Free SH Groups Research Articles

Coal and tire burning mixtures containing ultrafine and nanoparticulate materials induce oxidative stress and inflammatory activation in macrophages

Ultra-fine and nano-particulate materials resulting from mixtures of coal and non-coal fuels combustion for power generation release to the air components with toxic potential. We evaluated toxicological and inflammatory effects at cellular level that could be induced by ultrafine/nanoparticles-containing ashes from burning mixtures of coal and tires from an American power plant. Coal fly ashes (CFA) samples from the combustion of high-S coal and tire-derived fuel, the latter about 2–3% of the total fuel feed, in a 100-MW cyclone utility boiler, were suspended in the cell culture medium of RAW 264.7 macrophages. Cell viability, assessed by MTT reduction, SRB incorporation and contrast-phase microscopy analysis demonstrated that CFA did not induce acute toxicity. However, CFA at 1mg/mL induced an increase of approximately 338% in intracellular TNF-α, while release of this proinflammatory cytokine was increased by 1.6-fold. The expression of the inflammatory mediator CD40 receptor was enhanced by 2-fold, the receptor for advanced glycation endproducts (RAGE) had a 5.7-fold increase and the stress response protein HSP70 was increased nearly 12-fold by CFA at 1mg/mL. Although CFA did not induce cell death, parameters of oxidative stress and reactive species production were found to be altered at several degrees, such as nitrite accumulation (22% increase), DCFH oxidation (3.5-fold increase), catalase (5-fold increase) and superoxide dismutase (35% inhibition) activities, lipoperoxidation (4.2 fold-increase) and sulfhydryl oxidation (40% decrease in free SH groups). The present results suggest that CFA containing ultra-fine and nano-particulate materials from coal and tire combustion may induce sub-chronic cell damage, as they alter inflammatory and oxidative stress parameters at the molecular and cellular levels, but do not induce acute cell death.

Comparison of the gel-forming ability and gel properties of α-lactalbumin, lysozyme and myoglobin in the presence of β-lactoglobulin under high pressure

α-Lactalbumin (α-La) and lysozyme (LZM) each contain four disulfide bonds but no free SH group, whereas myoglobin (Mb) possesses no disulfide bond or free SH group. In this work, the pressure-induced gelation of α-La, LZM and Mb in the absence and in the presence of β-lactoglobulin (β-Lg) was studied. Solutions of α-La, LZM and Mb (1–24%, w/v) did not form a gel when subjected to a pressure of 800 MPa and circular dichroism analysis revealed that both α-La and LZM are pressure-resistant proteins. In the presence of β-Lg (5%, w/v), however, a pressure-induced gel formed for α-La and LZM (each 15%, w/v) but not for Mb (15%, w/v). One- and two-dimensional SDS-PAGE demonstrated the disulfide cross-linking of proteins was responsible for the gelation. Although α-La and LZM are homologous and have the same disulfide bond arrangement, the texture and appearance of the gels formed from α-La/β-Lg and LZM/β-Lg were markedly different even when induced under the same experimental conditions. Microscopic analysis indicated that phase separation occurs during the gelation of LZM/β-Lg but not during the gelation of α-La/β-Lg. NMR relaxation measurement revealed that the association of water molecules with the protein matrix in the α-La/β-Lg gel is tighter compared to that in the LZM/β-Lg gel. These results indicate that the gel-forming ability of a globular protein under high pressure is related to the primary structure of the protein, and that the gel properties depend on the cross-linking reaction and on the phase behavior of protein dispersion under high pressure.

Oxidized albumin. The long way of a protein of uncertain function

BackgroundProteins are extremely reactive to oxidants and should represent a potential target of instable reactive oxygen. This may represent a problem for plasma proteins since they may be directly modified in vivo in a compartment where antioxidant enzymatic systems are scarcely represented. On the other hand, it is possible that some plasma components have evolved over time to guarantee protection, in which case they can be considered as anti-oxidants. Scope of reviewTo present and discuss main studies which addressed the role of albumin in plasma antioxidant activity mainly utilizing in vitro models of oxidation. To present some advances on structural features of oxidized albumin deriving from studies carried out on in vitro models as well as albumin purified in vivo from patients affected by clinical conditions characterized by oxidative stress. Major conclusionsThere are different interaction with HOCl and chloramines. In the former case, HOCl produces an extensive alteration of 238Trp and 162Tyr, 425Tyr, 47Tyr, while thiol groups are only partially involved. Chloramines are extremely reactive with the unique free SH group of albumin (34Cys) with the formation of sulfenic and sulfinic acid as intermediates and sulfonic acid as end-product. Oxidized albumin has a modified electrical charge for the addition of an acidic residue and presents α-helix and random coil reorganization with subtle changes in domain orientation. General significanceAlbumin, is the major antioxidants in plasma with a concentration (0.8mM) higher than other antioxidants by an exponential factor. Functional and protective roles in the presence of oxidative stress must be defined. This article is part of a Special Issue entitled Serum Albumin.

