Reactive SH Groups Research Articles

Azospirillum baldaniorum improves acclimation, lipid productivity and oxidative response of a microalga under salt stress

There is a growing interest in using microalgae for a variety of purposes, including production of pharmaceuticals, wastewater treatment, CO2 capture from flue gases, and biomass as a feedstock for biofuels and feed/food supplements. In addition to environmental conditions, the local availability of suitable water for massive production microalgal biomass often compromises biomass productivity, and thus, techno-economic feasibility towards implementation. Although salinity tends to promote accumulation of carbon reserves in some freshwater microalgae, which could be advantageous for producing feedstocks for biofuels, the negative effect on growth usually off-sets this benefit, and results in a lower productivity of the target. The aim of this study was to analyse whether inoculation with a plant growth-promoting bacterium could ameliorate the detrimental effect of NaCl on growth of a microalga. We showed that inoculation with Azospirillum baldaniorum Sp245 improved the early response of Scenedesmus obliquus CS1 to NaCl addition, and its overall growth performance. As a result, co-cultures attained higher biomass and lipids productivities than microalgal axenic cultures. Lipid productivity raised up due to both: an increased biomass production and a higher lipids enrichment in the biomass. Similar results were obtained under constant light and temperature or under climate-simulated conditions in environmental photobioreactors (Phenometrics™). Inoculation with the bacterium lowered the levels of reactive oxygen species, SH groups, chlorophyll and carotenoids in the algal cells, as well as cell damage, induced by salt stress. Inoculation with Azospirillum strains genetically modified to produce contrasting levels of IAA, suggested that although under non-stress conditions most of the algal growth promotion is IAA-dependent, under salt stress other still-unidentified factor(s) might play a more prominent role in algal stimulation.This study increased the current understanding of the mechanisms underpinning bacterial-microalgal associations, and helps to design alternative strategies to increase algal productivity under suboptimal growth conditions, as most applications demand.

Modifying textural and functional characteristics of fish (Nemipterus japonicus) mince using high pressure technology.

Effect of high pressure in inducing textural and functional modification has been investigated in pink perch (Nemipterus japonicus) mince. Fish mince undergone pressurization at 200, 400 and 600MPa for a holding period of 10min and was compared against cooked mince (90°C; 40min). The treated mince at 400 and 600MPa lost its natural viscosity and behaved like cooked mince through denaturation and formation of protein aggregates. Textural characterisation showed the retention of tenderness in 200MPa treated samples, but become harder on application of higher pressures. Unlike heat gels, pressure induced gels were more smooth, white and elastic in nature. A decreased in reactive SH groups was observed in 400 and 600MPa treated samples due to the formation of disulfide bonds. Hydrophobic concentration was higher in cooked and 600MPa treatments whereas Ca2+-ATPase activity decreased after pressurization. On application of different pressures microbial reduction of 2-3 log cycles was achieved in the mince samples. Hence pressure treatments at lower ranges cannot alter the texture and functionality of protein and the mince can undergo processing as required. Besides extending shelf life, the treatments above 400MPa can make irreversible effect on texture quality and protein functionality which is similar to that of cooking.

Synergistic Effect of Enzyme Hydrolysis and Microwave Reactor Pretreatment as an Efficient Procedure for Gluten Content Reduction

In this study, we assessed the effects of microwave irradiation of wheat gluten proteins as a pretreatment performed in a microwave reactor that could accurately control process parameters as a function of power and temperature, as well as comparing it with conventional heat treatment. The aim was to identify suitable combinations of partial enzymatic hydrolysis and microwave pretreatment parameters to produce gluten hydrolysates with reduced allergenicity and conserved techno-functional features for food application. FTIR analysis, and total and reactive SH group contents confirmed that the microwave-controlled heating can significantly change the secondary structure and conformation of gluten protein. The microwave treatment had the largest effect at 200 W and 100 °C, at which the content of gluten has been reduced by about 2.5-fold. The microwave pretreatment also accelerated the enzymatic hydrolysis of gluten, changing the kinetic profile. The apparent hydrolysis rate constants (k2) were 1.00, 3.68, 3.48, 4.64 and 4.17 min−1 for untreated gluten, and those pretreated with microwave power of 200, 400, 600 and 800 W, respectively. Compared to the heat treatment, it appeared that microwave specific non-thermal effects had a significant influence on the gluten structure and allergenicity and, in combination with the enzymatic hydrolysis, ultimately yielded protein hydrolysates with enhanced antioxidant and functional properties.

