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βcysteine 93 residue plays a key role in oxygen (O2)-linked conformational changes in the hemoglobin (Hb) molecule. This solvent accessible residue is also a target for binding of thiol reagents that can remotely alter O2 affinity, cooperativity, and Hb's sensitivity to changes in pH. In recent years, βCys93 was assigned a new physiological role in the transport of nitric oxide (NO) through a process of S-nitrosylation as red blood cells (RBCs) travel from lungs to tissues. βCys93 is readily and irreversibly oxidized in the presence of a mild oxidant to cysteic acid, which causes destabilization of Hb resulting in improper protein folding and the loss of heme. Under these oxidative conditions, ferryl heme (HbFe4+), a higher oxidation state of Hb is formed together with its protein radical (.HbFe4+). This radical migrates to βCys93 and interacts with other "hotspot" amino acids that are highly susceptible to oxidative modifications. Oxidized βCys93 may therefore be used as a biomarker of oxidative stress, reflecting the deterioration of Hb within RBCs intended for transfusion or RBCs from patients with hemoglobinopathies. Site specific mutation of a redox active amino acid(s) to reduce the ferryl heme or direct chemical modifications that can shield βCys93 have been proposed to improve oxidative resistance of Hb and may offer a protective therapeutic strategy.

Aquatic invertebrates vary in methylmercury (MeHg) levels among systems which has been attributed, in part, to environmental conditions, but may also be linked to differences in their biochemical composition. As MeHg is known to bind to thiol-containing amino acids such as cysteine in proteins of fish, our objective was to determine if these amino acids explain MeHg variability among aquatic invertebrate taxa. Benthic macroinvertebrates from diverse functional feeding groups and bulk zooplankton were collected from six acidic lakes in Kejimkujik National Park, Nova Scotia, Canada, and analyzed for MeHg, cysteine (as cysteic acid), methionine (as methionine sulfone), and nitrogen (relative trophic level, δ 15 N) and carbon (carbon source, δ 13 C) isotopes. MeHg was significantly and positively related to cysteine or methionine in zooplankton, caddisfly and stonefly tissues (R 2 from 0.24 to 0.57). In addition, methionine or cysteine in combination with δ 15 N and/or δ 13 C were better predictors of MeHg levels in stoneflies, mayflies, caddisflies and zooplankton among these lakes (R 2 adj = 0.25–0.91). Overall, these novel findings suggest that the variability in MeHg of aquatic invertebrates can be explained, in part, by their tissue levels of thiol-containing amino acids.

Electrochemical oxidation of l ‑cysteine resulted in the formation of a film on glassy carbon electrode. In a solution of levodopa ( l ‑DOPA), l ‑tyrosine (Tyr) and uric acid (UA), three separated intense anodic peaks were appeared in differential pulse voltammetry regime. Experimental conditions were optimized for simultaneous determination of the three compounds. All experiments were carried out in phosphate buffer solution (0.1 M, pH 8). Calibration curves were obtained in the presence of various concentrations of l ‑DOPA, Tyr, and UA. Linear concentration ranges were 0.65–22 μM for l ‑DOPA, 3.5–96 μM for Tyr, and 1.0–19 μM for UA. The limits of detection (LODs) were calculated as 0.2, 1.1 and 0.36 μM for l ‑DOPA, Tyr, and UA, respectively. The electrochemical sensor was used successfully for the simultaneous determination of l ‑DOPA, Tyr, and UA species in human blood serum samples.

The rate at which taurine is synthesized in cells is unclear. This study reports the rate constants for taurine, hypotaurine, and other precursor molecules with hydrogen peroxide and superoxide. Raman spectroscopy permitted direct observation of reactions between hydrogen peroxide and the sulfinate and dithiol precursors of taurine. No observable reaction occurred between hydrogen peroxide and the sulfonates taurine or cysteate. Superoxide reacts with hypotaurine, taurine, and cysteate, although hypotaurine engages in rapid side reactions with a tetrazolium dye. Superoxide-produced radical intermediates for hypotaurine and taurine reacted with the nitroxyl radical-containing molecule TEMPONE. Hypotaurine oxidation by superoxide is calculated to occur at a rate sufficient to produce intracellular concentrations of taurine in humans. Hypotaurine's and taurine's reactions as antioxidants are predicted to occur at a fraction of the rate of enzyme-based antioxidant systems, but they may reach similar rates when hypotaurine is present at millimolar concentration in an intracellular compartment.