Thiol Containing Redox System and Blood Serum Inhibitory Properties Regarding IGM Antibodies from the Viewpoint of Pathophysiology: a Review

Redox system is a complex multi-level system. Various factors are involved in the regulation of its activity. Normally the system tends to the state of equilibrium. It is important to consider the balance mechanisms in this system and the approaches to the individual redox status evaluation of the concrete host. The literature was reviewed to examine the participation of the intracellular and extracellular thiol containing redox system (TCRS) in the physiological processes of the immune functioning regulation as well as to show the quantitative and qualitative changes of the TCRS properties under the influence of different endogenous and exogenous factors. А good example is the appearance of blood serum inhibitory properties regarding IgM abs. This phenomenon is a general problem in mammals and is connected with the increased blood free SH-groups level. The research shows the double effect of the TCRS: so the intensification of the antioxidant protection is not always favourable for the host. Thus, the individual evaluation of the TCRS state in patients having various pathologies is necessary for the choice of optimum treatment.

A water-soluble selenoxide reagent as a useful probe for the reactivity and folding of polythiol peptides

A water-soluble selenoxide (DHSox) having a five-membered ring structure enables rapid and selective conversion of cysteinyl SH groups in a polypeptide chain into SS bonds in a wide pH and temperature range. It was previously demonstrated that the second-order rate constants for the SS formation with DHSox would be proportional to the number of the free SH groups present in the substrate if there is no steric congestion around the SH groups. In the present study, kinetics of the SS formation with DHSox was extensively studied at pH 4–10 and 25 °C by using reduced ribonuclease A, recombinant hirudin variant (CX-397), insulin A- and B-chains, and relaxin A-chain, which have two to eight cysteine residues, as polythiol substrates. The obtained rate constants showed stochastic SS formation behaviors under most conditions. However, the rate constants for CX-397 at pH 8.0 and 10.0 were not proportional to the number of the free SH groups, suggesting that the SS intermediate ensembles possess densely packed structures under weakly basic conditions. The high two-electron redox potential of DHSox (375 mV at 25 °C) compared to l-cystine supported the high ability of DHSox for SS formation in a polypeptide chain. Interestingly, the rate constants of the SS formation jumped up at a pH around the pKa value of the cysteinyl SH groups. The SS formation velocity was slightly decreased by addition of a denaturant due probably to the interaction between the denaturant and the peptide. The stochastic behaviors as well as the absolute values of the second-order rate constants in comparison to dithiothreitol (DTTred) are useful to probe the chemical reactivity and conformation, hence the folding, of polypeptide chains.

The modified action of triphenyllead chloride on UVB-induced effects in albumin and lipids

Previously we have shown a toxic effect of the organometallic compound triphenyllead (TPhPb) on cells. In the present study we evaluated the destructive effect of TPhPb on model systems – serum albumin and liposome membranes – alone and under UVB irradiation. UVB irradiation of bovine serum albumin results in protein SS bond reduction, free SH and CO group formation and decrease in fluorescence intensity of tryptophans. Triphenyllead chloride alone and under UVB irradiation did not induce protein oxidation, measured as formation of carbonyl groups, in serum albumin; however, it decreased the content of SH groups in both cases (alone and under UVB radiation) in a dose-dependent manner. It was found that triphenyllead chloride alone did not induce lipid peroxidation of liposomes but increased their fluidity. However, under UVB irradiation TPhPb dramatically enhances the pro-oxidant action of UVB in a manner dependent on concentration and intensity of radiation, and these effects were suppressed by Trolox. These results suggest that the toxicity of TPhPb under UVB irradiation is due to formation of radical forms of the compound and its disordered effects on the membrane structure.

