Mechanism of S-nitrosation of recombinant human brain calbindin D28K

Mass spectrometry and UV-vis absorption results support a mechanism for NO donation by S-nitrosoglutathione (GSNO) to recombinant human brain calbindin D(28K) (rHCaBP) that requires the presence of trace copper, added as either Cu,Zn-superoxide dismutase (CuZnSOD) or CuSO(4). The extent of copper-catalyzed rHCaBP S-nitrosation depends on the ratio of protein to GSNO and on the reaction time, and NO-transfer is prevented when copper chelators are present. CuZnSOD is an efficient catalyst of rHCaBP S-nitrosation, and the mechanism of CuZnSOD-catalyzed S-nitrosation involves reduction of the active-site Cu(II) by a number of the five free thiols in rHCaBP, giving rise to thiyl radicals. The Cu(I)ZnSOD formed catalyzes the reductive cleavage of GSNO present in solution to give GSH and release NO. rHCaBP thiyl radicals react with NO to yield the S-nitrosoprotein. Cu(II)ZnSOD is also reduced by GSH in a concentration-dependent manner up to 5 mM but not at higher GSH concentrations. However, unlike the rHCaBP thiyl radicals, GS(*) radicals dimerize to GSSG faster than their reaction with NO. The data presented here provide a biologically relevant mechanism for protein S-nitrosation by small S-nitrosothiols. S-nitrosation is rapidly gaining recognition as a major form of protein posttranslational modification, and the efficient S-nitrosation of CaBP by CuZnSOD/GSNO is speculated to be of neurochemical importance given that CaBP and CuZnSOD are abundant in neurons.     

Putative denitrosylase activity of Cu,Zn-superoxide dismutase

The Cu,Zn-superoxide dismutase (SOD1) has been reported to exert an S-nitrosylated glutathione (GSNO) denitrosylase activity that was augmented by a familial amyotrophic lateral sclerosis (FALS)-associated mutation in this enzyme. This putative enzymatic activity as well as the spontaneous decomposition of GSNO has been reexamined. The spontaneous decomposition of GSNO exhibited several peculiarities, such as a lag phase followed by an accelerating rate plus a marked dependence on GSNO concentration, suggestive of autocatalysis, and a greater rate in polypropylene than in glass vessels. Dimedone caused a rapid increase in absorbance likely due to reaction with GSNO, followed by a slower increase possibly due to reaction with an intermediate such as glutathione sulfenic acid. SOD1 weakly increased the rate of decomposition of GSNO, but did so only when GSH was present; and FALS-associated mutant forms of SOD1 were not more active in this regard than was the wild type. Decomposed GSNO, when added to fresh GSNO, hastened its decomposition, in accord with autocatalysis, and when added to GSH, generated GSNO in accord with the presence of nitrite. A mechanism is proposed that is in accord with these observations.     

Heme nitrosylation of deoxyhemoglobin by s-nitrosoglutathione requires copper

NO reactions with hemoglobin (Hb) likely play a role in blood pressure regulation. For example, NO exchange between Hb and S-nitrosoglutathione (GSNO) has been reported in vitro. Here we examine the reaction between GSNO and deoxyHb (HbFe(II)) in the presence of both Cu(I) (2,9-dimethyl-1, 10-phenanthroline (neocuproine)) and Cu(II) (diethylenetriamine-N,N,N',N",N"-pentaacetic acid) chelators using a copper-depleted Hb solution. Spectroscopic analysis of deoxyHb (HbFe(II))/GSNO incubates shows prompt formation (<5 min) of approximately 100% heme-nitrosylated Hb (HbFe(II)NO) in the absence of chelators, 46% in the presence of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, and 25% in the presence of neocuproine. Negligible (<2%) HbFe(II)NO was detected when neocuproine was added to copper-depleted HbFe(II)/GSNO incubates. Thus, HbFe(II)NO formation via a mechanism involving free NO generated by Cu(I) catalysis of GSNO breakdown is proposed. GSH is a source of reducing equivalents because extensive GSSG was detected in HbFe(II)/GSNO incubates in the absence of metal chelators. No S-nitrosation of HbFe(II) was detected under any conditions. In contrast, the NO released from GSNO is directed to Cysbeta(93) of oxyHb in the absence of chelators, but only metHb formation is observed in the presence of chelators. Our findings reveal that the reactions of GSNO and Hb are controlled by copper and that metal chelators do not fully inhibit NO release from GSNO in Hb-containing solutions.     

