Reaction Of Nitrite Research Articles

Synthesis of Dimethyl Carbonate through Vapor‐Phase Carbonylation Catalyzed by Pd‐Doped Zeolites: Interaction of Lewis Acidic Sites and Pd Species

All doped up: The interaction between Lewis (L) acidic sites and Pd species in the vapor-phase carbonylation reaction of methyl nitrite over Pd/Faujasite (FAU) catalysts is determined quantitatively. By keeping the Pd species in an electron-deficient state, the Lewis acids of the Pd/FAU catalysts promote the synthesis of dialkyl carbonates.

Detection of Nitrite in Water Using Minoxidil as a Reagent

Water analysis is one of the most important issues in environmental chemistry. The increasing scale of water contamination owing to the presence of nitrate and nitrite in the environment has converted it into one of the most serious public health problems in modern society. Here an easy colorimetric method for determining nitrite in water is reported. The method is based on the reaction of nitrite with minoxidil in acidic media, which gives nitrosominoxidil as a product that shows an absorption band in the λ = 315–330 nm range. Minoxidil was the first drug approved by the U.S. Food and Drug Administration as a treatment against alopecia (hair loss), thus leading minoxidil to be one of the most popular and commonly used drugs that can be purchased freely at low cost on the market in the form of topical solutions. The experiment can be completed over 3.5 h, and it can be extended to include a kinetic mechanistic study of the nitrosation reaction. The nitrite detection range makes the method suitable for env...

Synthesis and structure of a new polyoxometalate-based inorganic–organic hybrid and application as a chemically bulk-modified electrode

A new Keggin-type polyoxometalate-based inorganic–organic hybrid, [Cu(H2O)2(daphen)]2[SiW12O40]·9H2O (1) (daphen = 5,6-diamino-1,10-phenanthroline), was hydrothermally synthesized, and characterized by single-crystal X-ray diffraction, elemental analysis, infrared spectroscopy, fluorescence spectra, and thermal analysis. Single-crystal X-ray diffraction analysis reveals that in 1, [SiW12O40]4− is a tetradentate ligand with its four terminal oxygens coordinating to four Cu(II)–daphen fragments to form a 2D sheet with (4,4) topology. On the basis of the insolubility of 1 in water and common organic solvent and its reversible multielectron redox processes, 1 was used to fabricate a bulk-modified carbon paste electrode (1-CPE) by direct mixing. Electrochemistry indicated that 1-CPE is stable over hundreds of cycles and possessed electrocatalytic activity toward the reduction reactions of nitrite.

P55: Effect of hematocrit and oxygen saturation on nitric oxide and nitrite interactions with red blood cell in a single RBC: A computational analysis

Background Nitrite acts as a reservoir of nitric oxide (NO) bioactivity in the vasculature. Catalytic reduction of nitrate, diet and in vivo NO production are major sources of nitrite. Nitrite reaction with RBC-encapsulated deoxy-hemoglobin can lead to formation of NO or vasoactive species that can be further exported to cause vasodilation. Quantification of nitrite formation from NO, NO–RBC interactions metabolites and nitrite–RBC interaction metabolites is necessary to understand the nitrite reductase activity of deoxyHb. Methods We developed an unsteady state model to quantify the formation of NO-RBC and nitrite-RBC reaction metabolites such as methemoglobin (metHb) and nitrosyl hemoglobin (HbNO) under oxygenated and deoxygenated conditions. The model geometry consists of two concentric spheres. The outer sphere represents plasma surrounding the RBC and the inner sphere represents the RBC. We estimated the concentrations of all the NO–RBC and nitrite–RBC reaction metabolites by solving the mass balance equations for each metabolite over a period of 1 min. Results and conclusions Using this model, we studied the effects of hematocrit and fractional oxygen saturation on NO–RBC and nitrite–RBC interactions. Our results showed that metHb, HbNO and nitrite concentrations increased with time. MetHb concentrations increased whereas HbNO and nitrite concentrations decreased with increase in fractional oxygen saturation. For a given oxygen saturation, MetHb, HbNO and nitrite increased with hematocrit. The results obtained from this model will assist in quantifying the amount and the distribution of NO or NO active species resulting from nitrite reductase activity in RBC, which can be exported to smooth muscles for vasodilation. Disclosure Supported by NIH Grant R01 HL084337.