Thiomer-coated liposomes harbor permeation enhancing and efflux pump inhibitory properties

An ideal oral drug carrier should facilitate drug delivery to the gastrointestinal tract and its absorption into the systemic circulation. To meet these requirements, we developed a thiomer-coated liposomal delivery system composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and a maleimide-functionalized lipid, to which chitosan-thioglycolic acid (CS-TGA) was covalently coupled. In addition to conventional 77kDa CS-TGA (CS-TGA77), we tested the 150kDa homologue (CS-TGA150) as well as an S-protected version of this polymer (CS-TGA150-MNA), in which some of the free SH-groups are conjugated with 6-mercaptonicotinamide to protect them from oxidation. Coupling of CS-TGA to the liposomal surface led to an increase in the particle size of at least 150nm and an increase in the zeta potential from approximately −33mV to a maximum of about +36mV, depending on the polymer. As revealed by fluorescence dequenching the formulations have a storage stability of at least two weeks without releasing any encapsulated compounds. In simulated gastric fluid, the system was shown to be stable over 24h, while in simulated intestinal fluid, a slow, sustained release of encapsulated compounds was observed. According to our experiments, thiomer-coated liposomes did not induce immunogenic reactions after an oral administration to mice. To evaluate the permeation enhancing and efflux pump inhibiting properties of CS-TGA coated liposomes we monitored the transport of fluoresceinisothiocyanate-dextran (FD4) and rhodamine-123 (Rho-123), respectively, through rat small intestine. Permeation studies showed a 2.8-fold higher permeation of FD4 in the presence of CS-TGA77 coated liposomes and an even 4-fold higher permeation in the presence of CSA-TGA150-MNA coated liposomes. The latter also performed best when we evaluated P-glycoprotein inhibiting properties by monitoring the transport of Rho-123, revealing a 4.2-fold enhancement respective to the buffer control. Taken together, thiomer-coated liposomes were shown to protect encapsulated drugs in the stomach, slowly release them in the small intestine and enhance their absorption through the intestinal tissue by opening tight junctions and inhibiting efflux pumps.

481 Mitigation of the Allergic Activity of Ovomucoid By Electrolysis

Ovomucoid (OMC) is the most prominent allergen causing egg allergy, and contains disulfide (S-S) bonds that may be responsible for its allergic action. As S-S bonds may be reduced during...

An Unusual Subtilisin-like Serine Protease Is Essential for Biogenesis of Quinohemoprotein Amine Dehydrogenase

Quinohemoprotein amine dehydrogenase (QHNDH), an αβγ heterotrimer present in the periplasm of several Gram-negative bacteria, catalyzes the oxidative deamination of various aliphatic amines such as n-butylamine for assimilation as carbon and energy sources. The γ subunit of mature QHNDH contains a protein-derived quinone cofactor, cysteine tryptophylquinone, and three intrapeptidyl thioether cross-links between Cys and Asp or Glu residues. In its cytoplasmic nascent form, the γ subunit has a 28-residue N-terminal leader peptide that is necessary for the production of active QHNDH but must be removed in the following maturation process. Here, we describe the role of a subtilisin-like serine protease encoded in the fifth ORF of the n-butylamine-utilizing operon of Paracoccus denitrificans (termed ORF5) in QHNDH biogenesis. ORF5 disruption caused bacterial cell growth inhibition in n-butylamine-containing medium and production of inactive QHNDH, in which the γ subunit retained the leader peptide. Supply of plasmid-encoded ORF5 restored the cell growth and production of active QHNDH, containing the correctly processed γ subunit. ORF5 expressed in Escherichia coli but not its catalytic triad mutant cleaved synthetic peptides surrogating for the γ subunit leader peptide, although extremely slowly. The cleaved leader peptide remained unstably bound to ORF5, most likely as an acyl enzyme intermediate attached to the active-site Ser residue. These results demonstrate that ORF5 is essential for QHNDH biogenesis, serving as a processing protease to cleave the γ subunit leader peptide nearly in a disposable manner.