Oxidation and nitrosation of thiols at low micromolar exposure to nitric oxide. Evidence for a free radical mechanism

Although the nitric oxide (.NO)-mediated nitrosation of thiol-containing molecules is increasingly recognized as an important post-translational modification in cell signaling and pathology, little is known about the factors that govern this process in vivo. In the present study, we examined the chemical pathways of nitrosothiol (RSNO) production at low micromolar concentrations of .NO. Our results indicate that, in addition to nitrosation by the .NO derivative dinitrogen trioxide (N2O3), RSNOs may be formed via intermediate one-electron oxidation of thiols, possibly mediated by nitrogen dioxide (.NO2), and the subsequent reaction of thiyl radicals with .NO. In vitro, the formation of S-nitrosoglutathione (GSNO) from .NO and excess glutathione (GSH) was accompanied by the formation of glutathione disulfide, which could not be ascribed to the secondary reaction of GSH with GSNO. Superoxide dismutase significantly increased GSNO yields and the thiyl radical trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), inhibited by 45 and 98% the formation of GSNO and GSSG, respectively. Maximum nitrosation yields were obtained at an oxygen concentration of 3%, whereas higher oxygen tensions decreased GSNO and increased GSSG formation. When murine fibroblasts were exposed to exogenous .NO, RSNO formation was sensitive to DMPO and oxygen tension in a manner similar to that observed with GSH alone. Our data indicate that RSNO formation is favored at oxygen concentrations that typically occur in tissues. Nitrosothiol formation in vivo depends not only on the availability of .NO and O2 but also on the degree of oxidative stress by affecting the steady-state concentration of thiyl radicals.     

The redox pathway of S-nitrosoglutathione, glutathione and nitric oxide in cell to neuron communications

Recent results demonstrated that S-nitrosoglutathione (GSNO) and nitric oxide (*NO) protect brain dopamine neurons from hydroxyl radical (*OH)-induced oxidative stress in vivo because they are potent antioxidants. GSNO and *NO terminate oxidant stress in the brain by (i) inhibiting iron-stimulated hydroxyl radicals formation or the Fenton reaction, (ii) terminating lipid peroxidation, (iii) augmenting the antioxidative potency of glutathione (GSH), (iv) mediating neuroprotective action of brain-derived neurotrophin (BDNF), and (v) inhibiting cysteinyl proteases. In fact, GSNO--S-nitrosylated GSH--is approximately 100 times more potent than the classical antioxidant GSH. In addition, S-nitrosylation of cysteine residues by GSNO inactivates caspase-3 and HIV-1 protease, and prevents apoptosis and neurotoxicity. GSNO-induced antiplatelet aggregation is also mediated by S-nitrosylation of clotting factor XIII. Thus the elucidation of chemical reactions involved in this GSNO pathway (GSH GS* + *NO-->[GSNO]-->GSSG + *NO-->GSH) is necessary for understanding the biology of *NO, especially its beneficial antioxidative and neuroprotective effects in the CNS. GSNO is most likely generated in the endothelial and astroglial cells during oxidative stress because these cells contain mM GSH and nitric oxide synthase. Furthermore, the transfer of GSH and *NO to neurons via this GSNO pathway may facilitate cell to neuron communications, including not only the activation of guanylyl cyclase, but also the nitrosylation of iron complexes, iron containing enzymes, and cysteinyl proteases. GSNO annihilates free radicals and promotes neuroprotection via its c-GMP-independent nitrosylation actions. This putative pathway of GSNO/GSH/*NO may provide new molecular insights for the redox cycling of GSH and GSSG in the CNS.     

