complex oxidative and nitrosative chemistry, which has been implicated in many pathophysiological conditions. The present study investigated the role of ascorbate in affecting the kinetics of nitrosative chemistry in a model dynamic snystem of coexisting nitric oxide and superoxide. SIN-1 (3-morpholinosydnonimine) was used to elicit various degrees of nitroxidative stress in a reaction buffer and DAN (2,3-diaminonaphthalene) was used as a probe for N-nitrosation reaction. The nitrosation kinetics in the absence and presence of ascorbate was followed by measuring the formation of the fluorescent product over time. Computational modelling was used to provide quantitative or semi-quantitative insights into the studied system. The results show that ascorbate effectively quenches N-nitrosation reaction, which could be partially attributed to the free radical scavenging and repairing effect of ascorbate. Computational modelling reveals an interesting temporal distribution of superoxide, nitric oxide and peroxynitrite. The model predicts that peroxynitrite is the most predominant species in the SIN-1 system. Furthermore, ascorbate might alter the system dynamics by removing superoxide and, thereby, increasing the availability of nitric oxide.     

Catalytic scavenging of peroxynitrite by catalase

Peroxynitrite (ONOO⁻/ONOOH), the product of the diffusion controlled reaction between nitric oxide (NO) and superoxide anion (), is a strong oxidizing and nitrating agent. Several heme proteins react rapidly with peroxynitrite, some of them catalyze its decomposition. In this work we found, contrary to previous reports, that catalase, a ferriheme enzyme, catalytically scavenges peroxynitrite. The second-order reaction rate constants of peroxynitrite decay catalyzed by catalase increase with decreasing pH and are equal to (2.7 ± 0.2) × 10⁶, (1.7 ± 0.1) × 10⁶ and (0.8 ± 0.1) × 10⁶ M⁻¹ s⁻¹ at pH 6.1, 7.1 and 8.0, respectively. This dependence suggests that peroxynitrous acid, ONOOH, is the species that reacts with heme center of catalase. The possible reaction mechanisms of the decay of peroxynitrite catalyzed by catalase and physiological relevance of this reaction are discussed.     

Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite

Background

Peroxynitrite, the product of the reaction between superoxide radicals and nitric oxide, is an elusive oxidant with a short half-life and a low steady-state concentration in biological systems; it promotes nitroxidative damage.

Scope of review

We will consider kinetic and mechanistic aspects that allow rationalizing the biological fate of peroxynitrite from data obtained by a combination of methods that include fast kinetic techniques, electron paramagnetic resonance and kinetic simulations. In addition, we provide a quantitative analysis of peroxynitrite production rates and conceivable steady–state levels in living systems.

Major conclusions

The preferential reactions of peroxynitrite in vivo include those with carbon dioxide, thiols and metalloproteins; its homolysis represents only < 1% of its fate. To note, carbon dioxide accounts for a significant fraction of peroxynitrite consumption leading to the formation of strong one-electron oxidants, carbonate radicals and nitrogen dioxide. On the other hand, peroxynitrite is rapidly reduced by peroxiredoxins, which represent efficient thiol-based peroxynitrite detoxification systems. Glutathione, present at mM concentration in cells and frequently considered a direct scavenger of peroxynitrite, does not react sufficiently fast with it in vivo; glutathione mainly inhibits peroxynitrite-dependent processes by reactions with secondary radicals. The detection of protein 3-nitrotyrosine, a molecular footprint, can demonstrate peroxynitrite formation in vivo. Basal peroxynitrite formation rates in cells can be estimated in the order of 0.1 to 0.5 μM s^− 1 and its steady-state concentration at ~ 1 nM.

General significance

The analysis provides a handle to predict the preferential fate and steady-state levels of peroxynitrite in living systems. This is useful to understand pathophysiological aspects and pharmacological prospects connected to peroxynitrite. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Kinetic studies of the reaction of heme-thiolate enzyme chloroperoxidase with peroxynitrite

