S-nitrosothiols react preferentially with zinc thiolate clusters of metallothionein III through transnitrosation

Metallothionein (MT) is a two-domain protein with zinc thiolate clusters that bind and release zinc depending on the redox states of the sulfur ligands. Since S-nitrosylation of cysteine is considered a prototypic cellular redox signaling mechanism, we here investigate the reactions of S-nitrosothiols with different isoforms of MT. MT-III is significantly more reactive than MT-I/II toward S-nitrosothiols, whereas the reactivity of all three isoforms toward reactive oxygen species is comparable. A cellular system, in which all three MTs are similarly effective in protecting rat embryonic cortical neurons in primary culture against hydrogen peroxide but where MT-III has a much more pronounced effect of protecting against S-nitrosothiols, confirms this finding. MT-III is the only isoform with consensus acid-base sequence motifs for S-nitrosylation in both domains. Studies with synthetic and zinc-reconstituted domain peptides demonstrate that S-nitrosothiols indeed release zinc from both the alpha- and the beta-domain of MT-III. S-Nitrosylation occurs via transnitrosation, a mechanism that differs fundamentally from that of previous studies of reactions of MT with NO*. Our data demonstrate that zinc thiolate bonds are targets of S-nitrosothiol signaling and further indicate that MT-III is biologically specific in converting NO signals to zinc signals. This could bear importantly on the physiological action of MT-III, whose biological activity as a neuronal growth inhibitory factor is unique, and for brain diseases that have been related to oxidative or nitrosative stress.     

N-dansyl-S-nitrosohomocysteine a fluorescent probe for intracellular thiols and S-nitrosothiols

The fluorescence emission spectrum of N-dansyl-S-nitrosohomocysteine was enhanced approximately 8-fold upon removal of the NO group either by photolysis or by transnitrosation with free thiols like glutathione. The fluorescence enhancement was reversible in that it could be quenched in the presence of excess S-nitrosoglutathione. Attempts were then made to utilize N-dansyl-S-nitrosohomocysteine as an intracellular probe of thiols/S-nitrosothiols. Fluorescence microscopy of fibroblasts in culture indicated that intracellular N-dansyl-S-nitrosohomocysteine levels reached a maximum within 5 min. N-Dansyl-S-nitrosohomocysteine fluorescence was directly proportional to intracellular GSH levels, directly determined with HPLC. N-Dansyl-S-nitrosohomocysteine preloaded cells were also sensitive to S-nitrosoglutathione uptake as the intracellular fluorescence decreased as a function of time upon exposure to extracellular S-nitrosoglutathione.     

Rocket fuel for the quantification of S-nitrosothiols. Highly specific reduction of S-nitrosothiols to thiols by methylhydrazine

Abstract

Reduction of S-nitrosothiols to the corresponding thiol function is the key step in analyzing S-nitrosocysteinyl residues in proteins. Though it has been shown to give low yields, ascorbate-dependent reduction is commonly performed in the frequently used biotin-switch technique. We demonstrate that the compound methylhydrazine can act as a specific and efficient reducing agent for S-nitrosothiols. The corresponding thiol function is exclusively generated from low molecular weight and proteinaceous S-nitrosothiols while methylhydrazine failed to reduce disulfides. It was possible to optimize the experimental conditions so that thiol autoxidation is excluded, and high reaction yields (> 90%) are obtained for the thiol function. The biotin-switch technique performed with methylhydrazine-dependent reduction shows remarkably improved sensitivity compared to the ascorbate-dependent procedure.     

Formation of S-nitrosothiols from regiospecific reaction of thiols with N-nitrosotryptophan derivatives

S-nitrosothiols transport nitric oxide in vivo, and so-called transnitrosation reactions (i.e. the transfer of the nitroso function from nitrosothiol to thiolate) are believed to be involved in this process. In the present study we examined the N-nitrosotryptophan derivative-dependent nitrosation of thiols, a hitherto ignored possibility for the formation of S-nitrosothiols. The corresponding products were identified by (15)N-NMR spectrometry. The fact that the reaction proceeded under hypoxic conditions as well as in non-aqueous solution strongly indicated the occurrence of a transnitrosation reaction. Interestingly, S-nitrosothiols could only very slowly transnitrosate N-terminal-blocked tryptophan derivatives like melatonin in non-aqueous solution but did not induce such a reaction in water. The indole moiety of the N-nitrosotryptophan derivatives was fully restituted during the reaction with thiols, as demonstrated by both capillary zone electrophoresis and fluorescence spectroscopy. A determination of the Arrhenius parameters demonstrated that the corresponding rate constants were comparable with the ones known for the transfer of the nitroso function from nitrosothiol to thiolate. Thus, N-nitrosotryptophan-dependent nitrosation of thiols may occur in vivo and might offer the possibility of developing a new class of vasodilative drugs.     