P26: Nitrite induced oxidation of oxygenated hemoglobin is accelerated by nitroxyl (HNO)

Angeli’s salt decomposes into nitrite and nitroxyl (HNO), compounds of physiological and therapeutic interest for their impact on biological signaling through both nitric oxide dependent and independent pathways. Both nitrite and nitroxyl oxidize oxygenated hemoglobin (oxyHb) to methemoglobin (metHb), though the nitroxyl reaction rate is approximately seven orders of magnitude larger, and the nitroxyl reaction is thus often considered independent of the nitrite reaction. Earlier work has shown that nitroxyl catalyzes the reduction of nitrite by deoxygenated hemoglobin. However, this research represents the first known report showing nitroxyl accelerates the nitrite reaction with oxyHb. We have demonstrated this nitroxyl mediated acceleration of the nitrite/oxyHb reaction with oxyHb in excess to nitroxyl and nitrite (as would be found under physiological conditions) by monitoring the formation of metHb in the presence of Angeli’s salt with and without added nitrite. In addition, this acceleration has been demonstrated using the nitroxyl donor 4-nitrosotetrahydro-2H-pyran-4-yl pivalate, a water-soluble acyloxy nitroso compound that does not release significant amounts of nitrite but does generate nitroxyl in the presence of esterase. This nitroxyl donor was used both with and without nitrite and acceleration of the nitrite induced formation of methemoglobin was observed. We found that the acceleration was not significantly affected by catalase, superoxide dismutase, or IHP, suggesting that it is not due to formation of peroxide, or superoxide, or to modulation of allosteric properties. In addition, we found that the acceleration is not likely to be related to nitroxyl binding to free reduced hemoglobin. We suggest that the mechanism of acceleration involves local propagation of autocatalysis in the nitrite/oxyHb reaction. This acceleration of the nitrite/oxyHb reaction could affect studies aimed at understanding physiological roles of nitroxyl and nitrite and the use of these agents in therapeutics for conditions such as hypertension, hemolytic anemia, heart failure, and ischemia reperfusion injury. Disclosure Supported by HL098032.

New detector tube for the detection of nitrite in water

Abstract A new colorimetric detector tube for the detection of trace concentrations of nitrite in water was developed. The detector tube is based on a diazo coupling reaction of nitrite with dapsone and N-(1-naphthyl) ethylenediamine dihydrochloride during the formation of red-violet azo dye. Spherical granules (pellets) made from the composite material were used as the carrier of chemical reagents. They were prepared by pelletizing the mixture of microcrystalline cellulose, lactose, kaolin and polyvinylpyrrolidone. Colouring is done on a layer of silica gel impregnated with citric acid. The detector tube is highly sensitive, selective and stable when stored. The detection limit is 0.1 μg.ml-1, i.e. 0.03 μg.ml-1 N(NO2 -). The optimal measuring range of nitrite concentration in the analyzed sample ranges from 0.5 to 80 μg.ml-1, i.e. 0.15-24.36 μg.ml-1 N(NO2 -). The detector tube can easily be structurally modified for analysis of nitrates with a detection limit of 1 μg/ml-1, i.e. 0.23 μg.ml-1 N(NO3 -).