Human S-nitroso oxymyoglobin is a store of vasoactive nitric oxide

Nitric oxide (.NO) regulates vascular function, and myoglobin (Mb) is a heme protein present in skeletal, cardiac, and smooth muscle, where it facilitates O(2) transfer. Human ferric Mb binds .NO to yield nitrosylheme and S-nitroso (S-NO) Mb (Witting, P. K., Douglas, D. J., and Mauk, A. G. (2001) J. Biol. Chem. 276, 3991-3998). Here we show that human ferrous oxy-myoglobin (oxyMb) oxidizes .NO, with a second order rate constant k = 2.8 +/- 0.1 x 10(7) M(-1).s(-1) as determined by stopped-flow spectroscopy. Mixtures containing oxyMb and S-nitrosoglutathione or S-nitrosocysteine added at 1.5-2 moles of S-nitrosothiol/mol oxyMb yielded S-NO oxyMb through trans-nitrosation equilibria as confirmed with mass spectrometry. Rate constants for the equilibrium reactions were k(forward) = 110 +/- 3 and k(reverse) = 16 +/- 3 M(-1).s(-1) for S-nitrosoglutathione and k(forward) = 293 +/- 5 and k(reverse) = 20 +/- 2 M(-1).s(-1) for S-nitrosocysteine. Incubation of S-NO oxyMb with Cu(2+) ions stimulated .NO release as measured with a .NO electrode. Similarly, Cu(2+) released .NO from Mb immunoprecipitated from cultured human vascular smooth muscle cells (VSMCs) that were pre-treated with diethylaminenonoate. No .NO release was observed from VSMCs treated with vehicle alone or immunoprecipitates obtained from porcine aortic endothelial cells with and without diethylaminenonoate treatment. Importantly, pre-constricted aortic rings relaxed in the presence of S-NO oxyMb in a cyclic GMP-dependent process. These data indicate that human oxyMb rapidly oxidizes .NO and that biologically relevant S-nitrosothiols can trans-(S)nitrosate human oxyMb. Furthermore, S-NO oxyMb can be isolated from cultured human VSMCs exposed to an exogenous .NO donor at physiologic concentration. The potential biologic implications of S-NO oxyMb acting as a source of .NO are discussed.     

Glutathione and other low-molecular-weight thiols relax guinea pig trachea ex vivo: interactions with nitric oxide?

The aim of this study was to determine the effects of glutathione (GSH) on trachea smooth muscle tension in view of previously reported interactions between GSH and nitric oxide (NO) (Gaston B. Biochim Biophys Acta 1411: 323-333, 1999; Kelm M. Biochim Biophys Acta 1411: 273-289, 1999; and Kharitonov VG, Sundquist AR, and Sharma VS. J Biol Chem 270: 28158-28164, 1995) and the high (millimolar) concentrations of GSH in trachea epithelium (Rahman I, Li XY, Donaldson K, Harrison DJ, and MacNee W. Am J Physiol Lung Cell Mol Physiol 269: L285-L292, 1995). GSH and other thiols (1.0-10 mM) dose dependently decreased the tension in isolated guinea pig tracheas. Relaxations by GSH were paralleled with sevenfold increased nitrite levels (P < 0.05) in the tracheal effluent, suggesting an interaction between GSH and NO. However, preincubation with a NO scavenger did not reduce the relaxations by GSH or its NO adduct, S-nitrosoglutathione (GSNO). Inhibition of guanylyl cyclase inhibited the relaxations induced by GSNO, but not by GSH. Blocking potassium channels, however, completely abolished the relaxing effects of GSH (P < 0.05). Preincubation of tracheas with GSH significantly (P < 0.05) suppressed hyperreactivity to histamine as caused by removal of tracheal epithelium. These data indicate that GSH plays a role in maintaining tracheal tone. The mechanism is probably an antioxidative action of GSH itself rather than an action of NO or GSNO.     

