Formation Of Cystine Research Articles

Multilevel Framework for Analysis of Protein Folding Involving Disulfide Bond Formation.

In this study, a three-layered multicenter ONIOM approach is implemented to characterize the naive folding pathway of bovine pancreatic trypsin inhibitor (BPTI). Each layer represents a distinct level of theory, where the initial layer, encompassing the entire protein, is modeled by a general all-atom force-field GFN-FF. An intermediate electronic structure layer consisting of three multicenter fragments is introduced with the state-of-the-art semiempirical tight-binding method GFN2-xTB. Higher accuracy, specifically addressing the breaking and formation of the three disulfide bonds, is achieved at the innermost layer using the composite DFT method r2SCAN-3c. Our analysis sheds light on the structural stability of BPTI, particularly the significance of interlinking disulfide bonds. The accuracy and efficiency of the multicenter QM/SQM/MM approach are benchmarked using the oxidative formation of cystine. For the folding pathway of BPTI, relative stabilities are investigated through the calculation of free energy contributions for selected intermediates, focusing on the impact of the disulfide bond. Our results highlight the intricate trade-off between accuracy and computational cost, demonstrating that the multicenter ONIOM approach provides a well-balanced and comprehensive solution to describe electronic structure effects in biomolecular systems. We conclude that multiscale energy landscape exploration provides a robust methodology for the study of intriguing biological targets.

Ferrihydrite synthesis in the presence of amino acids and artificial seawater.

Ferrihydrite is widespread in clays, soils, and living organisms and was found on Mars. This iron-mineral could be found on the prebiotic Earth, which also contained simple monomeric amino acids. For prebiotic chemistry, it is important to understand how amino acids have an effect on the process of iron oxide formations. There are three important results in this work: (a) preconcentration of cysteine and aspartic acid, (b) formation of cystine and probably the cysteine peptide occurred during ferrihydrite syntheses, and (c) amino acids have an effect on iron oxide synthesis. For samples containing aspartic acid and cysteine, their presence on the surface or mineral structure can be confirmed by FT-IR spectra. Surface charge analysis showed a relatively high decrease for samples synthesized with cysteine. Scanning electron microscopy did not show marked morphological differences among the samples, except for the seawater sample containing cysteine, which had a lamina-shaped morphology surrounded by circular iron particles, indicating the possible formation of a cysteine structure involving iron oxide particles. The thermogravimetric analysis of the samples indicates that the presence of salts and amino acids in the synthesis of ferrihydrite has an effect on the thermal behavior of the iron oxide/amino acids and modifying the water-loss temperature. The heating of the cysteine samples, synthesized in distilled water and artificial seawater, showed several peaks of degradation of cysteine. In addition, heating of the aspartic acid samples produced the polymerization of this amino acid and peaks of degradation of it. FTIR spectra and XRD patterns did not indicate the precipitation of methionine, 2-aminoisobutyric acid, lysine, or glycine with the iron oxide formations. However, the heating of the glycine, methionine and lysine samples, synthesized in artificial seawater, showed peaks that could be attributed to the degradation of them. Then this could be an indication that these amino acids precipitate with the minerals during the syntheses. Also, the dissolution of these amino acids in artificial seawater prevents the formation of ferrihydrite.

Intermediate Detection in the Casiopeina-Cysteine Interaction Ending in the Disulfide Bond Formation and Copper Reduction.

A strategy to improve the cancer therapies involves agents that cause the depletion of the endogenous antioxidant glutathione (GSH), increasing its efflux out of cells and inducing apoptosis in tumoral cells due to the presence of reactive oxygen species. It has been shown that Casiopeina copper complexes caused a dramatic intracellular GSH drop, forming disulfide bonds and reducing CuII to CuI. Herein, through the determination of the [CuII]–SH bond before reduction, we present evidence of the adduct between cysteine and one Casiopeina as an intermediate in the cystine formation and as a model to understand the anticancer activity of copper complexes. Evidence of such an intermediate has never been presented before.

Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) detection as a rapid and convenient screening test for cystinuria

BackgroundCystinuria is an inborn congenital disorder characterised by a defective cystine metabolism resulting in the formation of cystine stones.The Brand’s test, used for diagnosis, requires dangerous substances, so has been replaced with high-performance liquid chromatography with fluorimetric detection (HPLC-FL). However, this technique requires the use of complex equipment.Infrared spectroscopy, universally used for stone analysis, recently was employed to detect insoluble cystine in urine. The aim of this study is to evaluate Infrared Spectroscopy combined with chemometric analysis as screening method to identify those patients requiring confirmation by HPLC-FL analysis. MethodsWe examined 24 h urine specimens from 57 patients. The quantitative analysis was performed by HPLC-FL. The infrared spectroscopic urine sediment analysis was performed with an ATR accessory (ATR-FTIR). Urine is centrifuged, the supernatant is discarded, and the sediment is dried on to the ATR prism surface.Statistical analysis was performed using a custom-made software developed in MATLAB environment. ResultsThe HPLC-FL determination showed a normal excretion of cystine in 49 samples and an abnormal excretion in the remaining 8 samples.The ATR-FTIR analysis combined with a statistical approach gives a sensitivity of 1.0 and a specificity of 0.82 were obtained. ConclusionsThe introduction of the ATR-FTIR technique in our clinical laboratory setting may reduce time and cost analysis for diagnosis of cystinuria.

3D interaction homology: Hydropathic interaction environments of serine and cysteine are strikingly different and their roles adapt in membrane proteins.

Atomic-resolution protein structural models are prerequisites for many downstream activities like structure-function studies or structure-based drug discovery. Unfortunately, this data is often unavailable for some of the most interesting and therapeutically important proteins. Thus, computational tools for building native-like structural models from less-than-ideal experimental data are needed. To this end, interaction homology exploits the character, strength and loci of the sets of interactions that define a structure. Each residue type has its own limited set of backbone angle-dependent interaction motifs, as defined by their environments. In this work, we characterize the interactions of serine, cysteine and S-bridged cysteine in terms of 3D hydropathic environment maps. As a result, we explore several intriguing questions. Are the environments different between the isosteric serine and cysteine residues? Do some environments promote the formation of cystine S–S bonds? With the increasing availability of structural data for water-insoluble membrane proteins, are there environmental differences for these residues between soluble and membrane proteins? The environments surrounding serine and cysteine residues are dramatically different: serine residues are about 50% solvent exposed, while cysteines are only 10% exposed; the latter are more involved in hydrophobic interactions although there are backbone angle-dependent differences. Our analysis suggests that one driving force for –S–S– bond formation is a rather substantial increase in burial and hydrophobic interactions in cystines. Serine and cysteine become less and more, respectively, solvent-exposed in membrane proteins. 3D hydropathic environment maps are an evolving structure analysis tool showing promise as elements in a new protein structure prediction paradigm.