P45: Enhanced nitrite reaction rates of MP4OX, a novel ischemic rescue therapy

Background Hemoglobin-based oxygen carriers have been associated with vasoconstriction, attributed primarily to binding of nitric oxide (NO) as a heme ligand. MP4OX, an ischemic rescue therapy, designed with high O2 affinity using poly (ethylene glycol) (PEG) conjugation chemistry, has been observed not to cause vasoconstriction and to preserve microvascular blood flow, despite reducing perivascular levels of NO [1] . Recent evidence shows that reduction of nitrite to NO by deoxyhemoglobin has the ability to vasodilate blood vessels [2] . It also has been reported that the oxidative reaction of oxyhemoglobin with nitrite may facilitate the release of NO from heme [3] . We hypothesized that MP4OX may have enhanced reaction rates with nitrite and regeneration of NO, which may contribute to its lack of systemic vasoconstriction. Materials and methods MP4OX was prepared by reacting human stroma-free hemoglobin (SFH) with 2-iminothiolane and maleimide-activated PEG to produce a PEG-hemoglobin conjugate, with approximately 8 PEGs/Hb. Rates of the deoxyHb/nitrite reaction were measured by mixing deoxygenated SFH or MP4OX anaerobically with a 10-fold excess of sodium nitrite in a sealed cuvette in the presence of sodium dithionite. The reaction was monitored spectrophotometrically, and the spectra were deconvoluted using parent spectra for deoxyhemoglobin, iron-nitrosyl-hemoglobin (HbNO), and methemoglobin. Rates of the oxyHb/nitrite reaction were measured by mixing oxygenated SFH or MP4 with a 20-fold excess of nitrite. These spectra were deconvoluted using parent spectra for oxyhemoglobin, methemoglobin, and nitrite-bound methemoglobin. Results Time courses for both deoxy-and oxyHb reactions showed autocatalytic properties that deviated from pseudo first-order kinetics. Therefore, the maximum reaction rates were used for comparison purposes. Fully deoxygenated SFH and MP4OX (i.e., MP4DX) reduced nitrite to NO with maximum rates of 0.022 and 0.206 μM/s, respectively, showing a nearly 10-fold higher rate for MP4DX compared to deoxySFH. For the oxyHb/nitrite reaction, SFH and MP4OX showed maximum rates of 0.55 and 0.92 μM/s, respectively, a nearly 2-fold enhancement for MP4OX over SFH. Conclusions Our results demonstrate that deoxygenated MP4OX is more effective at reducing nitrite to NO than unmodified Hb. In addition, oxygenated MP4OX reacts more rapidly with nitrite, which may then accelerate the release of NO from heme sites. Taken together, these enhanced nitrite reaction rates for MP4 may compensate for NO scavenging and explain, at least in part, why MP4OX does not induce vasoconstriction in vivo. The increased reaction rates of MP4OX appear to be due to R-state stabilization due to the PEG conjugation. Further studies are required to understand the extent to which MP4OX’s interactions with nitrite contribute to its ability to improve perfusion and oxygenation of ischemic tissues. Disclosure This work was funded in part by Sangart Inc. and under US Army Contract No. W81XWH1020111 to Sangart.

Kinetics of N-Nitrosopiperazine Formation from Nitrite and Piperazine in CO2 Capture

Piperazine (PZ) is an efficient amine for carbon capture systems, but it can form N-nitrosopiperazine (MNPZ), a carcinogen, from nitrogen oxides (NO(x)) in flue gas from coal or natural gas combustion. The reaction of nitrite with PZ was studied in 0.1 to 5 mol/dm(3) PZ with 0.001 to 0.8 mol CO2/mol PZ at 50 to 135 °C. The reaction forming MNPZ is first order in nitrite, piperazine carbamate species, and hydronium ion. The activation energy is 84 ± 2 kJ/mol with a rate constant of 8.5 × 10(3) ± 1.4 × 10(3) dm(6) mol(-2) s(-1) at 100 °C. The proposed mechanism involves protonation of the carbamate species, nucleophilic attack of the carbamic acid, and formation of bicarbonate and MNPZ. These kinetics and mechanism will be useful in identifying inhibitors and other strategies to reduce nitrosamine accumulation in CO2 capture by scrubbing with PZ or other amines.