Production of Hydroxyl Free Radical in the Xanthine Oxidase System with Addition of 1-methyl-3-nitro-1-nitrosoguanidine

Recent results demonstrated that S-nitrosoglutathione (GSNO) and nitric oxide (^·NO) protect brain dopamine neurons from hydroxyl radical (^·OH)-induced oxidative stress in vivo because they are potent antioxidants. GSNO and ^·NO terminate oxidant stress in the brain by (i) inhibiting iron-stimulated hydroxyl radicals formation or the Fenton reaction, (ii) terminating lipid peroxidation, (iii) augmenting the antioxidative potency of glutathione (GSH), (iv) mediating neuroprotective action of brain-derived neurotrophin (BDNF), and (v) inhibiting cysteinyl proteases. In fact, GSNO — S-nitrosylated GSH — is approximately 100 times more potent than the classical antioxidant GSH. In addition, S-nitrosylation of cysteine residues by GSNO inactivates caspase-3 and HIV-1 protease, and prevents apoptosis and neurotoxicity. GSNO-induced antiplatelet aggregation is also mediated by S-nitrosylation of clotting factor XIII. Thus the elucidation of chemical reactions involved in this GSNO pathway (GSH → GS^· + ^·NO → [GSNO] → GSSG + ^·NO → GSH) is necessary for understanding the biology of ^·NO, especially its beneficial antioxidative and neuroprotective effects in the CNS. GSNO is most likely generated in the endothelial and astroglial cells during oxidative stress because these cells contain mM GSH and nitric oxide synthase. Furthermore, the transfer of GSH and ^·NO to neurons via this GSNO pathway may facilitate cell to neuron communications, including not only the activation of guanylyl cyclase, but also the nitrosylation of iron complexes, iron containing enzymes, and cysteinyl proteases. GSNO annihilates free radicals and promotes neuroprotection via its c-GMP-independent nitrosylation actions. This putative pathway of GSNO/GSH/^·NO may provide new molecular insights for the redox cycling of GSH and GSSG in the CNS.     

Comparison of nitric oxide-induced oxidation of recombinant oxyhemoglobin subunits using a competition experiment

A low reaction rate with nitric oxide (NO) is one of the important characteristics of hemoglobin (Hb)-based oxygen carriers. The reaction rate between oxyHb and NO is usually measured by stopped-flow spectrophotometry. However, the reported rates vary due to the difficulty of accurately determining the NO concentration and the limit of the instrument dead time. To circumvent these problems, we developed an experiment using oxymyoglobin (oxyMb) to compete with oxyHb for NO that is released from an NO donor. Determination of the rate constants in the competition experiment no longer depends on accurate measurement of time or NO concentration, since this approach instead measures the ratio of rate constants for the reaction of oxyHb and oxyMb with NO. For recombinant mutant Hb α(L29F)β the rates for α(L29F) and β are approximately 15- and 1.6-fold smaller than for wild-type Hb. In conclusion, the competition experiment provides an alternative method for determination of relative reaction rates of recombinant Hb subunits with NO.     

S-nitrosation of glutathione by nitric oxide,peroxynitrite, and (*)NO/O(2)(*-)

To elucidate potential mechanisms of S-nitrosothiol formation in vivo, we studied nitrosation of GSH and albumin by nitric oxide ((*)NO), peroxynitrite, and (*)NO/O(2)(*)(-). In the presence of O(2), (*)NO yielded 20% of S-nitrosoglutathione (GSNO) at pH 7.5. Ascorbate and the spin trap 4-hydroxy-[2,2,4,4-tetramethyl-piperidine-1-oxyl] (TEMPOL) inhibited GSNO formation by 67%. Electron paramagnetic resonance spectroscopy with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) demonstrated intermediate formation of glutathionyl radicals, suggesting that GSNO formation by (*)NO/O(2) is predominantly mediated by (*)NO(2). Peroxynitrite-triggered GSNO formation (0.06% yield) was stimulated 10- and 2-fold by ascorbate and TEMPOL, respectively. Co-generation of (*)NO and O(2)(*)(-) at equal fluxes yielded less GSNO than (*)NO alone, but was 100-fold more efficient (8% yield) than peroxynitrite. Moreover, in contrast to the reaction of peroxynitrite, GSNO formation by (*)NO/O(2)(*)(-) was inhibited by ascorbate. Similar results were obtained with albumin instead of GSH. We propose that sulfhydryl compounds react with O(2)(*)(-) to initiate a chain reaction that forms radical intermediates which combine with (*)NO to yield GSNO. In RAW 264.7 macrophages, S-nitrosothiol formation by (*)NO/O(2) and (*)NO/O(2)(*)(-) occurred with relative efficiencies comparable to those in solution. Our results indicate that concerted generation of (*)NO and O(2)(*)(-) may essentially contribute to nitrosative stress in inflammatory diseases.     