Effect of Iron–Nitric Oxide Complexes on the Reactivity of Hemoglobin Cysteines

Human erythrocyte hemoglobin (Hb) has two reactive cysteines located on the surface of β-subunits. These cysteines play an important role in the adjustment of Hb functions. It is known that they are involved in the transport of intracellular nitric oxide (NO), redox signaling, and the regulation of dimeric-tetrameric Hb equilibrium. It is shown in this work that the incorporation of Cys-93β as ligands in iron-NO complexes is another way to regulate of SH group reactivity. Such complexes stabilize the SH group as a thiolate anion (R–S–), the reactivity of which is significantly higher than that of the protonated form of thiol (Cys-SH). This is why the thiols included in the complexes show increased activity in relation to electrophilic agents, such as ThioGlo1. Conversely, as part of the complexes, thiols are protected from oxidation by tert-butyl hydroperoxide. The incorporation of SH groups into complexes of iron and NO can be considered a means of thiol protection from irreversible oxidation upon oxidative stress.

Dysregulated hydrogen sulfide metabolism links aberrant neuronal stress responses and neurodegeneration in Huntington's disease

Hydrogen sulfide (H2S) is a gaseous signaling molecule that plays central roles in a myriad of physiological processes ranging from response to oxidative stress and transcriptional regulation. Hydrogen sulfide is synthesized endogenously from the semi‐essential amino acid cysteine via the reverse transsulfuration pathway. Thus disrupted cysteine metabolism may also lead to aberrant H2S signaling. One of the modes by which hydrogen sulfide signals is by a posttranslational modification designated as sulfhydration/persulfidation, which occurs on reactive cysteine residues on target proteins, where the reactive SH group is converted to an SSH group. Sulfhydration is a highly prevalent modification which modifies the structure and/or activity of target proteins. Research on H2S and persulfidation is still in its infancy. We show here that H2S is generated in response to stress stimuli such as Golgi stress and oxidative stress in neurons, which modulates sulfhydration levels to modulate signaling pathways. We further show that these cytoprotective stress responses are compromised in striatal cell culture model and the R6/2 and Q175 mouse models of Huntington’s disease (HD). We have shown previously that the biosynthetic enzyme for cysteine and H2S in neurons, cystathionine g‐lyase (CSE) is depleted in HD and mediates neurodegeneraton. HD is a neurodegenerative disease which is triggered by expansion of polyglutamine repeats in the protein huntingtin, which causes it to aggregate and elicit multi‐system toxicity. Restoring H2S balance in cells mitigates neuronal dysfunction in neurodegenerative diseases such as HD. A screen for regulators of the transsulfuration pathway has resulted in identification of compounds that modulate cysteine balance and disease progression in HD.Support or Funding InformationUnited States Public Health Service (USPHS) grants DA000266 and MH18501 to S.H.S.

Effects of different ultrasound power on physicochemical property and functional performance of chicken actomyosin

The aim of present study was to investigate the effect of different ultrasound power on physicochemical property and functional performance of chicken actomyosin (CAM). The treated CAM had the lowest particle size and the highest absolute zeta potential at 150 W. The conformation changes of treated CAM exhibited the reduction in α-helix as well as the improvement of fluorescence intensity. Ultrasound power at 150 W could significantly increase protein hydrophobicity and reactive SH groups compared to the others (P < 0.05). The disappearance of specific peaks and lower endothermic peaks implied that the treated CAM became thermally instable. The microstructure of CAM revealed small and homogeneous sub-bunches treated by 100–150 W. Furthermore, the solubility and emulsion property of CAM was significantly increased at 150 W (P < 0.05). These results demonstrated that ultrasound treatment at the power of 100–150 W may be an appropriate range to modify CAM for enhancing its functional properties.

Green synthesis with enhanced magnetization and life cycle assessment of Fe3O4 nanoparticles

Magnetic nanoparticles were synthesized by a novel eco-friendly method using glutathione as a reductant and stabilizer. A careful study of the life cycle in the synthesis process was performed to assess the environmental impact caused by the production of this material. From the results, it is possible to conclude that the environmental impact is much less significant than that of conventional routes, specifically the co-precipitation method with which it was compared. Nanoparticles with a saturation magnetization of 85.4 emu/g were obtained, which is similar to the maximum value obtained through the co-precipitation method. One of the main advantages of this method is the formation of magnetic nanoparticles with a coating of free and reactive SH groups, which offers interesting possibilities for its use in environmental remediation tasks, specifically in adsorbing heavy metals such as mercury.