INFLUENCE OF L-CYSTEINE AND N-ACETYL-L-CYSTEINE ON REDUCING ACTIVITY OF THIOUREA DIOXIDE IN AQUEOUS SOLUTIONS

The influence of L-cysteine and N-acetyl-L-cysteine on the stability and reducing activity of thiourea dioxide (NH2)2CSO2 (TDO) in the reaction with azo dye Orange II has been studied. The addition of L-cysteine and N-acetyl-L-cysteine leads to the increase in reducing activity of TDO in weakly acidic, neutral and weakly alkaline solutions, but does not influence its reducing activity in strongly alkaline solutions. In alkaline solutions, the rate of reduction of Orange II is increasing, but the influence of additives of thiol acids is decreasing. The activating effect of N-ace-tyl-L-cysteine is weaker than the effect of L-cysteine, that can be explained by the differences of pKa of these acids. Indeed, the reducing activity of thiols depends on the concentration of thiolate-ions in solutions. Since pKa of cysteine (8.30) is significantly less than pKa of N-acetyl-L-cysteine (9.52), concentration of thiolate-ions in cysteine solutions is higher. Therefore, reducing activity of L-cysteine is higher. The influence of L-cysteine and N-acetyl-L-cysteine on the reaction of TDO with Orange II is different from the influence of aminoacid glycine: in neutral solutions glycine decreases the rate of reaction. The interaction of TDO with L-cysteine in weakly acidic, neutral and weakly alkaline solutions is accompanied by the oxidation of L-cysteine to cysteinesulfenic acid and reduction of TDO to thiourea monoxide (NH2)2CSO. Then cysteinesulfenic acid reacts with L-cysteine with formation of cystine. The reaction studied here is the first example of reduction of TDO by sulfur-containing compounds.

Differential diagnostics and correction of metabolic and immunological disorders in patients with hepatic cirrhosis, complicated hepatosplenomegalia and portal hypertension

The study of the role of the spleen in the pathogenesis of liver cirrhosis is important for treatment. Currently available literature data indicate the role of altered spleen functions in the pathogenesis of liver cirrhosis. The aim of this study was to clarify the features of the etiology and pathogenetic disorders in patients with hepatosplenomegaly. Materials and methods. We investigated the uniform elements of blood and blood serum from 58 patients with hepatosplenomegaly amid cirrhosis, portal hypertension complicated. Patients were divided into two groups depending on the antigenic load: I - patients with liver cirrhosis against the background of hepatitis viruses HBV/HCV and II - patients with autoimmune hepatitis against the background of herpes viruses CMV/VEB. Treatment of patients included the use of antiherpetic immunoglobulins and hematopoietic cord blood progenitor cells. Results. In patients of group I was detected an increase in the spontaneous oxidative activity of neutrophils and depletion of the oxidative reserve in oxygen-dependent phagocytosis. It activated the signaling pathways of pro-inflammatory cytokines IL-6 (an increase of 60 times). We found an increased content of CD22+ -plasma B-lymphocyte that elevated synthesized concentrations of IgA. In patients of group II were found an increase in the activity of C3 and a decrease in the C4-components of complement; thrombocytopenia due to the presence of platelet autoantibodies; increased synthesis of immunoglobulins IgM, IgG, increased synthesis of IFN-. All patients showed disturbances in the metabolism of amino acids in various metabolic cycles - ornithine, which disrupted the formation of urea and indicates the accumulation of ammonia, which can enhance the cytotoxic effect in group I; methionine with a violation of the formation of cystine from cysteine and the accumulation of homocysteine, which enhances the synthesis of IFN-γ, which was increased in group II by 4 times. Conclusions. Some etiological and pathogenetic features of the formation of liver cirrhosis complicated by portal hypertension and recurrent bleeding from varicose veins of the esophagus were revealed. The immunological and metabolic changes verified by us correlated with the disappearance of the clinical manifestations of the disease, improved well-being, and the absence of recurrence of bleeding.

Association between cystine urolithiasis and neuter status of dogs within the UK

The objectives of the study were to examine the association between diagnosis of cystine urolithiasis and entire versus neutered status in male dogs and whether the strength of association varies among breeds. A previously reported canine urolithiasis database was used, documenting all urolith submissions to Hill's Pet Nutrition UK over a 10-year period. Uroliths were classified as cystine or non-cystine, and only male dogs with known neuter status were included in the analysis. Breeds of dog (and an additional crossbreed group), for which there was a minimum of 10 cystine urolith submissions, were analysed individually, with all other breeds combined together to form a reference group. Results were analysed using chi-squared and Fisher's exact tests. Logistic regression was used to assess associations between breed and neuter status and formation of cystine calculi. In multiple breeds, dogs with cystine uroliths were significantly more likely to be entire than dogs forming other types of urolith. Being an entire male, regardless of breed, was associated with an increased risk of cystine urolithiasis (odds ratio=4·5; 95% confidence interval: 3·22 to 6·37; P<0·001). Increased odds of cystine formation in entire dogs supports further investigation of castration as a method to prevent cystine urolithiasis.