Reactions of nitrite with hemoglobin measured by membrane inlet mass spectrometry

Membrane inlet mass spectrometry was used to observe nitric oxide in the well-studied reaction of nitrite with hemoglobin. The membrane inlet was submerged in the reaction solutions and measured NO in solution via its flux across a semipermeable membrane leading to the mass spectrometer detecting the mass-to-charge ratio m/z 30. This method measures NO directly in solution and is an alternate approach compared with methods that purge solutions to measure NO. Addition to deoxy-Hb(Fe(II)) (near 38 microM heme concentration) of nitrite in a range of 80 microM to 16 mM showed no accumulation of either NO or N(2)O(3) on a physiologically relevant time scale with a sensitivity near 1 nM. The addition of nitrite to oxy-Hb(Fe(II)) and met-Hb(Fe(III)) did not accumulate free NO to appreciable extents. These observations show that for several minutes after mixing nitrite with hemoglogin, free NO does not accumulate to levels exceeding the equilibrium level of NO. The presence of cyanide ions did not alter the appearance of the data; however, the presence of 2 mM mercuric ions at the beginning of the experiment with deoxy-Hb(Fe(II)) shortened the initial phase of NO accumulation and increased the maximal level of free, unbound NO by about twofold. These experiments appear consistent with no role of met-Hb(Fe(III)) in the generation of NO and an increase in nitrite reductase activity caused by the presumed binding of mercuric to cysteine residues. These results raise questions about the ability of reduction of nitrite mediated by deoxy-Hb(Fe(II)) to play a role in vasodilation.     

Reaction mechanism between nitric oxide and glutathione mediated by Fe(III) myoglobin.

Ferrimyoglobin at pH 7.4 binds nitric oxide to yield nitric oxide adducts. In the presence of glutathione (GSH), nitrosoadducts of Mb(III) react with it to give nitrosoglutathione, whose concentration has been determined with an apparatus based on a specific and sensitive solid-state amperometric gas sensor. The reaction constant between the adduct and glutathione, kGSH = (47 +/- 1) M(-1) x s(-1), obtained by UV-Vis spectroscopy kinetic measurements, is about one-eighth of the constant with OH- determined by other authors. We can explain this fact with the higher nucleophilicity of OH- compared to GSH, due to the bulkiness and charge of the species. It is known that the formation of nitrosothiols starting from nitrite or NO (nitrogen monoxide) and glutathione, in the absence of oxygen, is impossible. Thus, from a biological point of view, it is important to point out that GSH reacts with NO in the presence of ferrimyoglobin, even at physiological pH, to form nitrosoglutathione.     

Determination of S-nitrosoglutathione in human and rat plasma by high-performance liquid chromatography with fluorescence and ultraviolet absorbance detection after precolumn derivatization with o-phthalaldehyde.

An analytical method is described for the quantification of S-nitrosoglutathione (GSNO), a potent physiological vasodilator and inhibitor of platelet aggregation, in the presence of a high excess of reduced glutathione (GSH). The method is based on the quantitative elimination of GSH by N-ethylmaleimide, the conversion of GSNO by 2-mercaptoethanol to GSH, its reaction with o-phthalaldehyde (OPA) to form a highly fluorescent and UV-absorbing tricyclic isoindole derivative, and subsequent high-performance liquid chromatographic (HPLC) separation with fluorescence and/or UV absorbance detection. The OPA derivatives of GSH and GSNO obtained by this method were found to be identical by mass spectrometry. GSH (up to 50 microM) did not interfere with the analysis of GSNO (up to 1000 nM). The limits of detection of the method for buffered aqueous solutions of GSNO were determined as 3 nM using fluorescence and 70 nM using UV absorbance detection. Isolation of GSNO by HPLC analysis (pH 7.0) of plasma ultrafiltrate samples (200 microl) prior to derivatization allows specific and artifact-free quantification of GSNO in human and rat plasma. Reduced and oxidized glutathione, nitrite, and cysteine did not interfere with the measurement of GSNO in human and rat plasma. The limit of quantitation (LOQ) of the combined method was determined as 100 nM of GSNO in human plasma ultrafiltrate using fluorescence detection. No endogenous GSNO could be detected in ultrafiltrate samples of plasma of 10 healthy humans at concentrations exceeding the LOQ of the method. After iv infusion of GSNO (125 micromol/kg body wt) in a rat for 20 min GSNO and GSH were detected in rat plasma at 60 and 130 microM, respectively. The method should be useful to investigate formation, metabolism, and reactions of GSNO in vitro and in vivo at physiologically relevant concentrations.     