Selective Chemical Labeling of Type II Cannabinoid Receptor CB2 for 19F-NMR and EPR Studies

Human cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) is implicated in an array of health related systemic body functions including the immune response, inflammation, and pain sensing, and it is an important target for pharmaceutical drug development. Structural and functional studies of GPCR by NMR, EPR and fluorescence techniques require non-intrusive, site-specific labeling of the protein. This can be achieved by chemical labeling of cysteines of the receptor. In this study we explored feasibility of labeling of selected cysteines by minimizing the number of reactive SH groups through systematic replacement of the majority of the 13 naturally-occurring cysteines of CB2. The replacement of the six water-exposed cysteines resulted in a well-expressed, fully functional receptor as evidenced by ligand binding- and G protein activation studies. While the replacement of four additional cysteines in the transmembrane domain of the protein lowered ligand binding affinity several fold, the receptor could still be activated by an excess of a high affinity agonists and performed competently in an in vitro G protein activation assay. The reduced cysteine templates of CB2 were either labeled directly or used to introduce new cysteines in selected positions at the extracellular and intracellular surfaces of the protein. The cysteine mutants of CB2 were chemically labeled with 2,2,2-trifluoroethanethiol (TET) and their spectra recorded by 19F NMR in micelles composed of dodecyl maltoside and CHAPS or in particles formed by the NVoy polymer. The latter was advantageous since it yielded monomeric receptor suitable for recording solution-state 19F NMR spectra with decent resolution. This study is a precursor for exploring the structure-function relationship of CB2 by NMR and EPR.

Changes in Some Biochemical Indices of Stability of Broiler Chicken Actomyosin at Different Levels of Sodium Bicarbonate in Presence and Absence of Sodium Chloride

This study examined some physicochemical properties of actomyosin isolated from pre-rigor chicken breast muscle (Pectoralis major) to gain insight on its interaction with 0.2–1.0M sodium bicarbonate (NaHCO3) either with or without 3% sodium chloride (NaCl). NaHCO3 has been a frequent quality raiser with respect to water holding capacity of meat, which is adversely affected at low pH. This study document a remarkable inactivation of ATPases and the weakening of actin-myosin interaction with increasing molarity of NaHCO3, irrespective of the presence or absence of NaCl. Similarly, there was a concomitant increase in turbidity of soluble fraction, increase in total protein solubility and reactive SH groups. The observations on protein solubility/insolubility are supported by SDS-PAGE profiles. Therefore, the conformational changes induced by NaHCO3 are concentration dependent and appear different from those during thermal transitions. The findings suggest that low concentration of NaHCO3 may be more suitable component of marinades, since it affects the main myofibrillar contractile complex less adversely.

Modification of placenta blood serum proteins under low temperature effect

Changes in environmental physical and chemical factors upon freeze-thawing and low temperature storage of biological samples can result in impairments of protein structures. This work specifies spontaneous and diamide-induced protein aggregations of placenta blood serum stored at -20 degrees and -196 degrees C during 2 years with SDS-PAGE. It was shown that storage of placenta blood serum at low temperatures did not cause any quantitative and qualitative changes in fraction distribution of proteins denatured with SDS in comparison to the native samples which were not frozen. Application of beta-mercaptoethanol revealed that placenta blood serum proteins upon freeze-thawing did not form spontaneous aggregates linked by disulphide bridges. Oxidation of amino acid sulfhydryl groups induced by diamide and accompanied by high molecular aggregate formation proved to be a quite effective way for indirect estimation of structural changes in protein upon low temperature effects. In samples thawed after low temperature storage the protein aggregation with 4 microM diamide was significantly higher than in native serum. These discrepancies between native and frozen-thawed samples are stipulated by impairments of protein structure under low temperature and increased in accessibility of reactive SH-groups of proteins for oxidation with diamide. Structural changes in placenta blood serum proteins, which caused by low temperatures and revealed by elevated sensibility to diamide-induced aggregate formation, did not depend on temperature (-20 degrees and -196 degrees C) and storage terms (2 years and 3 weeks). They reflect protein reaction to freeze-thawing processes and could be sequence of ice crystal formation which takes place in unprotected media.