Switching from immediate- to extended-release cysteamine in nephropathic cystinosis patients: a retrospective real-life single-center study.

Nephropathic cystinosis is a rare lysosomal storage disease which is characterized by the accumulation of free cystine in lysosomes and subsequent intracellular crystal formation of cystine throughout the body. If not treated with cysteamine, a cystine-depleting agent, end-stage renal disease will develop early, followed by multiple organ failure as the disease progresses. The established cysteamine formulation requires a strict dosing regimen at 6-h intervals. An extended release (ER) twice-daily formulation has recently been developed. The aim of our study was to evaluate the implementation and outcomes of this option in routine care. All pediatric cystinosis patients' records in Hannover Medical School were screened, and data on cysteamine therapy, tolerability, dosing, estimated glomerular filtration rates (eGFR), white blood cell cystine levels, and proton pump inhibitor (PPI) use were extracted for the period January 2014 to January 2016. The median age of the 12 patients enrolled in the study was 12.5 (range 1-18) years. At the end of the study period ten of these patients received ER-cysteamine. There were no additional side effects. Halitosis/bad breath was often subjectively judged as improved or eliminated, and PPI use could be stopped in one of three patients. The main reasons for switching to the ER formulation were difficult night-time administration and uncontrolled disease. Mean eGFR values remained stable with a median of 67ml/min/1.73m2 before and after the transition. White blood cell (WBC) cystine values remained low after the switch (1nmol/mg protein before and after transition; p = 0.64). In this single-center cohort, the switch from IR- to ER-cysteamine was safe and effective over the short term and provided advantages in terms of frequency of administration and less halitosis/bad breath. The long-term benefit of this option needs to be evaluated in future studies.

Electrochemical Studies of L-Cysteine

Introduction L-Cysteine is an important amino acid because of its role in various biochemical pathways (1). Considering that L-cysteine is one of the few amino acids with significant redox characteristics, it was considered worthwhile to investigate its electrochemical behavior in various media and electrode surfaces. Experimental L-Cysteine was obtained from Sigma-Aldrich. Electrochemical experiments were carried out using a Gamry Interface 1000 potentiostat and Gamry Framework software. Potentials are referenced against a Ag/AgCl reference electrode. Platinum and glassy carbon electrodes were obtained from BASi. Results and Discussion At a platinum electrode, L-cysteine in aqueous 0.10 M KNO3 produced a cyclic voltammogram with two merged oxidation processes (Figure 1), which were much more clearly resolved using differential pulse voltammetry. Preparative electrolysis at the potential of the first oxidation process (+0.80 V) resulted in the precipitation of cystine, which is the coupling product formed as a result of one-electron L-cysteine oxidation. The current for the reduction peak at -0.60 V (dotted line in Figure 1) increased during the electrolysis, suggesting that this reduction process is related to cystine formation. Similar results, including cystine formation, were also obtained in aqueous phosphate buffer at pH 7. It is likely that the platinum oxide layer on the electrode surface plays a role in L-cysteine oxidation under these conditions (2). The results of additional studies of L-cysteine oxidation in other media, particularly dimethylsulfoxide (DMSO), are also planned for presentation. References C. K. Mathews, K. E. Van Holde, D. R. Appling, and S. J. Anthony-Cahill, Biochemistry, 4th Edition, Pearson Canada, Toronto, 2013.J. Koryta and J. Pradac, J. Electroanal. Chem., 17, 185-189 (1968). Figure 1. Cyclic voltammogram of 10 mM L-cysteine at platinum in aqueous 0.10 M KNO3. 100 mV/s. First sweep (solid line), second sweep (dotted line). Figure 1

Substrate and pH-Dependent Kinetic Profile of 3-Mercaptopropionate Dioxygenase from Pseudomonas aeruginosa.