In reply

In reply: We read with interest the article by Feys and colleagues1 comparing nitrite/nitrate (NO2−/NO3−) levels during storage and preparation of platelet concentrate (PC) from pooled buffy coats. Although they did not measure nitrite separately, their results suggest no substantial production of nitric oxide (NO) in stored PCs since NO produced in large quantities would likely affect the nitrite/nitrate levels. It is also consistent with our report2 that apheresis PCs did not produce NO by endogenous nitric oxide synthase (NOS) during storage. However, based on our electron paramagnetic resonance results, NO level in PCs was higher than that in plasma. Furthermore, we also showed that there was a slight decrease in nitrite levels during storage. We believe that our results suggest a possibility of a nitrite reduction system in PCs or nitrite reaction with residual hemoglobin, cell free or in red blood cells (RBCs), possibly indicative of some NO metabolic processes. Interestingly, Feys and colleagues observed an increase in nitrite/nitrate levels immediately after the procedures of PC preparation from buffy coats in which centrifugations and dilution with additive solution are used. As they suggested, it is possible that RBCs and white blood cells in buffy coats contribute to this increase in nitrite/nitrate levels as a native mechanism to prevent overt platelet (PLT) activation during preparation. Some authors have suggested the presence of active NOS in RBCs, although the definitive proof of functional NOS in RBCs is still under debate.3 In addition, it is still not clear if PLTs themselves express NOS.4 Considering the fact that buffy coat PCs show the lowest level of PLT activation by in vitro assays compared with apheresis PCs or PLT-rich plasma (PRP)-derived PCs,5 it would be interesting to see if the procedures of apheresis or PRP PC preparations also increase nitrite and nitrate levels. As Feys and colleagues note, production of NO in these preparations may be important for the maintenance of PLT function during in vitro storage, allowing clinical utility of PLT transfusions. ANS is a co-inventor of a patent issued to NIH for the use of sodium nitrite in the treatment of cardiovascular diseases. The authors declare that they have no conflicts of interest relevant to the manuscript submitted to TRANSFUSION.

Nitrite determination in water samples based on a modified Griess reaction and central composite design

A simple, sensitive, selective and rapid spectrophotometric method for determination of trace amounts of nitrite was proposed. The method is based on the diazotization-coupling reaction of nitrite by 3-nitroaniline and 1-naphthylamine in hydrochloric acid solution at pH 1.0 (Griess reaction). The final product (an azo dye) has a purple color with maximum absorption at 515 nm. Optimum conditions of the variables influencing the reaction were explored by central composite design (CCD). The variables chosen were pH, reaction temperature, concentration of 3-nitroaniline and concentration of 1-naphthylamine. The univariate calibration curve was linear in the range of 0.01–1.70 mg L−1 of nitrite. A detection limit of 5 × 10−4 mg L−1 was obtained for the determination of nitrite by the proposed method. The modified Griess method was successfully applied to determine nitrite in tap, rain and wastewater samples. The results of the analysis of the real samples showed that percent relative standard deviation of the method (RSD%) is below 4% and percent recoveries vary between 99.0% and 102%.

A novel route to organonitrites by Pd-catalyzed cross-coupling of sodium nitrite and potassium organotrifluoroborates

Microwave irradiated palladium-catalyzed cross-coupling reaction of potassium styryltrifluoroborates and sodium nitrite gives the corresponding styryl nitrites in high yields. Potassium aryltrifluoroborates also furnish aryl nitrites under same reaction condition. This unprecedented cross-coupling is an interesting development and has the potential to lead to new nitration protocols.

Poly-o-toluidine cobalt supported on ordered mesoporous carbon as an efficient electrocatalyst for oxygen reduction

Polymer–metal complex of poly-o-toluidine (POT) with cobalt supported on ordered mesoporous carbon (OMC) was prepared by the reaction of cobalt nitrite and POT in the presence of OMC. This new POT–Co–OMC composite was used as a cathode material for oxygen reduction reaction (ORR). The electroconductive polymer of the as-obtained composite provides active sites (Co–N), and in addition OMC has a high specific surface area for achieving high dispersion of POT–Co. This unique structure facilitates the reduction of oxygen via a four-electron pathway in alkaline conditions. The POT–Co–OMC catalyst also exhibits excellent anti-crossover effect to methanol oxidation in the oxygen reduction reaction (ORR), superior to the commercial Pt/C catalyst. Such a novel polymer–metal complex can provide a platform for a new generation of non-precious metal catalyst for fuel cell applications.