An infrared study of adsorbed metal ions on modified silica: Comparative chelation between mercury, cadmium, and lead divalent ions to silica functionalized with ortho- and para-aminothiophenoles

Chelating selectivity and capacity of silica functionalized 2- and 4-aminothiophenoles (2-ASP-[silica] and 4-ASP-[silica]) toward mercury, lead, and cadmium ions in aqueous medium are studied. In this comparative study, the three metal ions were allowed to interact individually and simultaneously with two aminothiophenol (ASP) derivatives namely, 2- and 4-ASP once as free chelates in solution and secondly as immobilized chelates on silica. Upon individually or simultaneously interacting the three metal ions with 4-ASP-[silica], Hg(II) ions are preferentially adsorbed where 100% of Hg(II) is removed compared to 83.0% of Pb(II) and 76% of Cd(II) ions. In solution, Hg(II) ions are found to be preferentially adsorbed by 2-ASP when compared to 4-ASP. Whereas, anchoring 4-ASP to a silica surface via amide linkage provides a significant enhancement in selectivity and extent of chelation toward Hg(II) over Cd(II) and Pb(II) ions. In the case of 4-ASP-[silica], the existence of a free SH group allows an easy-accessible and strain-free binding site for the incoming Hg(II) ions. Whereas, in 2-ASP-[silica], the SH group is sterically hindered due to proximity to the point of attachment with the surface. As a result, 2-ASP-[silica] showed less potential for Hg(II) binding compared to the modified analogue, 4-ASP-[silica] with less chelation extent observed in solution compared to that observed at the surface.

Multiple Actions of the Anthracycline Daunorubicin on Cardiac Ryanodine Receptors

Our aim was to examine the molecular basis for acute effects of the anthracycline daunorubicin on cardiac ryanodine receptor (RyR2) channels and cardiac calsequestrin (CSQ2). Cardiotoxic effects of anthracyclines preclude their chemotherapeutic use in patients with pre-existing heart conditions. To address this significant problem, the mechanisms of anthracycline toxicity must be defined but at present are poorly understood. RyR2 channel activity was assessed by measuring Ca²⁺ release from cardiac sarcoplasmic reticulum vesicles and by examining single RyR2 channels inserted into artificial lipid bilayers. We show that 0.5 to 10 μM daunorubicin increases the activity of RyR2 channels after 5 to 10 min and that activity then declines to very low levels when channels are exposed to daunorubicin concentrations of ≥ 2.5 μM for a further 10 to 20 min. Extensive dissection of these effects shows for the first time that the activation results from a redox-independent binding of daunorubicin to the RyR2 complex. Novel data include the demonstration of daunorubicin binding to RyR2. We provide compelling evidence that RyR2 channel inhibition is due to the oxidation of free SH groups. The oxidation reaction is prevented by the presence of 1 mM dithiothreitol. We also present novel data showing that CSQ2 modifies the response of RyR2 to daunorubicin, but that the response of RyR2 is not dependent on daunorubicin binding to CSQ2. We suggest that binding of daunorubicin to RyR2 and CSQ2, and oxidation of RyR2, are all likely to contribute to anthracycline-induced cardiotoxicity during chemotherapy.

Alimentary induced fatty liver and adjuvant therapy with effective natural bioactive molecules

Changes of redox-homeostasis generate cytokines, and free radicals influence many intracellular signaling pathways in different liver diseases. Liophylised table beet and carrot powder (GPS Powder Kft. 1361/004/2003BFÁÉÉÁ) containing bioactive components such as betaine, betanins, betaxanthins, flavonoids, polyphenols, glutamine, beta carotene, vitamins and folic acid may produce changes various cellular pathways. Aim: The aim of this study was to determine the protecting effects of bioactive agents of the liophylised table beet and carrot powder on fatty liver in a “short term” experiment. Method: Male Wistar rats were fed with chow with or without high fat (2% cholesterol, 0.5% cholic acid, 20% sunflower oil) and treated with 0.1 or 1 g/bwkg/day natural product for ten days parallel with the feedings. Cyclooxygenase-2, inducible nitric oxide synthase and tumor necrosis factor-α mRNA levels were determined using molecular biologic methods. Free radicals, H-donating activity, reducing power and free SH-group concentrations were determined by luminometry and spectrophotometry. Mobilized methyl groups were assayed by over pressure liquid chromatography method in liver homogenates. Results: It was found that the higher dose of the natural product better decreased the induced free radical reactions, cyclooxygenase-2, inducible nitric oxide synthase and tumor necrosis factor-α mRNA-levels both in normal and fatty liver tissues. Although treatments failed to exert significant changes in all global antioxidant parameters, mobilized methyl group concentrations were higher after treatments in fatty liver. Favorable tendencies were also noted in the redox-homeostasis of the fatty liver after treatment. Conclusions: As expected, lyophylised table beet and carrot proved to be a “functional food” in rats with alimentary fat induced fatty liver. It cannot be ruled out that this beneficial effect may have clinical relevance. Orv. Hetil., 2011, 152, 1035–1042.