In vitro oxidation of ascorbic acid and its prevention by GSH

The interaction of glutathione (GSH) with ascorbic acid and dehydroascorbic acid was examined in in-vitro experiments in order to examine the role of GSH in protecting against the autoxidation of ascorbic acid and in regenerating ascorbic acid by reaction with dehydroascorbic acid. If a buffered solution (pH 7.4) containing 1.0 mM ascorbic acid was incubated at 37 degrees C, there was a rapid loss of ascorbic acid in the presence of oxygen. When GSH was added to this solution, ascorbic acid did not disappear. Maximum protection against ascorbic acid autoxidation was achieved with as little as 0.1 mM GSH. Cupric ions (0.01 mM) greatly accelerated the rate of autoxidation of ascorbic acid, an effect that was inhibited by 0.1 mM GSH. Other experiments showed that GSH complexes with cupric ions, resulting in in a lowering of the amount of GSH in solution as measured in GSH standard curves. These results suggest that the inhibition of ascorbic acid autoxidation by GSH involves complexation with cupric ions that catalyze the reaction. When ascorbic acid was allowed to autoxidize at 37 degrees C the subsequent addition of GSH (up to 10 mM) did not lead to the regeneration of ascorbic acid. This failure to detect a direct reaction between GSH and the dehydroascorbic acid formed by oxidation of ascorbic acid under this condition was presumably due to the rapid hydrolysis of dehydroascorbic acid. When conditions were chosen, i.e., low temperature, that promote stability of dehydroascorbic acid, the direct reaction between GSH and dehydroascorbic acid to form ascorbic acid was readily detected. The marked instability of dehydroascorbic acid at 37 degrees C raises questions regarding the efficiency of the redox couple between GSH and dehydroascorbic acid in maintaining the concentration of ascorbic acid in mammalian cells exposed to an oxidative challenge.     

Iron catalyzed conversion of NO into nitrosonium (NO+) and nitroxyl (HNO/NO-) species.

The conversion of NO into its congeners, nitrosonium (NO+) and nitroxyl (HNO/NO-) species, has important consequences in NO metabolism. Dinitrosyl iron complex (DNIC) combined with thiol ligands was shown to catalyze the conversion of NO into NO+, resulting in the synthesis of S-nitrosothiols (RSNO) both in vitro and in vivo. The formation mechanism of DNIC was proposed to involve the intermediate release of nitroxyl. Since the detection of hydroxylamine (as the product of a rapid reaction of HNO/NO- with thiols) is taken as the evidence for nitroxyl generation, we examined the formation of hydroxylamine, RSNO, and nitrite (the product of a rapid reaction of NO+ with water) in neutral solutions containing iron ions and thiols exposed to NO under anaerobic conditions. Hydroxylamine was detected in NO treated solutions of iron ions in the presence of cysteine, but not glutathione (GSH). The addition of urate, a major "free" iron-binding agent in humans, to solutions of GSH and iron ions, and the subsequent treatment of these solutions with NO increased the synthesis of GSNO and resulted in the formation of hydroxylamine. This caused a loss of urate and yielded a novel nitrosative/nitration product. GSH attenuated the urate decomposition to such a degree that it could be reflected as the function of GSH:urate. Results described here contribute to the understanding of the role of iron ions in catalyzing the conversion of NO into HNO/NO- and point to the role of uric acid not previously described.     