The oxidative denitrosylation mechanism and nitric oxide release from human fetal and adult hemoglobin, an experimentally based model simulation study

Generation of unbound nitric oxide (NO) via the oxidative denitrosylation (ODN) mechanism is proposed to involve the simultaneous reaction of nitrite with oxy and deoxy hemoglobin (Hb(O2) (k1) and Hb (k13)) to yield respectively, *NO2 and Hb(+2)(NO). These two reaction pathways are coupled when *NO2 reacts with Hb(+2)(NO) to yield Hb(+3)(NO) (k22), a species that releases NO rapidly. Here, I have constructed an experimentally based molecular model of the ODN mechanism (k1-k31), focusing on the high nitrite reductase activity of R-state hemoglobin. This model was used to test the hypothesis that human fetal hemoglobin (HbF) can generate unbound NO faster and to a greater extent than HbA, consequent to a 25-fold larger value of k1, which was determined in an earlier study. The results show that despite the use of identical values for k22, there was a 44-fold larger apparent rate of reaction of *NO2 with HbF(NO) compared to HbA(NO), for reactions simulated at 410 μM nitrite and 100 μM hemoglobin (heme basis), 50% oxygen saturation at pH 7.4 and 37°C. This faster reaction was associated with the generation of about 11 μM peak unbound NO. In contrast, HbA failed to generate unbound NO rapidly under the same conditions. However, raising the concentration of nitrite into the millimolar range did generate unbound NO in the HbA simulation, in agreement with the experimental literature, and that result was associated with acceleration in the rate of reaction of NO2 with HbA(NO). Unbound NO could be generated at 410 μM nitrite in the HbA simulation by lowering the pH. This too was associated with an acceleration in the rate of reaction of NO2 with HbA(NO). Furthermore, generation of unbound NO could be assigned to the pH-dependent increase in k1, independent of the associated increase in k(13). Finally, selective exchange of the HbA value of k1 for the HbF value, keeping all other constants and conditions unchanged, generated kinetic patterns for the various species of the "k1-modified" HbA simulation, which were virtually indistinguishable from those seen in the HbF simulation. Taken together, these findings show that rapid and extensive generation of unbound NO within the ODN mechanism is controlled by the value of k1. The faster and more extensive generation of unbound NO by HbF at micromolar nitrite concentration suggests a possible second function for HbF in sickle cell disease, namely enhanced vasodilation. The failure of 410 μM nitrite to generate unbound NO in the HbA simulation at pH 7.4, contrasts with evidence in the literature showing that exposure of intact red cells to 100 to 200 μM nitrite in PBS, promoted NO release into the gas phase. I point out that this difference in outcome may be due to the higher activity of HbA when bound to the cytoplasmic domain of the red cell membrane anion transport protein SLC4A1 (band 3) and to the demonstrated capacity of band 3 to transport nitrite.

Abstract 195: Nitro-Oleic Acid is a Dual Inhibitor of Nox1 and Nox2

Nitrated fatty acids (NFAs) are α,β-unsaturated keto derivatives of ω-3 fatty acids formed from redox reactions of nitric oxide and nitrite and have recently emerged as anti-inflammatory agents that act as electrophiles. NFAs activate Nrf2 and PPARγ signaling and inhibit NF-κB. Recent data suggest a role in inhibition of reactive oxygen species (ROS) production. This function however, remains largely unexplored. The Nox family of oxidases is a major source of ROS implicated in hypertension and other cardiovascular diseases (CVDs). Here we postulate that the NFA nitro-oleic acid (OA-NO 2 ) specifically inhibits ROS production by the Nox1 and Nox2 oxidases. Treatment of canonical Nox1-expressing COS-7 cells with OA-NO 2 vs its oleic acid (OA) control resulted in a concentration-dependent inhibition of ROS production (101.5, 98.9, 91.7, 94.8, 54.2, and 45.5 % of vehicle treated groups for 0.1, 1, 2, 4, 8, and 10 μM OA-NO 2 , respectively), with an IC 50 of 6.5 μM. Treatment of phorbol ester (PMA)-stimulated Nox2-expressing COS-7 cells yielded similar results (103.6, 106.6, 112.2, 102.1, 51.8 and 35.1 % vehicle treated groups for the same OA-NO 2 concentration range) with an IC 50 of 7.0 μM. Treatment with the same OA-NO 2 concentrations of Nox4- or Nox5-expressing COS-7 cells did not result in any inhibition. The effects of OA-NO 2 on Nox1 and Nox2 were unique to its electrophilic unsaturated keto structure, as OA (lacking NO 2 group), palmitic acid (lacking both double bond and NO 2 group) and linoleic acid (containing two cis double bonds) did not result in any inhibition, demonstrating that fatty acids other than NFAs are incapable of this inhibition. Moreover, experiments using potassium superoxide (O 2 •- ) ruled out any scavenging effects of OA-NO 2 on O 2 •- . Finally, 10 μM OA-NO 2 inhibited both angiotensin II (AngII)- and PMA-stimulated O 2 •- in rat aortic smooth muscle cells (4.4 ± 0.9, 10.8 ± 1.7 and 9.0 ± 2.5 in OA-treated vehicle, AngII and PMA groups vs. 5.8 ± 0.6, 5.7 ± 0.3 and 3.3 ± 1.0 pmol O 2 •- /min/mg protein in OA-NO 2 -treated vehicle, AngII and PMA groups, respectively). Taken together, these results identify NFAs as potent inhibitors of Nox1- and Nox2-derived ROS raising the possibility for their use as therapeutic agents to treat hypertension and other CVDs.