Novel 1,4 substituted piperidine derivatives. Synthesis and correlation of antioxidant activity with structure and lipophilicity.

A series of novel piperidine derivatives was prepared and their lipophilicity was determined (as RM values). These compounds as well as two intermediate alpha-keto-esters were tested for antioxidant activity. It was found that the cysteamine derivatives were efficient antioxidants, i.e. they could inhibit lipid peroxidation, act as hydroxyl radical scavengers and interact with 2,2-diphenyl-1-picrylhydrazyl radicals. This interaction could be attributed to the free SH group and this activity seemed to be favoured by increased lipophilicity. Replacement of SH by NH2 or OH resulted in a decreased antioxidant activity of the compounds. However, the described activities seem not to be connected with any O2-.scavenging ability, at least under the experimental conditions applied. Furthermore, cysteamine derivatives seem to induce O2-.generation, a phenomenon often observed with thiol compounds. The antioxidant activity of the intermediate alpha-keto-esters varied and is probably mediated by different mechanisms.

DBU-Catalyzed transprotection of N-Fmoc-cysteine di- and tripeptides into S-Fm-cysteine di- and tripeptides

The transprotection of N-Fmoc-cysteine containing di- and tripeptides possessing a free SH group to produce the corresponding S-Fm-cysteine di- and tripeptides bearing a free amino group is accomplished efficiently with DBU in dry THF. The N-Fmoc to S-Fm transformation mechanism is discussed. S-Fm-Cysteine di- and tripeptides readily form amide bonds on coupling with N-(Pg-α-aminoacyl)benzotriazoles and N-(Pg-α-dipeptidoyl)benzotriazoles to give larger peptides.

Effect of glucose oxidase and mixing time on soluble and insoluble wheat flour protein fractions: Changes on SH groups and H2O2 consumption

Glucose oxidase (GOX) has been recognized as an alternative to substitute chemical oxidants in bread making, however the action exerted on wheat flour proteins by the H2O2 produced is still unclear. Looking for a better understanding of the role of GOX and H2O2 in the bread making process, the effect of varying GOX levels and mixing time on soluble and insoluble wheat flour protein fractions was studied. Results showed that GOX promoted a decrement in free SH groups in both proteins, being the most evident effect on the soluble fraction at the beginning of the mixing process, remaining almost constant afterwards. H2O2 was continuously produced during mixing, showing its largest consumption during the first 6 min without significant SH changes afterwards. Results suggest that H2O2 must be involved in other reactions as it remains in the sample; this also suggests that oxygen is not a limiting factor in this reaction.

Biosynthesis and Control of Keratinase in Recalcitrant Feather-Degrading Bacillus megaterium F7-1

This study was performed to investigate the nutritional conditions controlling keratinase activity in Bacillus megaterium F7-1. B. megaterium F7-1 produced keratinase using chicken feather as a sole source of carbon, nitrogen and sulfur. Addition of the feather medium with glucose enhanced keratinase production (68.9 U/ml), compared to control without glucose (63.2 U/ml). The synthesis of keratinase was repressed by addition of <TEX>$NH_4Cl$</TEX> in B. megaterium F7-1. The highest keratinase production (70.9 U/ml) was obtained with the feather medium containing glucose and <TEX>$MgSO_4{\cdot}7H_2O$</TEX>. Keratinase was produced in the absence of feather (4.9 U/ml), indicating its constitutive synthesis. Feather degradation resulted in free SH group formation. B. megaterium F7-1 effectively degraded chicken feather meal (86%), whereas duck feather, human nail, human hair and sheep wool displayed relatively low degradation rates (8-34%).

Changes of protein solutions during storage: a study of albumin pharmaceutical preparations