Immuno-spin trapping of hemoglobin and myoglobin radicals derived from nitrite-mediated oxidation

The reaction of nitrite with hemoglobin has become of increasing interest due to the realization that plasma nitrite may act as an NO congener that is activated by interaction with red blood cells. Using a combination of spectrophotometry, immuno-spin trapping, and EPR, we have examined the formation of radicals during the oxidation of oxyhemoglobin (oxyHb) and oxymyoglobin (oxyMb) by inorganic nitrite. The proposed intermediacy of ferryl species during this oxidation was confirmed by spectrophotometry using multiple linear regression analysis of kinetic data. Using EPR/spin trapping, a protein radical was observed in the case of oxyMb, but not oxyHb, and was inhibited by catalase. When DMPO spin trapping was combined with Western blot analysis using an anti-DMPO-nitrone antibody, globin/DMPO adducts of both oxyHb and oxyMb were detected, and their formation was inhibited by catalase. Catalase effects confirm the intermediacy of hydrogen peroxide as a heme oxidant in this system. Spectrophotometric kinetic studies revealed that the presence of DMPO elongated the lag phase and decreased the maximal rate of oxidation of both oxyHb and oxyMb, which suggests that the globin radical plays an active role in the mechanism of autocatalysis. Interestingly, the oxidation of oxyHb or oxyMb by nitrite, but not by hydrogen peroxide, produced a diffusible radical that was able to generate spin adducts on a bystander protein. This indicates that the oxidation of oxyhemeproteins by nitrite may cause more widespread oxidative damage than the corresponding oxidation by hydrogen peroxide. The immuno-spin trapping technique represents an important new development for the study of the range and extent of protein oxidation by free radicals and oxidants.     

Glutaraldehyde fixation of the cAMP-dependent Na+/H+ exchanger in trout red cells

It has been shown that the addition of a beta-adrenergic catecholamine to a trout red blood cell suspension induces a 60-100-fold increase of sodium permeability resulting from the activation of a cAMP-dependent Na+/H+ antiport. Subsequent addition of propranolol almost instantaneously reduces the intracellular cAMP concentration, and thus the Na permeability, to their basal values (Mahé et al., 1985). If glutaraldehyde (0.06-0.1%) is added when the Na+/H+ exchanger is activated after hormonal stimulation, addition of propranolol no longer inhibits Na permeability: once activated and fixed by glutaraldehyde, the cAMP dependence disappears. Glutaraldehyde alone causes a rapid decrease in the cellular cAMP concentration. In its fixed state the antiporter is fully amiloride sensitive. The switching on of the Na+/H+ exchange by cAMP is rapidly (2 min) followed by acute but progressive desensitization of the exchanger (Garcia-Romeu et al., 1988). The desensitization depends on the concentration of external sodium, being maximal at a normal Na concentration (145 mM) and nonexistent at a low Na concentration (20 mM). If glutaraldehyde is added after activation in nondesensitizing conditions (20 mM Na), transfer to a Na-rich medium induces only a very slight desensitization: thus the fixative can "freeze" the exchanger in the nondesensitizing conformation. NO3- inhibits the activity of the cAMP-dependent Na+/H+ antiporter of the trout red blood cell (Borgese et al., 1986). If glutaraldehyde is added when the cells are activated by cAMP in a chloride-containing medium, the activity of the exchanger is no longer inhibited when Cl- is replaced by NO3-. Conversely, after fixation in NO3- medium replacement of NO3- by Cl- has very little stimulatory effect. This indicates that the anion dependence is not a specific requirement for the exchange process but that the anion environment is critical for the switching on of the Na+/H+ exchanger and for the maintenance of its activated configuration.     