Influence of the microenvironment of thiol groups in low molecular mass thiols and serum albumin on the reaction with methylglyoxal

Methylglyoxal (MG), a reactive α-oxoaldehyde that is produced in higher quantities in diabetes, uremia, oxidative stress, aging and inflammation, reacts with the thiol groups (in addition to the amino and guanidino groups) of proteins. This causes protein modification, formation of advanced glycated end products (AGEs) and cross-linking. Low molecular mass thiols can be used as competitive targets for MG, preventing the reactions mentioned above. Therefore, this paper investigated how the microenvironment of the thiol group in low molecular mass thiols (cysteine, N-acetylcysteine (NAcCys), carboxymethylcysteine (CMC) and glutathione (GSH)) and human serum albumin (HSA) affected the thiol reaction with MG. The SH group reaction course was monitored by 1H-NMR spectroscopy and spectrophotometric quantification. Changes in the HSA molecules were monitored by SDS-PAGE. The microenvironment of the SH group had a major effect on its reactivity and on the product yield. The reactivity of SH groups decreased in the order Cys > GSH > NAcCys. CMC did not react. The percentages of the reacted SH groups in the equilibrium state were almost equal, regardless of the ratio of thiol compound/MG (1:1, 1:2, 1:5): 38.1 ± 0.9%; 38.2 ± 0.7% and 39.0 ± 0.8% for Cys; 26.5 ± 0.6%; 26.6 ± 2.6% and 27.4 ± 2.5% for GSH; 10.8 ± 0.9%; and 11.2 ± 0.7% and 12.2 ± 0.9% for NAcCys, respectively. Our results explain why substances containing α-amino-β-mercapto-ethane as a pharmacophore are successful scavengers of MG. In equilibrium, HSA SH reacted in high percentages both with an insufficient amount and with an excess of MG (55% and 65%, respectively). An analysis of the hydrophobicity of the microenvironment of the SH group on the HSA surface showed that it could contribute to high levels of SH modification, leading to an increase in the scavenging activity of the albumin thiol.

Investigation of Physical−Chemical Properties of Agarose Hydrogels with Embedded Emulsions

Composite agarose hydrogels with embedded tetradecane emulsions stabilized by cetylpyridinium chloride were studied. The absorption efficiency of 4-nitrophenyl ethers of carbonic acids by the composite agarose gels increases with the length of the hydrocarbon tail of the ester. The diffusion rate of amphiphilic substances in the composite gels was demonstrated to be much less that than in the standard agarose gels. The reaction kinetics between the esters and dodecylmercaptan dissolved in tetradecane droplets of composite hydrogel was studied. In the region of physiological pH, the reactivity of SH groups embedded in the composite agarose gel in the reaction with the esters is significantly higher than that in a homogeneous solution. Hydrogels with embedded emulsion droplets are of considerable practical importance as drug delivery systems, microreactors, and absorbers. Composite gels filled with emulsions incorporating lipophilic mercaptanes are effective absorbers of heavy metal ions.

Spectroscopic evaluation of surface functionalization efficiency in the preparation of mercaptopropyltrimethoxysilane self-assembled monolayers on glass

Four synthetic protocols have been examined for the preparation of a self-assembled monolayer of propanethiol functions on glass surfaces, through the wet reaction of mercaptopropyltrimethoxysilane (MPTS) on glass slides, in order to determine an optimal method to obtain dense SH monolayers for further reaction with maleimide-bearing molecules. We have also demonstrated that the surface density of reactive SH groups can be indagated by the coupling reaction with a maleimide-functionalized rhodamine dye and by the examination of the absorption spectra of the glass slides. The reaction of MPTS in dry toluene gives the most dense surface of reactive thiols, among the examined protocols. We have also evidenced that thermal curing of the MPTS functionalized slides induces thiol coupling and S S formation, resulting in a dramatically lowered availability of SH groups.