The kinetics of the reaction of chloroperoxidase with peroxynitrite was studied under neutral and acidic pH by stopped-flow spectrophotometry. Chloroperoxidase catalyzed peroxynitrite decay with the rate constant, k_c, increasing with decreasing pH. The values of k_c obtained at pH 5.1, 6.1 and 7.1 were equal to: (1.96 ± 0.03) × 10⁶, (1.63 ± 0.04) × 10⁶ and (0.71 ± 0.01) × 10⁶ M⁻¹ s⁻¹, respectively. Chloroperoxidase was converted to compound II by peroxynitrite with pH-dependent rate constants: (12.3 ± 0.4) × 10⁶ and (3.8 ± 0.3) × 10⁶ M⁻¹ s⁻¹ at pH 5.1 and 7.1, respectively. After most of peroxynitrite had disappeared, the conversion of compound II into the ferric form of chloroperoxidase was observed. The recovery of the native enzyme was completed within 1 s and 5 s at pH 5.1 and 7.1, respectively. The possible reaction mechanisms of the catalytic decomposition of peroxynitrite by chloroperoxidase are discussed.     

Reactive nitroxidative species and nociceptive processing: determining the roles for nitric oxide, superoxide, and peroxynitrite in pain

Pain is a multidimensional perception and is modified at distinct regions of the neuroaxis. During enhanced pain, neuroplastic changes occur in the spinal and supraspinal nociceptive modulating centers and may result in a hypersensitive state termed central sensitization, which is thought to contribute to chronic pain states. Central sensitization culminates in hyperexcitability of dorsal horn nociceptive neurons resulting in increased nociceptive transmission and pain perception. This state is associated with enhanced nociceptive signaling, spinal glutamate-mediated N-methyl-d-aspartate receptor activation, neuroimmune activation, nitroxidative stress, and supraspinal descending facilitation. The nitroxidative species considered for their role in nociception and central sensitization include nitric oxide (NO), superoxide ( \text O₂^·-{\text {O}_2}^{{\cdot }^{-}}), and peroxynitrite (ONOO⁻). Nitroxidative species are implicated during persistent but not normal nociceptive processing. This review examines the role of nitroxidative species in pain through a discussion of their contributions to central sensitization and the underlying mechanisms. Future directions for nitroxidative pain research are also addressed. As more selective pharmacologic agents are developed to target nitroxidative species, the exact role of nitroxidative species in pain states will be better characterized and should offer promising alternatives to available pain management options.     

The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg) and organic mercury (CH3Hg): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC₅₀) = 250 nM and k_inact = 1.4 × 10⁴ M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 1.4 μM and k_inact = 2 × 10² M⁻¹ s⁻¹ for CH₃Hg⁺). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC₅₀ = 3 and 20 μM for Hg²⁺ and CH₃Hg⁺, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.     

KatG from Salmonella Typhimurium is a peroxynitritase

Pathogenic bacteria elicit protective responses to oxidative and nitrosative stresses. Although such responses are generally distinct, it was recently reported in Mycobacterium tuberculosis that catalase-peroxidase (KatG), a classical defence against peroxides, also exhibits peroxynitritase activity. Here, the katG gene from Salmonella Typhimurium was cloned and protein purified and characterised. An increase in the rate of decomposition of peroxynitrite was observed for KatG from the enterobacterium with a second-order rate constant of 4.2 × 10⁴ M⁻¹ s⁻¹ at pH 7.4, 25 °C. This enzyme was able to reduce dihydrorhodamine oxidation by peroxynitrite to ∼83%. Given the peroxynitritase activity demonstrated here it is likely that KatG may play a wider role in the detoxification of oxidative stresses than previously thought.     

Peroxynitrite formation and function in plants

Peroxynitrite (ONOO⁻) is a reactive nitrogen species formed when nitric oxide (NO) reacts with the superoxide anion (O₂⁻). It was first identified as a mediator of cell death in animals but was later shown to act as a positive regulator of cell signaling, mainly through the posttranslational modification of proteins by tyrosine nitration. In plants, peroxynitrite is not involved in NO-mediated cell death and its physiological function is poorly understood. However, it is emerging as a potential signaling molecule during the induction of defense responses against pathogens and this could be mediated by the selective nitration of tyrosine residues in a small number of proteins. In this review we discuss the general role of tyrosine nitration in plants and evaluate recent evidence suggesting that peroxynitrite is an effector of NO-mediated signaling following pathogen infection.     

Viral mutation accelerated by nitric oxide production during infection in vivo.