Metalloprotein-dependent decomposition of S-nitrosothiols: studies on the stabilization and measurement of S-nitrosothiols in tissues

The stabilization of S-nitrosothiols is critical for the development of assays to measure their concentration in tissues. Low-molecular-weight S-nitrosothiols are unstable in tissue homogenates, even in the presence of thiol blockers or metal-ion chelators. The aim of this study was to try and stabilize low-molecular-weight S-nitrosothiols in tissue and gain insight into the mechanisms leading to their decomposition. Rat tissues (liver, kidney, heart, and brain) were perfused and homogenized in the presence of a thiol-blocking agent (N-ethylmaleimide) and a metal-ion chelator (DTPA). Incubation of liver homogenate with low-molecular-weight S-nitrosothiols (L-CysNO, D-CysNO, and GSNO) resulted in their rapid decomposition in a temperature-dependent manner as measured by chemiluminescence. The decomposition of L-CysNO requires a cytoplasmic factor, with activity greatest in liver > kidney > heart > brain > plasma, and is inhibitable by enzymatic proteolysis or heating to 80 degrees C, suggesting that a protein catalyzes the decomposition of S-nitrosothiols. The ability of liver homogenate to catalyze the decomposition of L-CysNO is up-regulated during endotoxemia and is dependent on oxygen, with the major product being nitrate. Multiple agents were tested for their ability to block the decomposition of L-CysNO without success, with the exception of potassium ferricyanide, which completely blocked CysNO decomposition in liver homogenates. This suggests that a ferrous protein (or group of ferrous proteins) may be involved. We also show that homogenization of tissues in ferricyanide-containing buffers in the presence of N-ethylmaleimide and DTPA can stabilize both low- and high-molecular-weight S-nitrosothiols in tissues before the measurement of their concentration.     

Equilibrium and kinetics studies of heavy metal ions biosorption on green algae waste biomass

The biosorption of Pb(II), Cd(II), and Co(II), respectively, from aqueous solution on green algae waste biomass was investigated. The green algae waste biomass was obtained from marine green algae after extraction of oil, and was used as low-cost biosorbent. Batch shaking experiments were performed to examine the effects of initial solution pH, contact time and temperature. The equilibrium biosorption data were analyzed using two isotherm models (Langmuir and Freundlich) and two kinetics models (pseudo-first order and pseudo-second order). The results indicate that Langmuir model provide best correlation of experimental data, and the pseudo-second order kinetic equation could best describe the biosorption kinetics of considered heavy metals.     

Equilibrium partition studies of the interaction between aldolase and myofibrils

The adsorption of aldolase to myofibrils derived from rabbit skeletal muscle has been investigated by partition equilibrium studies at pH 6.8, I = 0.158 M, and the results interpreted in terms of an intrinsic association constant of 410,000 m^?1 for the interaction of four sites on aldolase with myofibrillar sites, there being one such site for every 10–12 heptameric repeat units of F-actin-tropomyosin-troponin thin filament. Involvement of the active site of the enzyme in the adsorption process is indicated by the fact that competitive inhibition of the phenomenon by phosphate may be accounted for by an intrinsic association constant of 400 m^?1 for the aldolase-phosphate interaction, a value in good agreement with that describing phosphate inhibition of the enzymatic hydrolysis of fructose-1,6-bisphosphate under similar conditions. On the basis of these equilibrium constants plus the aldolase and thin filament contents of muscle, resting muscle is indicated as containing a significant proportion (25–30%) of aldolase in the bound form, with changes in the subcellular distribution of the enzyme being likely during exercise due to the increased concentrations of Ca²⁺ and fructose-1,6-bisphosphate that then prevail.     