A Spectrophotometric Flow Injection Method for Determination of Ultratrace Amounts of Phenylhydrazine by Its Inhibition Effect on the Reaction of Thionin and Nitrite

A simple flow injection colorimetric procedure for determining phenylhydrazine was established. It is based on the reaction of phenylhydrazine in sulfuric acid with thionin and sodium nitrite. Reaction was monitored spectrophotometrically by measuring thionin absorbance at λmax = 602 nm. A standard or sample solution was injected into the sulfuric acid stream, which was then merged with sodium nitrite stream and thionin stream. Optimum conditions for determining phenylhydrazine were investigated by univariate method. Under the optimum conditions, a linear calibration graph was obtained over the range 0.05–0.60 μmol L−1, and the detection limit was 0.027 μmol L−1(s/n = 3). The proposed method has been satisfactorily applied to the determination of phenylhydrazine in human serum and water samples.

Rapid determination of nitrites in food using a diffuse UV-visible reflectance method

A simple, low-cost, sensitive and selective method for the determination of trace quantities of nitrite in foods such as cheese and cured meat using diffuse ultraviolet-visible reflectance was developed. It is based on the reaction of nitrite with sulphadiazine and α-naphthol, which produces a coloured product in basic medium. The reaction is carried out directly in the measuring cell. For cheese the limit of detection (LOD), expressed as NaNO2, was estimated to be about 2.0 × 10−2 mg l−1 (2.9 × 10−7 mol l−1) in the final measuring solution and 0.17 mg kg−1 in cheese (2.5 × 10−6 mol kg−1). The relative standard deviation (RSD) varied from 5% to 8% depending on the sample. For meat the LOD was estimated to be about 2.0 × 10−2 mg l−1 (2.9 × 10−7 mol l−1) in the final measuring solution and 0.13 mg kg−1 in meat (1.9 × 10−6 mol kg−1). The RSD varied from 3% to 6% depending on the sample. The results of the proposed method were also compared with those obtained with the official method using the statistical Student's t-test and F-test procedures.

Sodium nitrite protects against kidney injury induced by brain death and improves post-transplant function

Renal injury induced by brain death is characterized by ischemia and inflammation and limiting it is a therapeutic goal that could improve outcomes in kidney transplantation. Brain death resulted in decreased circulating nitrite levels and increased infiltrating inflammatory cell infiltration into the kidney. Since nitrite stimulates nitric oxide signaling in ischemic tissues, we tested whether nitrite therapy was beneficial in a rat model of brain death followed by kidney transplantation. Nitrite, administered over 2 hours of brain death, blunted the increased inflammation without affecting brain death-induced alterations in hemodynamics. Kidneys were transplanted after 2 hours of brain death and renal function followed over 7 days. Allografts collected from nitrite-treated brain dead rats showed significant improvement in function over the first 2 to 4 days post transplantation compared to untreated brain dead animals. Gene microarray analysis after 2 hours of brain death without or with nitrite therapy showed the latter significantly altered the expression of about 400 genes. Ingenuity Pathway analysis indicated multiple signaling pathways were affected by nitrite, including those related to hypoxia, transcription and genes related to humoral immune responses. Thus, nitrite-therapy attenuates brain death-induced renal injury by regulating responses to ischemia and inflammation, ultimately leading to better post-transplant kidney function.