Nucleotide-induced and actin-induced structural changes in SH1-SH2-modified myosin subfragment 1

We compared the structural properties of myosin subfragment 1 (S1) modified at both reactive SH-groups, SH1 (Cys707) and SH2 (Cys697), with the properties of unmodified S1 and SH1-modified S1. It is shown using differential scanning calorimetry (DSC) that SH1 modification has no noticeable influence on the changes in S1 thermal unfolding induced by the formation of S1 ternary complexes with ADP and P(i) analogs (V(i), AlF(4)(-), and BeF(x)). These changes, however, normally expressed in a significant increase of S1 thermal stability, are almost fully prevented by modification of both SH1 and SH2. In contrast, SH2 modification had no effect on the changes induced by the formation of the ternary complexes S1-ADP-V(i), S1-ADP-AlF(4)(-), and S1-ADP-BeF(x) in EPR spectra of S1 spin-labeled at SH1 group. Interaction of S1 with F-actin substantially increased the thermal stability of S1; a similar effect was observed by DSC with both SH1- and SH1-SH2-modified S1. Overall, our results demonstrate that modification of both reactive SH-groups on S1 has no influence on the actin-induced changes of S1 and on the local nucleotide-induced conformational changes in the SH1 group region, but strongly prevents the global nucleotide-induced structural changes in the entire S1 molecule. The results suggest that modification of SH1 and SH2 impairs the spread of nucleotide-induced conformational changes from the ATPase site throughout the structure of the entire S1 molecule, thus disturbing a coupling between the motor and regulatory domains in the myosin head.

Properties of Edible Surimi Film as Affected by Heat Treatment of Film-Forming Solution

The effect of heat treatment of film-forming solutions on the properties of edible surimi films was investigated. The film-forming solutions prepared at pH 3 from frozen Alaska pollack surimi were heated to 45, 70 or 100°C to promote unfolding of surimi protein molecules. As a result, solubility, surface hydrophobicity, and reactive SH group of surimi proteins increased. After 45°C -treatment, the mechanical properties, film solubility, and protein solubility of surimi films were not affected and myosin heavy chain (MHC) of surimi proteins was degraded by endogenous acid proteinases. Conversely, at higher heating temperatures (70°C, 100°C), degradation of MHC was effectively inhibited and mechanical properties were improved, while the film solubility and protein solubility of surimi films decreased. It is revealed that the prevention of MHC degradation by heat treatment could improve mechanical properties of surimi films. The optimum condition was found to be heating the film-forming solutions (pH 3) at 70°C for 20min.

A Study on p-Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens

Conventional methods to prepare the apoenzyme of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens yield an inhomogeneous apoenzyme with respect to the reconstitutable activity. To overcome this problem the holoenzyme was bound covalently to a Sepharose–5,5′-dithio-bis(2-nitrobenzoate) column via the reactive sulfhydryl group of the enzyme. The prosthetic group FAD was removed at neutral pH by addition of a high concentration of KBr and urea to the elution buffer. The holoenzyme can be reconstituted while the apoenzyme is still bound to the column or the apoenzyme can be isolated in the free state. The yield of apoenzyme was between 90% and 95% of the starting enzyme. The degree of reconstitution of holoenzyme is better than 95% of the original activity. The apoenzyme is stable for a long period of time as an ammonium sulfate precipitate. The relative molecular mass of the apoenzyme is 43000 per polypeptide chain. The apoenzyme exists mainly as a dimer in solution. During the preparation of the apoenzyme the substrate p-hydroxybenzoate protects the apoenzyme from inactivation, indicating complex formation. The dissociation constant for the apoenzyme-substrate complex was determined to be about 30 μM. The apoenzyme also forms a complex with NADPH. The dissociation constant (∼ 12 μM) of this complex is about a factor of ten smaller than that of the holoenzyme (∼ 120 μM). The dissociation constant of the apoenzyme-NADPH complex is strongly dependent on pH and ionic strength of the solution. The dissociation constant of the holoenzyme into its constituents is 45 nM. The association rate constant calculated from kinetic experiments is 4.1 × 104 M−1 s−1. The calculated dissociation rate constant is 1.85 × 10−3 s−1, indicating that the holoenzyme possesses a high stability which is governed by the low dissociation rate constant. The method of preparation of apoenzyme can also be used to separate enzyme molecules where the reactive SH group has been oxidized by air from enzyme molecules still containing the original SH group. This separation technique is in particular useful when quantitative labelling of the SH group is required.