Thiol dioxygenases catalyze the synthesis of sulfinic acids in a range of organisms from bacteria to mammals. A thiol dioxygenase from the bacterium Pseudomonas aeruginosa oxidizes both 3-mercaptopropionic acid and cysteine, with a ∼70 fold preference for 3-mercaptopropionic acid over all pHs. This substrate reactivity is widened compared to other thiol dioxygenases and was exploited in this investigation of the residues important for activity. A simple model incorporating two protonation events was used to fit profiles of the Michaelis-Menten parameters determined at different pH values for both substrates. The pKs determined using plots of k(cat)/Km differ at low pH, but not in a way easily attributable to protonation of the substrate alone and share a common value at higher pH. Plots of k(cat) versus pH are also quite different at low pH showing the monoprotonated ES complexes with 3-mercaptopropionic acid and cysteine have different pKs. At higher pH, k(cat) decreases sigmoidally with a similar pK regardless of substrate. Loss of reactivity at high pH is attributed to deprotonation of tyrosine 159 and its influence on dioxygen binding. A mechanism is proposed by which deprotonation of tyrosine 159 both blocks oxygen binding and concomitantly promotes cystine formation. Finally, the role of tyrosine 159 was further probed by production of a G95C variant that is able to form a cysteine-tyrosine crosslink homologous to that found in mammalian cysteine dioxygenases. Activity of this variant is severely impaired. Crystallography shows that when un-crosslinked, the cysteine thiol excludes tyrosine 159 from its native position, while kinetic analysis shows that the thioether bond impairs reactivity of the crosslinked form.

The Cysteine-rich Region of Type VII Collagen Is a Cystine Knot with a New Topology

Collagens are a group of extracellular matrix proteins with essential functions for skin integrity. Anchoring fibrils are made of type VII collagen (Col7) and link different skin layers together: the basal lamina and the underlying connective tissue. Col7 has a central collagenous domain and two noncollagenous domains located at the N and C terminus (NC1 and NC2), respectively. A cysteine-rich region of hitherto unknown function is located at the transition of the NC1 domain to the collagenous domain. A synthetic model peptide of this region was investigated by CD and NMR spectroscopy. The peptide folds into a collagen triple helix, and the cysteine residues form disulfide bridges between the different strands. The eight cystine knot topologies that are characterized by exclusively intermolecular disulfide bridges have been analyzed by molecular modeling. Two cystine knots are energetically preferred; however, all eight disulfide bridge arrangements are essentially possible. This novel cystine knot is present in type IX collagen, too. The conserved motif of the cystine knot is CX3CP. The cystine knot is N-terminal to the collagen triple helix in both collagens and therefore probably impedes unfolding of the collagen triple helix from the N terminus.

The Structural Model of Salmonella typhimurium Ethanolamine Ammonia-Lyase Directs a Rational Approach to the Assembly of the Functional [(EutB–EutC)2]3 Oligomer from Isolated Subunits