Nitric oxide (NO), superoxide (O(2)(-)), and their reaction product peroxynitrite (ONOO(-)) are generated in excess during a host's response against viral infection, and contribute to viral pathogenesis by promoting oxidative stress and tissue injury. Here we demonstrate that NO and peroxynitrite greatly accelerates the mutation of Sendai virus (SeV), a nonsegmented negative-strand RNA virus, by using green fluorescent protein (GFP) inserted into and expressed by a recombinant SeV (GFP-SeV) as an indicator for mutation. GFP-SeV mutation frequencies were much higher in the wild-type mice than in those lacking inducible NO synthase, suggesting that mutation of the virus in vivo is NO dependent. High levels of NO and NO-mediated oxidative stress were induced by GFP-SeV infection in the lung of the wild-type mice, but not in the iNOS-deficient mice, as evidenced by electron spin resonance spectroscopy and immunohistochemical analysis for nitrotyrosine formation as well as histopathological examination. Furthermore, peroxynitrite, an NO-derived reactive nitrogen intermediate, enhanced viral mutation in vitro. These results indicate that the oxidative stress induced by NO produced during the natural course of viral infection increases mutation, expands the quasispecies spectrum, and facilitates evolution of RNA viruses.     

A facile one-pot synthesis of a fluorescent agarose-O-naphthylacetyl adduct with slow release properties

A microwave assisted facile synthesis of a fluorescent 6-O-naphthylacetyl agarose (NA-agarose) employing carbodiimide chemistry (dicyclohexylcarbodiimide/4-dimethylaminopyridine) has been described. NA-agarose was characterized by TGA, GPC, UV spectrophotometry, fluorescence spectroscopy, FT-IR, ¹H and ¹³C NMR spectra, exhibiting that in NA-agarose the naphthylacetyl group was attached to the backbone of the agarose polymer. The hydrolysis of NA-agarose in heterogeneous aqueous phase showed that the 1-naphthyl acetic acid (NAA), a plant growth regulator, got released in a controlled manner, the release rate being dependent on the hydrophilicity of the polymer adduct as well as on pH and temperature. The fluorescence emission (λ_max 332 nm) of NA-agarose (1 × 10⁻³ M) in ethylene glycol was significantly higher (ca. 82%) than that of the molar equivalent of NAA content in the product i.e. 0.08 mg in 1 × 10⁻³ M solution. The resulting polymer would be of potential utility as a sustained release plant growth regulator and sensory applications.     

Rapid determination of nitrite by reversed-phase high-performance liquid chromatography with fluorescence detection

Measurement of nitrite and nitrate, the stable oxidation products of nitric oxide (NO), provides a useful tool to study NO synthesis in vivo and in cell cultures. A simple and rapid fluorometric HPLC method was developed for determination of nitrite through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite, in standard solution, cell culture medium, or biological samples, readily reacted with DAN under acidic conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). For analysis of nitrate, it was converted to nitrite by nitrate reductase, followed by the derivatization of nitrite with DAN to form NAT. NAT was separated on a 5-μm reversed-phase C₈ column (150×4.6 mm, I.D.) guarded by a 40-μm reversed-phase C₁₈ column (50×4.6 mm, I.D.), and eluted with 15 mM sodium phosphate buffer (pH 7.5) containing 50% methanol (flow-rate, 1.3 ml/min). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. Mean retention time for NAT was 4.4 min. The fluorescence intensity of NAT was linear with nitrite or nitrate concentrations ranging from 12.5 to 2000 nM in water, cell culture media, plasma and urine. The detection limit for nitrite and nitrate was 10 pmol/ml. Because NAT is well separated from DAN and other fluorescent components present in biological samples, our HPLC method offers the advantages of high sensitivity and specificity as well as easy automation for quantifying picomole levels of nitrite and nitrate in cell culture medium and biological samples.     

The oxidative and nitrosative chemistry of the nitric oxide/superoxide reaction in the presence of bicarbonate

The primary product of the interaction between nitric oxide (NO) and superoxide () is peroxynitrite (ONOO-), which is capable of either oxidizing or nitrating various biological substrates. However, it has been shown that excess NO or can further react with ONOO- to form species which mediate nitrosation. Subsequently, the controlled equilibrium between nitrosative and oxidative chemistry is critically dependent on the flux of NO and. Since ONOO- reacts not only with NO and but also with CO2, the effects of bicarbonate () on the biphasic oxidation profile of dihydrorhodamine-123 (DHR) and on the nitrosation of both 2,3-diaminonaphthalene and reduced glutathione were examined. Nitric oxide and were formed with DEA/NO [NaEt2NN(O)NO] and xanthine oxidase, respectively. The presence of did not alter either the oxidation profile of DHR with varying radical concentrations or the affinity of DHR for the oxidative species. This suggests that the presence of CO2 does not affect the scavenging of ONOO- by either NO or. However, an increase in the rate of DHR oxidation by ONOO- in the presence of suggests that a CO2-ONOO- adduct does play a role in the interaction of NO or with a product derived from ONOO-. Further examination of the chemistry revealed that the intermediate that reacts with NO is neither ONOO- nor cis-HOONO. It was concluded that NO reacts with both trans-HOONO and a CO2 adduct of ONOO- to form nitrosating species which have similar oxidation chemistry and reactivity with and NO.     