During the production of air-filled albumin microspheres, to be used as an ultrasound contrast agent, it was observed that some albumin lots could not be used owing to albumin precipitation. In order to understand the reason for these lot-to-lot variations, 24 lots of 5% (w/v) human albumin pharmaceutical preparations were analysed. The results revealed that the good albumin lots all contained <0.03 mol of free SH groups per mol of albumin. The precipitation observed with other lots was most probably due to higher amounts of free SH groups. The lower amount of free SH groups in the good lots correlated with: (i) a yellow colour of the solutions and a UV-visible spectrum similar to that observed for non-enzymatic glycosylation; (ii) a decreased fructosamine content; (iii) an increased mobility against the anode in isoelectric focusing; and (iv) an increased truncation of the two N-terminal amino acids. No, or only small, differences were observed for the amounts of albumin dimer, albumin aggregates and protein impurities, and these could not account for the albumin precipitation. The differences observed between the albumin lots were most probably due to varying storage times and/or storage conditions, and incubation experiments revealed changes in all parameters that differed between the good and bad lots. Increasing the storage temperature or exposing the solutions to light resulted in a faster decrease of free SH groups and increase of the yellow colouration. It is likely that at least some of the changes observed were due to reactive degradation products formed from the stabilizer N-acetyl-L-tryptophan. The results presented should also be of interest regarding the storage of monoclonal antibodies and other proteins used in pharmaceuticals.

The effect of gaseous ozone treatment on egg components

Because of the salmonella problem in poultry, disinfection technologies are necessary. Ozone is a strong oxidant used for the disinfection of surfaces, drinking water and foods. However, since ozone not only destroys bacteria but may also damage eggs, it is necessary to clarify the effects of ozone treatment on hatching egg components. In this study, doses of gaseous ozone ranging from 10 to 50 mL L(-1) were tested. The vitamin A and E contents and fatty acid composition of the egg yolk were determined. To detect possible damage to the DNA of the germ disc, single-cell gel electrophoresis was used. Moreover, free SH groups were measured in the egg white. The soluble cuticula proteins were analysed by polyacrylamide gel electrophoresis. The yolk was not significantly affected by ozone treatment. However, the DNA of the germ disc was attacked and a significant decrease in free SH groups in the egg white was recorded at 50 mL L(-1) ozone. Even at low ozone doses the soluble cuticula proteins were completely destroyed. Significant alterations of egg components were caused by 50 mL L(-1) ozone. At lower ozone doses the oxidative processes occurred mainly at the egg surface and are therefore probably harmless to the developing embryo.

Physicochemical, functional and structural properties of vicilin-rich protein isolates from three Phaseolus legumes: Effect of heat treatment

The physicochemical, functional and structural properties of vicilin-rich protein isolates from kidney, red and mung beans (KPI, RPI and MPI) were investigated, and the influences of heating (at 95 °C for 30 min) were also evaluated and compared. In the untreated samples, the thermal stability, free SH contents and surface hydrophobicity were different. The differences seemed to be closely related to the differences in extent of aggregation and/or tertiary and secondary conformational structures among proteins. The heating resulted in extensive denaturation of the protein, significant decreases in free SH groups and increases in surface hydrophobicity, but to a varying extent, depending on the type of protein isolates. The protein solubility and emulsifying activities were significantly improved by the heating. The tertiary and secondary structures of these proteins were also to a various extent affected. The conformational structures of proteins in RPI were most flexible, and susceptible to the heating, while the proteins in KPI were most heat-stable in structures. The results clearly indicated close relationships between functional properties of these vicilin-rich protein isolates and their conformational structures.

Formation of nitrosothiols by the reaction of different forms of hemoglobin with (tetranitrosyl)bis(pyrimidin-2-ylthio)diiron

The reaction of hemoglobin (Hb), oxyhemoglobin (HbO2), and methemoglobin (metHb) with the tetranitrosyl iron complex of the fu2-S type [Fe2(SC4H3N2)2(NO)4] (1) was studied. The reaction results in the nitrosylation of the free SH group of 93-β-cysteine in these forms of hemoglobin. The change in the Hb, HbO2, and metHb concentrations was monitored by spectrophotometry, recording the difference absorption spectra of the experimental systems with these forms of hemoglobin and the buffer containing complex 1 in the same concentration. The absorption spectra were processed to obtain the components using the MATHCAD method. The nitrosothiol concentration was determined by the Saville reaction. In a protic medium containing 3.3% DMSO, complex 1 spontaneously generates NO due to hydrolysis (k = 3.7 · 10-4 s-1). Oxyhemoglobin reacts with evolved NO to form metHb. Complex 1 reduces metHb with a high rate to yield Hb (k = 6.7 · 10-3 s-1) followed by the formation of HbNO (k = 6.5 · 10-3 s-1). Oxidized complex 1 yields NO with a higher rate than the starting complex does. The reaction of HbO2 and metHb (0.02 mmo1 L-1) with complex 1 affords nitrosothiols in micromolar concentration during 5 min, and no nitrosothiol is formed in the case of Hb.