Ethanolamine ammonia-lyase (EAL) is a 5'-deoxyadenosylcobalamin-dependent bacterial enzyme that catalyzes the deamination of the short-chain vicinal amino alcohols, aminoethanol and (S)- and (R)-2-aminopropanol. The coding sequence for EAL is located within the 17-gene eut operon, which encodes the broad spectrum of proteins that comprise the ethanolamine utilization (eut) metabolosome suborganelle structure. A high-resolution structure of the ∼500 kDa EAL [(EutB-EutC)₂]₃ oligomer from Escherichia coli has been determined by X-ray crystallography, but high-resolution spectroscopic determinations of reactant intermediate-state structures and detailed kinetic and thermodynamic studies of EAL have been conducted for the Salmonella typhimurium enzyme. Therefore, a statistically robust homology model for the S. typhimurium EAL is constructed from the E. coli structure. The model structure is used to describe the hierarchy of EutB and EutC subunit interactions that construct the native EAL oligomer and, specifically, to address the long-standing challenge of reconstitution of the functional oligomer from isolated, purified subunits. Model prediction that the (EutB₂)₃ oligomer assembly will occur from isolated EutB, and that this hexameric structure will template the formation of the complete, native [(EutB-EutC)₂]₃ oligomer, is verified by biochemical methods. Prediction that cysteine residues on the exposed subunit-subunit contact surfaces of isolated EutB and EutC will interfere with assembly by cystine formation is verified by activating effects of disulfide reducing agents. Angstrom-scale congruence of the reconstituted and native EAL in the active site region is shown by electron paramagnetic resonance spectroscopy. Overall, the hierarchy of subunit interactions and microscopic features of the contact surfaces, which are revealed by the homology model, guide and provide a rationale for a refined genetic and biochemical approach to reconstitution of the functional [(EutB-EutC)₂]₃ EAL oligomer. The results establish a platform for further advances in understanding the molecular mechanism of EAL catalysis and for insights into therapy-targeted manipulation of the bacterial eut metabolosome.

Kinetics and mechanism of the [RuIII(edta)(H2O)]−-mediated oxidation of cysteine by H2O2

The kinetics and mechanism of the [Ru(III)(edta)(H(2)O)](-)-mediated oxidation of cysteine (RSH) by hydrogen peroxide (edta(4-) = ethylenediaminetetraacetate), were studied in detail as a function of both the hydrogen peroxide and cysteine concentrations at pH 5.1 and room temperature. The kinetic traces reveal clear evidence for a catalytic process in which hydrogen peroxide reacts directly with cysteine coordinated to the Ru(III)(edta) complex in the form of [Ru(III)(edta)SR](2-). A parallel process in which [Ru(III)(edta)(H(2)O)](-) first reacts with H(2)O(2) to produce [Ru(V)(edta)O](-) and subsequently oxidizes cysteine, is orders of magnitude slower than the [Ru(III)(edta)(H(2)O)](-)-mediated oxidation in which cysteine rapidly coordinates to [Ru(III)(edta)(H(2)O)](-) prior to the reaction with H(2)O(2). HPLC product analyses revealed the formation of cystine (RSSR) as major product along with cysteine sulfinic acid (RSO(2)H) in the reaction system, and established the catalytic role of [Ru(III)(edta)(H(2)O)](-). Simulations were performed to account for the rather complex kinetic traces in terms of the suggested reaction mechanism. The results of the simulations support the proposed reaction mechanism that involves the oxidation of coordinated cysteine to cysteine sulfenic acid (RSOH), which subsequently rapidly reacts with H(2)O(2) and RSH to form RSO(2)H and RSSR, respectively.

Experimental and Computational Thermochemical Study of Sulfur-Containing Amino Acids: l-Cysteine, l-Cystine, and l-Cysteine-Derived Radicals. S−S, S−H, and C−S Bond Dissociation Enthalpies