Equilibrium and kinetics studies of adsorption of copper (II) on chitosan and chitosan/PVA beads

The adsorption of Cu(II) ions from aqueous solution by chitosan and chitosan/PVA beads was studied in a batch adsorption system. Chitosan solution was blended with poly(vinyl alcohol) (PVA) in order to obtain sorbents that are insoluble in aqueous acidic and basic solution. The adsorption capacities and rates of Cu(II) ions onto chitosan and chitosan/PVA beads were evaluated. The Langmuir, Freundlich and BET adsorption models were applied to describe the isotherms and isotherm constants. Adsorption isothermal data could be well interpreted by the Langmuir model. The kinetic experimental data properly correlated with the second-order kinetic model, which indicates that the chemical sorption is the rate-limiting step. The Cu(II) ions can be removed from the chitosan and chitosan/PVA beads rapidly by treatment with an aqueous EDTA solution. Results also showed that chitosan and chitosan/PVA beads are favourable adsorbers.     

Detection of S-nitrosothiols

Background

S-nitrosothiols have been recognized as biologically-relevant products of nitric oxide that are involved in many of the diverse activities of this free radical.

Scope of review

This review serves to discuss current methods for the detection and analysis of protein S-nitrosothiols. The major methods of S-nitrosothiol detection include chemiluminescence-based methods and switch-based methods, each of which comes in various flavors with advantages and caveats.

Major conclusions

The detection of S-nitrosothiols is challenging and prone to many artifacts. Accurate measurements require an understanding of the underlying chemistry of the methods involved and the use of appropriate controls.

General significance

Nothing is more important to a field of research than robust methodology that is generally trusted. The field of S-nitrosation has developed such methods but, as S-nitrosothiols are easy to introduce as artifacts, it is vital that current users learn from the lessons of the past. 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.     

Equilibrium amide hydrogen exchange and protein folding kinetics

The classical Linderstrøm-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (IUN). On the other hand, in an on-pathway three-state system (UIN), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments.     

Complex-formation between cytochrome c and cytochrome c peroxidase. Equilibrium and titration studies

1. Physical studies of complex-formation between cytochrome c and yeast peroxidase are consistent with kinetic predictions that these complexes participate in the catalytic activity of yeast peroxidase towards ferrocytochrome c. Enzyme-ferricytochrome c complexes have been detected both by the analytical ultracentrifuge and by column chromatography, whereas an enzyme-ferrocytochrome c complex was demonstrated by column chromatography. Estimated binding constants obtained from chromatographic experiments were similar to the measured kinetic values. 2. The physicochemical study of the enzyme-ferricytochrome c complex, and an analysis of its spectrum and reactivity, suggest that the conformation and reactivity of neither cytochrome c nor yeast peroxidase are grossly modified in the complex. 3. The peroxide compound of yeast cytochrome c peroxidase was found to have two oxidizing equivalents accessible to cytochrome c but only one readily accessible to ferrocyanide. Several types of peroxide compound, differing in available oxidizing equivalents and in reactivity with cytochrome c, seem to be formed by stoicheiometric amounts of hydrogen peroxide. 4. Fluoride combines not only with free yeast peroxidase but also with peroxidase-peroxide and accelerates the decomposition of the latter compound. The ligand-catalysed decomposition provides evidence for one-electron reduction pathways in yeast peroxidase, and the reversible binding of fluoride casts doubt upon the concept that the peroxidase-peroxide intermediate is any form of peroxide complex. 5. A mechanism for cytochrome c oxidation is proposed involving the successive reaction of two reversibly bound molecules of cytochrome c with oxidizing equivalents associated with the enzyme protein.     

Coenzyme binding by triphosphopyridine nucleotide dependent isocitrate dehydrogenase from beef liver. Equilibrium and kinetics studies.

The binding of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP) dependent isocitrate dehydrogenase from beef liver cytoplasm was studied by several equilibrium techniques (ultracentrifugation, molecular sieving, ultrafiltration, fluorescence). Two binding sites (per dimeric enzyme molecule) were found with slightly different dissociation constants (0.5 and 0.12 muM) and fluorescence yields (7.7 and 6.3). A ternary complex was formed between enzyme, isocitrate, and NADPH, in which NADPH dissociation constant was 5 muM. On the contrary, no binding of NADPH to the enzyme took place in the presence of magnesium isocitrate. Dialysis experiments showed the existence of 1 NADP binding site/dimer, with a dissociation constant of 26 muM. When NADPH was present with the enzyme in the proportion of 1 molecule/dimer, the dissociation constant of NADP was decreased fourfold, reaching a value quantitatively comparable to the Michaelis constant. The kinetics of coenzyme binding was followed using the stopped-flow technique with fluorescence detection. NADPH binding to the enzyme occurred through one fast reaction (k1 = 20 muM-1 s-1). Dissociation of NADPH took place upon NADP binding; however, equilibrium as well as kinetic data were incompatible with a simple competition scheme. Dissociation of NADPH from the enzyme upon magnesium isocitrate binding was preceded by the formation of a transitory ternary complex in which the fluorescence of NADPH was only about 30% of that in the enzyme-NADPH complex. Then interaction between the conenzymes and the involvement of ternary complexes in the catalytic mechanism are discussed in relation with what is known about the regulatory role of the coenzyme (Carlier, M. F., and Pantaloni, D. (1976), Biochemistry, 15, 1761-1766).     