Enhanced 2,3-butanediol production by Klebsiella oxytoca using a two-stage agitation speed control strategy

Batch fermentative production of 2,3-butanediol by Klebsiella oxytoca was investigated using various oxygen supply methods though varying agitation speed. Based on the analysis of three kinetic parameters including specific cell growth rate (μ), specific glucose consumption rate (q_s) and specific 2,3-butanediol formation rate (q_p), a two-stage agitation speed control strategy, aimed at achieving high concentration, high yield and high productivity of 2,3-butanediol, was proposed. At the first 15 h, agitation speed was controlled at 300 rpm to obtain high μ for cell growth, subsequently agitation speed was controlled at 200 rpm to maintain high q_p for high 2,3-butanediol accumulation. Finally, the maximum concentration of 2,3-butanediol reached 95.5 g l⁻¹ with the yield of 0.478 g g⁻¹ and the productivity of 1.71 g l⁻¹ h⁻¹, which were 6.23%, 6.22% and 22.14% over the best results controlled by constant agitation speeds.     

Targeting peroxynitrite driven nitroxidative stress with synzymes: A novel therapeutic approach in chronic pain management

Morphine sulfate and other opiate/narcotic analgesics are the most effective treatments for acute and chronic severe pain. However, their clinical utility is often hampered by the development of analgesic tolerance. This complex pathophysiological cycle contributes significantly to decreased quality of life in the growing population of subjects with chronic pain due to oversedation, reduced physical activity, respiratory depression, constipation, potential for addiction, and other side-effects. Accordingly, there is growing interest in new approaches that would maintain opiate efficacy during repetitive dosing without engendering tolerance or unacceptable side-effects. Considerable evidence implicates nitroxidative stress in the development of pain of several etiologies and importantly in opiate antinociceptive tolerance, caused by the presence of superoxide, O₂^?, (SO) nitric oxide, NO (NO) and more recently peroxynitrite, ONOO^? or its conjugate acid ONOOH, (PN) that is the product of their interaction. To this end, several antioxidant synthetic enzymes (synzymes) have been developed to effectively prevent the formation of PN (superoxide dismutase mimetics, SODms) or to decompose PN once it is formed (PN decomposition catalysts). The objectives of this mini-review written on PN and morphine antinociceptive tolerance are to 1) summarize recent advances made in the development of novel synzymes as therapeutics, 2) discuss the importance of nitroxidative stress in opiate anatinociceptive tolerance and 3) argue that PN is a rational target for therapeutic intervention in pain management. These concepts provide a pharmacological basis for developing inhibitors of PN biosynthesis as novel non-narcotic analgesics, thus addressing a large and currently unmet medical need with major socioeconomic consequences.     

Nitric oxide diffusion to red blood cells limits extracellular,but not intraphagosomal,peroxynitrite formation by macrophages