This paper reports an experimental and theoretical study of the standard (p(degrees) = 0.1 MPa) molar enthalpies of formation at T = 298.15 K of the sulfur-containing amino acids l-cysteine [CAS 52-90-4] and l-cystine [CAS 56-89-3]. The standard (p(degrees) = 0.1 MPa) molar enthalpies of formation of crystalline l-cysteine and l-cystine were calculated from the standard molar energies of combustion, in oxygen, to yield CO2(g) and H2SO4.115H2O, measured by rotating-bomb combustion calorimetry at T = 298.15 K. The vapor pressures of l-cysteine were measured as function of temperature by the Knudsen effusion mass-loss technique. The standard molar enthalpy of sublimation, at T = 298.15 K, was derived from the Clausius-Clapeyron equation. The experimental values were used to calculate the standard (p(degrees) = 0.1 MPa) enthalpy of formation of l-cysteine in the gaseous phase, DeltafH(degrees)m(g) = -382.6 +/- 1.8 kJ x mol-1. Due to the low vapor pressures of l-cystine and since this compound decomposes at the temperature range required for a possible sublimation, it was not possible to determine its enthalpy of sublimation. Standard ab initio molecular orbital calculations at the G3(MP2)//B3LYP and/or G3 levels were performed. Enthalpies of formation, using atomization and isodesmic reactions, were calculated and compared with experimental data. A value of -755 +/- 10 kJ x mol-1 was estimated for the enthalpy of formation of cystine. Detailed inspections of the molecular and electronic structures of the compounds studied were carried out. Finally, bond dissociation enthalpies (BDE) of S-H, S-S, and C-S bonds, and enthalpies of formation of l-cysteine-derived radicals, were also computed.

Adsorption of cysteine on hematite, magnetite and ferrihydrite: FT-IR, Mössbauer, EPR spectroscopy and X-ray diffractometry studies

In the present paper, the adsorption of cysteine on hematite, magnetite and ferrihydrite was studied using FT-IR, electron paramagnetic resonance (EPR), Mössbauer spectroscopy and X-ray diffractometry. Cysteine was dissolved in artificial seawater (two different pHs) which contains the major constituents. There were two main findings described in this paper. First, after the cysteine adsorption, the FT-IR spectroscopy and X-ray diffractometry data showed the formation of cystine. Second, the Mössbauer spectroscopy did not show any increase in the amount of Fe(2+) as expected due the oxidation of cysteine to cystine. An explanation could be that Fe(2+) was oxidized by the oxygen present in the seawater or there occurred a reduction of cystine by Fe(2+) generating cysteine and Fe(3+). The specific surface area and pH at point of zero charge of the iron oxides were influenced by adsorption of cysteine. When compared to other iron oxides, ferrihydrite adsorbed significantly (p < 0.05) more cysteine. The pH has a significant (p < 0.05) effect only on cysteine adsorption on hematite. The FT-IR spectroscopy results showed that cystine remains adsorbed on the surface of the iron oxides even after being mixed with KCl and the amine and carboxylic groups are involved in this interaction. X-ray diffractometry showed no changes on iron oxides mineralogy and the following precipitated substances were found along with the iron oxides after drying the samples: cysteine, cystine and seawater salts. The EPR spectroscopy showed that cysteine interacts with iron oxides, changing the relative amounts of iron oxides and hydroxide.

One-Step Synthesis and Characterization of Ultrastable and Amorphous Fe3O4 Colloids Capped with Cysteine Molecules

A one-step sonochemical synthesis of a uniformly dispersed aqueous solution of amorphous Fe3O4 nanoparticles capped with dl-cysteine (cys) has been achieved. During the sonication, several processes or chemical reactions occurred: the decomposition of iron pentacarbonyl (Fe(CO)5), the formation of amorphous Fe3O4 nanoparticles, the capping of the Fe3O4 with cysteine molecules, the formation of a surface chemical bond of Fe−S between Fe3O4 particles and sulfur from cysteine, and the formation of cystine by linking two cysteine molecules. These capping cystine and cysteine molecules acted both as antioxidants and stabilizers, and the capped Fe3O4 colloids could be stable as long as 3 months at room temperature. Usually, the size of the amorphous Fe3O4 nanoparticles capped with Cys was found to be 20−30 nm. The products were characterized by a number of analytical techniques, such as XRD, DSC, TGA, XPS, TEM, HRTEM, Raman Spectroscopy, magnetic measurements, and spot tests.

Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors

P2X receptors are ATP-gated cation channels and assembled as homotrimers or heterotrimers from seven cloned subunits. Each subunit contains two transmembrane domains connected by a large extracellular loop. We have previously shown that replacement of two conserved residues, K68 and F291, by cysteine residues leads to disulfide cross-linking between neighbouring P2X(1) subunits. Since mutation of these residues results in a reduced ATP potency and cysteine cross-linking is prevented in the presence of ATP, we suggested an inter-subunit ATP binding site. To investigate whether the proximity of these residues is preserved in other P2X subtypes, we tested for spontaneous cystine formation between the corresponding P2X(2 )(K69C, F289C), P2X(3 )(K63C, F280C), and P2X(4 )(K67C, F294C) mutants upon pairwise expression in Xenopus laevis oocytes. Non-reducing SDS-PAGE analysis of the purified receptors revealed a specific dimer formation between P2X(2)K69C and P2X(2)F289C mutants. Likewise, co-expression of P2X(1)K68C and P2X(2)F289C, but not P2X(1)F291C and P2X(2)K69C, mutants resulted in dimer formation between the respective subunits. Cross-linked P2X(1/2) heteromers showed strongly reduced or absent function that was selectively recovered upon treatment with DTT. Cross-linking was less efficient between P2X(3) or P2X(4) mutants but could be enhanced by the short cysteine-reactive cross-linker MTS-2-MTS. These results show that the spatial proximity and/or orientation of residues analogous to positions K68 and F291 in P2X(1) are preserved in P2X(2) receptors and at one of two possible interfaces in heteromeric P2X(1/2) receptors but appears to be redundant for P2X(3) and P2X(4) receptor function.

Functional Interaction of Phospholipid Hydroperoxide Glutathione Peroxidase with Sperm Mitochondrion-associated Cysteine-rich Protein Discloses the Adjacent Cysteine Motif as a New Substrate of the Selenoperoxidase

The mitochondrial capsule is a selenium- and disulfide-rich structure enchasing the outer mitochondrial membrane of mammalian spermatozoa. Among the proteins solubilized from the sperm mitochondrial capsule, we confirmed, by using a proteomic approach, the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPx) as a major component, and we also identified the sperm mitochondrion-associated cysteine-rich protein (SMCP) and fragments/aggregates of specific keratins that previously escaped detection (Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J., and Flohé, L. (1999) Science 285, 1393-1396). The evidence for a functional association between PHGPx, SMCP, and keratins is further supported by the identification of a sequence motif of regularly spaced Cys-Cys doublets common to SMCP and high sulfur keratin-associated proteins, involved in bundling hair shaft keratin by disulfide cross-linking. Following the oxidative polymerization of mitochondrial capsule proteins, catalyzed by PHGPx, two-dimensional redox electrophoresis analysis showed homo- and heteropolymers of SMCP and PHGPx, together with other minor components. Adjacent cysteine residues in SMCP peptides are oxidized to cystine by PHGPx. This unusual disulfide is known to drive, by reshuffling oxidative protein folding. On this basis we propose that oxidative polymerization of the mitochondrial capsule is primed by the formation of cystine on SMCP, followed by reshuffling. Occurrence of reshuffling is further supported by the calculated thermodynamic gain of the process. This study suggests a new mechanism where selenium catalysis drives the cross-linking of structural elements of the cytoskeleton via the oxidation of a keratin-associated protein.

The electro-oxidative activity of cysteine on the Au electrode as evidenced by surface enhanced Raman scattering

It is demonstrated from the surface-enhanced Raman scattering that cysteine is adsorbed on the Au electrode via its carboxylate moiety. The mechanism is mainly via the charge transfer involving the π electron-rich carboxylate moiety. The adsorption of cysteine is weak that its diffusion on the Au surface is possible. The collision of two cysteine molecules may lead to the electro-oxidative formation of cystine in an irreversible way due to that the disulfide bond is not in the close vicinity of the electrode and its rupture is hampered. The physical difference as compared to its adsorption on the Ag electrode is stressed.