Effect of Oxidized Myofibrils Protein Subjected to Mutiple Freeze-Thaw Cycles on N-Nitrosamine Formation in <i>In Vitro</i> Model System

N-nitrosamine is a kind of carcinogenic substance, which is possibly formed in the reaction of nitrites with amino acids or secondary amines. Two in vitro model systems were designed to evaluate the influence of oxidized myofibrils protein subjected to repeated freeze-thaw cycles (0, 1, 2, 3, 4, 7, 10 times) on N-nitrosamine formation. Model system I contains diethylamine and sodium nitrite, while model system II contains only sodium nitrite as reaction solution. Oxidized myofibrils protein were added to both systems. The results revealed that as the number of freeze-thaw cycles increased, cross-linking of myosin heavy chains and the content of protein carbonyl increased, but the content of protein sulfydryl decreased, which indicates oxidization of protein occurred. The concentration of N-nitrosodiethylamine increased as the number of freeze-thaw cycles increased, especially after four cycles. Oxidized myofibrils protein promoted the formation of N-nitrosodiethylamine. The more the times of freeze-thaw cycles were subjected, the more oxidization of myofibrils protein occurred and the higher yield of the N-nitrosodiethylamine.

Nitrite Reductase Activity of Nonsymbiotic Hemoglobins from Arabidopsis thaliana

Plant nonsymbiotic hemoglobins possess hexacoordinate heme geometry similar to that of the heme protein neuroglobin. We recently discovered that deoxygenated neuroglobin converts nitrite to nitric oxide (NO), an important signaling molecule involved in many processes in plants. We sought to determine whether Arabidopsis thaliana nonsymbiotic hemoglobins classes 1 and 2 (AHb1 and AHb2, respectively) might function as nitrite reductases. We found that the reaction of nitrite with deoxygenated AHb1 and AHb2 generates NO gas and iron-nitrosyl-hemoglobin species. The bimolecular rate constants for reduction of nitrite to NO are 19.8 ± 3.2 and 4.9 ± 0.2 M(-1) s(-1), respectively, at pH 7.4 and 25 °C. We determined the pH dependence of these bimolecular rate constants and found a linear correlation with the concentration of protons, indicating the requirement for one proton in the reaction. The release of free NO gas during the reaction under anoxic and hypoxic (2% oxygen) conditions was confirmed by chemiluminescence detection. These results demonstrate that deoxygenated AHb1 and AHb2 reduce nitrite to form NO via a mechanism analogous to that observed for hemoglobin, myoglobin, and neuroglobin. Our findings suggest that during severe hypoxia and in the anaerobic plant roots, especially in species submerged in water, nonsymbiotic hemoglobins provide a viable pathway for NO generation via nitrite reduction.

Effect of Different Carbon Substrates on Nitrate Stable Isotope Fractionation During Microbial Denitrification

In batch experiments, we studied the isotope fractionation in N and O of dissolved nitrate during dentrification. Denitrifying strains Thauera aromatica and "Aromatoleum aromaticum strain EbN1" were grown under strictly anaerobic conditions with acetate, benzoate, and toluene as carbon sources. (18)O-labeled water and (18)O-labeled nitrite were added to the microcosm experiments to study the effect of putative backward reactions of nitrite to nitrate on the stable isotope fractionation. We found no evidence for a reverse reaction. Significant variations of the stable isotope enrichment factor ε were observed depending on the type of carbon source used. For toluene (ε(15)N, -18.1 ± 0.6‰ to -7.3 ± 1.4‰; ε(18)O, -16.5 ± 0.6‰ to -16.1 ± 1.5‰) and benzoate (ε(15)N, -18.9 ± 1.3‰; ε(18)O, -15.9 ± 1.1‰) less negative isotope enrichment factors were calculated compared to those derived from acetate (ε(15)N, -23.5 ± 1.9‰ to -22.1 ± 0.8‰; ε(18)O, -23.7 ± 1.8‰ to -19.9 ± 0.8‰). The observed isotope effects did not depend on the growth kinetics which were similar for the three types of electron donors. We suggest that different carbon sources change the observed isotope enrichment factors by changing the relative kinetics of nitrate transport across the cell wall compared to the kinetics of the intracellular nitrate reduction step of microbial denitrification.