One- and two-electron oxidation of thiols: mechanisms,kinetics and biological fates

The oxidation of biothiols participates not only in the defense against oxidative damage but also in enzymatic catalytic mechanisms and signal transduction processes. Thiols are versatile reductants that react with oxidizing species by one- and two-electron mechanisms, leading to thiyl radicals and sulfenic acids, respectively. These intermediates, depending on the conditions, participate in further reactions that converge on different stable products. Through this review, we will describe the biologically relevant species that are able to perform these oxidations and we will analyze the mechanisms and kinetics of the one- and two-electron reactions. The processes undergone by typical low-molecular-weight thiols as well as the particularities of specific thiol proteins will be described, including the molecular determinants proposed to account for the extraordinary reactivities of peroxidatic thiols. Finally, the main fates of the thiyl radical and sulfenic acid intermediates will be summarized.     

Double edge redox-implications for the interaction between endogenous thiols and copper ions: In vitro studies

The present study investigated the redox-consequences of the interaction between various endogenous thiols (RSH)-glutathione, cysteine, homocysteine, gamma-glutamyl-cysteine, and cysteinyl-glycine- and Cu(2+) ions, in terms of their free radical-scavenging, ascorbate-oxidizing and O2(*-)-generating properties of the resulting mixtures. Upon a brief incubation (3-30 min) with Cu(2+), the free radical-scavenging properties (towards ABTS(*)(+) and DPPH(*)) and thiol-titratable groups of the RSH added to the mixtures decreased significantly. Remarkably, both effects were only partial, even in the presence of a large molar Cu(2+)-excess, and were unaffected despite increasing the incubation time. At equimolar concentrations, the RSH/Cu(2+) mixtures led to the formation of (EPR paramagnetic) Cu(II)-complexes that were time-stable and ascorbate-reducible, but redox-inactive towards oxygen. In turn, at a slight molar thiol-excess (3:1), the mixtures resulted in the formation of time-stable Cu(I)-complexes (EPR silent) that were unreactive towards ascorbate and oxygen. The only exception was seen for the thiol, glutathione, whose mixture with Cu(2+) mixture displayed a O2(*-)-generating capacity (cytochrome c- and lucigenin-reduction). The data indicate that, depending on their molar ratio, the interaction between Cu(2+) and the tested thiols would give place to mixtures containing either: (i) time-stable and ascorbate-reducible Cu(II)-complexes which display free radical-scavenging properties, or (ii) time-stable but redox-inactive towards oxygen Cu(I)-complexes. Among the latter, the only exception was that of glutathione.     

Competition between glutathione and protein thiols for disulphide-bond formation

It has long been assumed that the oxidized form of glutathione, the tripeptide glutamate-cysteine-glycine, is a source of oxidizing equivalents needed for the formation of disulphide bonds in proteins within the endoplasmic reticulum (ER), although the in vivo function of glutathione in the ER has never been studied directly. Here we show that the major pathway for oxidation in the yeast ER, defined by the protein Ero1, is responsible for the oxidation of both glutathione and protein thiols. However, mutation and overexpression studies show that glutathione competes with protein thiols for the oxidizing machinery. Thus, contrary to expectation, cellular glutathione contributes net reducing equivalents to the ER; these reducing equivalents can buffer the ER against transient hyperoxidizing conditions.     

The reaction between thiols and 8-azidoadenosine derivatives.

Thiols react at room temperature in dilute solution with 8-azidoadenosine and its nucleotides to give the corresponding 8-aminoadenosine derivatives. The reaction which takes place in the dark is base-catalysed and is particularly rapid when dithiols, e.g. dithiothreitol are used.