Macrophage-derived nitric oxide (^•NO) participates in cytotoxic mechanisms against diverse microorganisms and tumor cells. These effects can be mediated by ^•NO itself or ^•NO-derived species such as peroxynitrite formed by its diffusion-controlled reaction with NADPH oxidase-derived superoxide radical anion (O₂^•−). In vivo, the facile extracellular diffusion of ^•NO as well as different competing consumption routes limit its bioavailability for the reaction with O₂^•− and, hence, peroxynitrite formation. In this work, we evaluated the extent by which ^•NO diffusion to red blood cells (RBC) can compete with activated macrophages-derived O₂^•− and affect peroxynitrite formation yields. Macrophage-dependent peroxynitrite production was determined by boron-based probes that react directly with peroxynitrite, namely, coumarin-7-boronic acid (CBA) and fluorescein-boronate (Fl-B). The influence of ^•NO diffusion to RBC on peroxynitrite formation was experimentally analyzed in co-incubations of ^•NO and O₂^•−-forming macrophages with erythrocytes. Additionally, we evaluated the permeation of ^•NO to RBC by measuring the intracellular oxidation of oxyhemoglobin to methemoglobin. Our results indicate that diluted RBC suspensions dose-dependently inhibit peroxynitrite formation, outcompeting the O₂^•− reaction. Computer-assisted kinetic studies evaluating peroxynitrite formation by its precursor radicals in the presence of RBC are in accordance with experimental results. Moreover, the presence of erythrocytes in the proximity of ^•NO and O₂^•--forming macrophages prevented intracellular Fl-B oxidation pre-loaded in L1210 cells co-cultured with activated macrophages. On the other hand, Fl-B-coated latex beads incorporated in the macrophage phagocytic vacuole indicated that intraphagosomal probe oxidation by peroxynitrite was not affected by nearby RBC. Our data support that in the proximity of a blood vessel, ^•NO consumption by RBC will limit the extracellular formation (and subsequent cytotoxic effects) of peroxynitrite by activated macrophages, while the intraphagosomal yield of peroxynitrite will remain unaffected.     

The acute-phase protein serum amyloid A induces endothelial dysfunction that is inhibited by high-density lipoprotein

The acute-phase protein serum amyloid A (SAA) is elevated during inflammation and may be deposited in atheroma where it promotes atherosclerosis. We investigated the proatherogenic effects of SAA on the vascular endothelium and their regulation by high-density lipoprotein (HDL). Exposure of human aortic endothelial cells (HAEC) to SAA (0.25-25 μg/ml) decreased nitric oxide (^•NO) synthesis/bioavailability, although the endothelial NO synthase monomer-to-dimer ratio was unaffected. SAA (10 μg/ml) stimulated a Ca²⁺ influx linked to apocynin-sensitive superoxide radical anion (O₂^•−) production. Gene expression for arginase-1, nuclear factor κB (NF-κB), interleukin-8, and tissue factor (TF) increased within 4 h of SAA stimulation. Enzymatically active Arg-1/2 was detected in HAEC cultured with SAA for 24 h. Therefore, in addition to modulating ^•NO bioavailability by stimulating O₂^•− production in the endothelium, SAA modulated vascular l-Arg bioavailability. SAA also diminished relaxation of preconstricted aortic rings induced by acetylcholine, and added superoxide dismutase restored the vascular response. Preincubation of HAEC with HDL (100 or 200, but not 50, μg/ml) before (not after) SAA treatment ameliorated the Ca²⁺ influx and O₂^•− production; decreased TF, NF-κB, and Arg-1 gene expression; and preserved overall vascular function. Thus, SAA may promote endothelial dysfunction by modulating ^•NO and l-Arg bioavailability, and HDL pretreatment may be protective. The relative HDL to SAA concentrations may regulate the proatherogenic properties of SAA on the vascular endothelium.     

Design of selective substrates of proteinase 3 using combinatorial chemistry methods

In this study, chemical synthesis of the selective chromogenic/fluorogenic substrates for proteinase 3 is described. The substrates’ sequence was obtained using combinatorial chemistry methods. Deconvolution of the tripeptide library against proteinase 3 with general formula ABZ-X₃-X₂-X₁-ANB-NH₂ yielded the active sequence. Selected peptide was further modified on its C terminus to investigate the impact of chromophore moiety modification on enzyme-substrate interaction. To determine specificity, activity of selected substrates was characterized against proteinase 3 and neutrophil elastase. Finally, the peptide ABZ-Tyr-Tyr-Abu-ANB-NH₂ displayed the highest value of specificity constant (k_cat/K_M = 189 × 10³ M⁻¹ s⁻¹) for proteinase 3. To the best of our knowledge, this is the first short peptide that undergoes selective proteolysis by proteinase 3 and displays no significant hydrolysis in the presence of human neutrophil elastase and cathepsin G.     