Critical Redox and Allosteric Aspects of Nitric Oxide Interactions with Hemoglobin

Nitric oxide (NO) is an important signaling molecule. Relatively long-lived NO adducts at the heme and SH groups of hemoglobin (Hb) could enable NO to carry out long-range signaling functions. In spite of significant advances, there remain as yet unresolved issues regarding the possible role of Hb in moderating NO-signaling events that affect blood pressure regulation. In this review, we summarize recent reports concerning the redox and allosteric aspects of NO/Hb interactions that have advanced our understanding of the physiological significance of NO binding to heme groups (forming NO-Hb) and of reactions promoting formation of S-nitrosated Hb (SNO-Hb). Allosteric mechanisms modify the bioactivity of NO/Hb complexes by altering the lifetime of NO-Hb and the properties of SNO-Hb. Redox reactions are significant because of the complex chemistry possible for NO and its oxidation products. Reactions at ferrous and ferric heme sites have differing consequences and affinities for interactions with NO. Moreover, redox changes at heme groups affect reactivity of SH groups and vice versa. In spite of low levels of NO-Hb and SNO-Hb found in vivo, recent findings do not rule out participation of NO-Hb or SNO-Hb in NO-dependent signaling reactions.

Highly reactive cysteine residues are part of the substrate binding site of mammalian dipeptidyl peptidases III

Dipeptidyl peptidase III (DPP III) is a cytosolic zinc-exopeptidase involved in the intracellular protein catabolism of eukaryotes. Although inhibition by thiol reagents is a general feature of DPP III originating from various species, the function of activity important sulfhydryl groups is still inadequately understood. The present study of the reactivity of these groups was undertaken in order to clarify their biological significance. The inactivation kinetics of human and rat DPP III by sulfhydryl reagent p-hydroxy-mercuribenzoate ( pHMB) was monitored by determination of the enzyme’s residual activity with fluorimetric detection. Inactivation of this human enzyme exhibited pseudo-first-order kinetics, suggesting that all reactive SH-groups have equivalent reactivity, and the second-order rate constant was calculated to be 3523±567 M −1 min −1. Rat DPP III was hyperreactive to pHMB and showed biphasic kinetics indicating two classes of reactive SH-groups. The second-order rate constants of 3540 M −1 s −1 for slower reacting sulfhydryl, and 21,855 M −1 s −1 for faster reacting sulfhydryl were obtained from slopes of linear plots of pseudo-first-order constants versus reagent concentration. Peptide substrates protected both mammalian DPPs III from inactivation by pHMB. Physiological concentrations of biological thiols and H 2O 2 inactivated the rat DPP III. Human enzyme was resistant to H 2O 2 attack and less affected by reduced glutathione (GSH) than the rat homologue. A significantly lower DPP III level, determined by activity measurement and Western blotting, was found in the cytosols of highly oxygenated rat tissues. These results provide kinetic evidence that cysteine residues are involved in substrate binding of mammalian DPPs III.

S-nitrosation of Cys-800 of HIF-1α protein activates its interaction with p300 and stimulates its transcriptional activity

Hypoxia inducible factor 1 (HIF-1) is a heterodimeric transcriptional complex that plays pivotal role in the regulation of cellular utilization of oxygen as well as glucose and is an essential regulator of angiogenesis in solid tumor and ischemic disorders. Recently HIF-1α, a subunit of HIF-1 complex, was characterized as a potential target for S-nitrosation, providing no information about the impact of this posttranslational modification on the protein transactivation. Cys-800 of HIF-1α protein has reactive SH-group, which is critical for the recruitment of p300 co-activator that is necessary for transcriptional activity of HIF-1 complex. Here we report that S-nitrosation of Cys-800 activates HIF-1α–p300 interaction and stimulates protein transactivation. We have found that S-nitrosation of the HIF-1α C-terminal domain by nitric oxide derived from donors and nitric oxide synthase increases protein transcriptional activity. The increase of HIF-1 transcriptional activity was not observed in the case of Cys-800 substitution to Ala, though other protein thiol groups were nitrosated. Experiments with GST pull-down assay suggest that S-nitrosation of Cys-800 stimulates the recruitment of p300 co-activator protein to the HIF-1α C-terminal domain.

Coagulation of Zinc-modified Hemoglobin

At a sufficiently high concentration of bovine oxyhemoglobin, the effect Zn(II) ions exert on its coagulation compares with that of Hg(II) ions. It is suggested that the center of preferential stoichiometric binding with Zn(II) ions in the protein is the sulfur atom of reactive SH groups. However, the binding of Zn(II) ions with groups other than thiolic equally affects the protein aggregation. Analysis of the pH dependence of the protein aggregation rate showed that the most likely alternative binding centers are histidine residues of the protein.