Kinetics of the reaction between nitric oxide and glutathione: implications for thiol depletion in cells

Nitric oxide in the absence of oxygen was suggested to react with 5-50 mM glutathione (GSH) over many minutes when [NO*] < [GSH] (N. Hogg et al., FEBS Lett. 382:223-228; 1996). However, Aravindakumar et al. (J. Chem. Soc. Perkin Trans. 2:663-669; 2002) provided data suggesting approximately 200-fold higher reactivity under conditions of [NO*] > [GSH]. To help resolve these differences, the rate of loss of NO* ( approximately 9 microM) in aqueous solutions of GSH (2.5-20 mM) was measured by chemiluminescence. An apparent second-order rate constant of 0.080 +/- 0.008 M(-1) s(-1) at pH 7.4, 37 degrees C, was calculated based on the total [GSH] and "pseudo-first-order" kinetics; thiolate anion was much more reactive than undissociated thiol. These data imply a half-life of approximately 30 min for low concentrations of NO* with 5 mM GSH, 37 degrees C, pH 7.4, in the absence of oxygen. Possible kinetic schemes that can partially explain the divergent literature reports are discussed, notably an equilibrium in the reaction between NO* and GSH. Human breast carcinoma MCF-7 cells were exposed to NO* (initially approximately 18 microM) in alidded six well plate in an anaerobic chamber in vitro; intracellular GSH levels decreased by half in approximately 60 min. Aerobic exposure depletes GSH in cells in vitro much faster because of autoxidation of NO* to NO2*, >10(8) times more reactive toward GSH.     

Improved antimicrobial efficacy with nitric oxide releasing nanoparticle generated S-nitrosoglutathione

Nitric oxide (NO) plays a vital role in mammalian host defense through a variety of mechanisms. In particular, NO can oxidize to form reactive nitrogen species or interact with protein thiols and metal centers, blocking essential microbial processes. S-nitrosoglutathione (GSNO), a potent NO donor formed by the interaction of NO with intracellular glutathione (GSH), is a major factor in this pathway and is considered one of the strongest naturally occurring nitrosating agent. We previously described the broad-spectrum antimicrobial activity of a nanoparticulate platform capable of controlled and sustained release of NO (NO-np). Interestingly, in vivo efficacy of the NO-np surpassed in vitro data generated. We hypothesized that the enhanced activity was in part achieved via the interaction between the generated NO and available GSH, forming GSNO. In the current study, we investigated the efficiency of NO-np to form GSNO in the presence of GSH was evaluated, and assessed the antimicrobial activity of the formed GSNO against methicillin resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. When GSH was combined with NO-np, GSNO was rapidly produced and significant concentrations of GSNO were maintained for >24h. The GSNO generated was more effective compared to NO-np alone against all bacterial strains examined, with P. aeruginosa being the most sensitive and K. pneumoniae the most resistant. We conclude that the combination of NO-np with GSH is an effective means of generating GSNO, and presents a novel approach to potent antimicrobial therapy.     

Structure-reactivity studies of the Cu(2+)-catalyzed decomposition of four S-nitrosothiols based around the S-Nitrosocysteine/S-nitrosoglutathione structures.

S-Nitrosoglutathione (GSNO) and the dipeptide derivative S-nitrosoglutamylcysteine (SNO-GluCys) both at 1 x 10(-3) M in pH 7. 4 buffer containing added Cu(2+) (1 x 10(-5) M) are very unreactive toward decomposition (measured spectrophotometrically), and in both cases reaction stops at very low conversion. S-Nitrosocysteine (SNC) and the dipeptide derivative S-nitrosocysteinylglycine (SNO-CysGly), on the other hand, are orders of magnitude more reactive under the same conditions, and reaction proceeds to completion. Initially, we interpreted these results in terms of the requirement of a suitably positioned free NH(2) group (which is available in both SNC and SNO-CysGly, but not in GSNO and SNO-GluCys) for efficient complexation of Cu(+), the effective reagent. However, later results measured at much lower substrate concentration (1 x 10(-6) M) using the NO electrode system showed that at this concentration, all four S-nitrosothiols react at approximately the same rate and yield NO quantitatively. For GSNO the rate and percentage conversion were shown to drop progressively as the substrate concentration increases. All reactions are effectively halted in the presence of the metal ion chelator EDTA. The results can readily be explained in terms of complexation of Cu(2+) by the product disulfides from GSNO (i.e., GSSG) and SNO-GluCys, involving the glutamate residue, which is not present in SNC and SNO-CysGly. This is confirmed by the observed progressive reduction in yield and percentage conversion of GSNO decomposition as GSSG is added, at micromolar substrate concentrations.