Roles of reactive oxygen and nitrogen species in pain

Peroxynitrite (PN; ONOO⁻) and its reactive oxygen precursor superoxide (SO; O₂^•−) are critically important in the development of pain of several etiologies including pain associated with chronic use of opiates such as morphine (also known as opiate-induced hyperalgesia and antinociceptive tolerance). This is now an emerging field in which considerable progress has been made in terms of understanding the relative contributions of SO, PN, and nitroxidative stress in pain signaling at the molecular and biochemical levels. Aggressive research in this area is poised to provide the pharmacological basis for development of novel nonnarcotic analgesics that are based upon the unique ability to selectively eliminate SO and/or PN. As we have a better understanding of the roles of SO and PN in pathophysiological settings, targeting PN may be a better therapeutic strategy than targeting SO. This is because, unlike PN, which has no currently known beneficial role, SO may play a significant role in learning and memory [1]. Thus, the best approach may be to spare SO while directly targeting its downstream product, PN. Over the past 15 years, our team has spearheaded research concerning the roles of SO and PN in pain and these results are currently leading to the development of solid therapeutic strategies in this important area.     

Mechanism and biological implications of the NO release of cis-[Ru(bpy)2L(NO)](n+) complexes: a key role of physiological thiols

Nitric oxide (NO) has a critical role in several physiological and pathophysiological processes. In this paper, the reactions of the nitrosyl complexes of [Ru(bpy)₂L(NO)]ⁿ⁺ type, where L = SO₃²⁻ and imidazole and bpy = 2,2′-bipiridine, with cysteine and glutathione were studied. The reactions with cysteine and glutathione occurred through the formation of two sequential intermediates, previously described elsewhere, [Ru(bpy)₂L(NOSR)]ⁿ⁺ and [Ru(bpy)₂L(NOSR)₂] (SR = thiol) leading to the final products [Ru(bpy)₂L(H₂O)]ⁿ⁺ and free NO. The second order rate constant for the second step of this reaction was calculated for cysteine k₂(SR⁻) = (2.20 ± 0.12) × 10⁹ M^− 1 s^− 1 and k_2(RSH) = (154 ± 2) M^− 1 s^− 1 for L = SO₃²⁻ and k₂(SR⁻) = (1.30 ± 0.23) × 10⁹ M^− 1 s^− 1 and k₂(RSH) = (0.84 ± 0.02) M^− 1 s^− 1 for L = imidazole; while for glutathione they were k₂(SR⁻) = (6.70 ± 0.32) × 10⁸ M^− 1 s^− 1 and k₂(RSH) = 11.8 ± 0.3 M^− 1 s^− 1 for L = SO₃²⁻ and k₂(SR⁻) = (2.50 ± 0.36) × 10⁸ M^− 1 s^− 1 and k₂(RSH) = 0.32 ± 0.01 M^− 1 s^− 1 for L = imidazole. In all reactions it was possible to detect the release of NO from the complexes, which it is remarkably distinct from other ruthenium metallocompounds described elsewhere with just N₂O production. These results shine light on the possible key role of NO release mediated by physiological thiols in reaction with these metallonitrosyl ruthenium complexes.     

Hydrolysis of the soluble fluorescent molecule carboxyumbelliferyl-beta-D-glucuronide by E. coli beta-glucuronidase as applied in a rugged, in situ optical sensor

Techniques utilizing β-glucuronidase (GUS) activity as an indicator of Escherichia coli (E. coli) presence use labeled glucuronides to produce optical signals. Carboxyumbelliferyl-β-d-glucuronide (CUGlcU) is a fluorescent labeled glucuronide that is soluble and highly fluorescent at natural water pHs and temperatures and, therefore, may be an ideal reagent for use in an in situ optical sensor. This paper reports for the first time the Michaelis-Menten kinetic parameters for the binding of E. coli GUS with CUGlcU as K_m = 910 μM, V_max = 41.0 μM min⁻¹, V_max/K_m 45.0 μmol L⁻¹ min⁻¹, the optimal pH as 6.5 ± 1.0, optimal temperature as 38 °C, and the Gibb's free energy of activation as 61.40 kJ mol⁻¹. Additionally, it was found CUGlcU hydrolysis is not significantly affected by heavy solvents suggesting proton transfer and solvent addition that occur during hydrolysis are not limiting steps. Comparison studies were made with the more common fluorescent molecule methylumbelliferyl-β-d-glucuronide (MUGlcU). Experiments showed GUS preferentially binds to MUGlcU in comparison to CUGlcU. CUGlcU was also demonstrated in a prototype optical sensor for the detection of E. coli. Initial bench testing of the sensor produced detection of low concentrations of E. coli (1.00 × 10³ CFU/100 mL) in 230 ± 15.1 min and high concentrations (1.05 × 10⁵ CFU/100 mL) in 8.00 ± 1.01 min.