We examined the performance of an ultra-high performance liquid chromatography method to quantify protein-bound sulphur amino acids in zooplankton. Both cysteic acid and methionine sulfone were linear from 5 to 250 pmol (r2 = 0.99), with a method detection limit of 13 pmol and 9 pmol, respectively. Although there was no matrix effect on linearity, adjacent peaks and co-eluting noise from the invertebrate proteins increased the detection limits when compared to common standards. Overall, performance characteristics were reproducible and accurate, and provide a means for quantifying sulphur amino acids in aquatic invertebrates, an understudied group.

Brown human hair was bleached three times (45 min × 3) and four times (45 min × 3 + 15 min) with commercial formulations containing persulfate salts and hydrogen peroxide. The hair samples were characterized by Raman and IR spectroscopy in the Attenuated Total Reflectance (ATR) mode to gain more insights into the possible secondary structure and Cα-Cβ-S-S-Cβ-Cα conformational changes induced by bleaching. The latter were evaluated through band-fitting procedures; the relative content of the disulfide bridges and oxidized sulfur species (cysteic acid, Bunte salt, cystine oxides) was assessed. The observed conformational changes could be significant in developing restoring agents to be used after hair decoloration. The use of two different spectroscopic techniques allowed to discriminate the information coming from the cortical region of hair (Raman) and the cuticle (ATR/IR). This article refers to “Structural investigation on damaged hair keratin treated with α,β-unsaturated Michael acceptors used as repairing agents” (Di Foggia et al., Int. J. Biol. Macromol. 167 (2021) 620–632 https://doi.org/10.1016/j.ijbiomac.2020.11.194 ).

A new facile, rapid, sensitive and selective colorimetric assay is proposed for the determination of manganese ions (Mn 2+ ) utilizing cysteic acid (CA)-capped silver nanoparticles (CA-AgNPs) as colorimetric probes. The CA-AgNPs were prepared by reducing AgNO 3 with NaBH 4 in the presence of CA as the capping ligand. The presence of Mn 2+ induces the quick aggregation of CA-AgNPs, associated with notable color changes of the CA-AgNPs solution from yellow to dark green. The Mn 2+ can form a coordinated structure with CA capping on the AgNPs and leads to formation of large particles aggregation. We also used density functional theory (DFT) to calculate the change of the Gibbs free energy (ΔG) of the interactions between the CA-AgNPs and various metal ions, which shows that CA-AgNPs have high specificity for Mn 2+ . The high sensitivity and selectivity for Mn 2+ were achieved and the detection limit is as low as 5 nM. Furthermore, the proposed method was successfully applied in detecting Mn 2+ in a rat model of focal ischemia and the results indicate that our proposed method has great potential for practical applications.

Au nanoparticles (AuNPs) were electrodeposited onto glassy carbon electrode (GCE) and used as the scaffold to combine with l -cysteine (L-Cys) through Au S bonding, and polypyrrole (PPy) films templated with L-Cys were obtained by in situ polymerization of pyrrole (Py) on the L-Cys self-assembled monolayers. The L-Cys templates were removed via two successive steps: (a) electrooxidation of L-Cys templates to l -cysteic acid; (b) removal of l -cysteic acid through electrostatic repulsion between l -cysteic acid and overoxidized PPy (OPPy). The molecularly imprinted OPPy-AuNPs were applied for chiral electrochemical recognition of Cys enantiomers for the first time, and the recognition efficiency was superior to that at the molecularly imprinted OPPy. The enhanced recognition efficiency was attributed to the existence of AuNPs, which could adsorb L-Cys templates effectively and increase the number of recognition sites. Various important experimental factors influencing the performance of the molecularly imprinted OPPy-AuNPs were also investigated in the present work.

A novel peptide (Cya-Phe-Leu-Ala-Pro, SCP) was formulated through non-protein amino acid-cysteic acid (Cya) modification of collagen peptide (Phe-Leu-Ala-Pro, CP) from Acaudina molpadioides. Introduction of this Cya showed remarkable improvement in the scavenging activities of OH·. SCP exhibited stronger effects than CP in preventing H2O2-induced oxidative damage due to lower levels of ROS and MDA, and higher activities of antioxidant enzymes, such as SOD, GSH-Px, HO-1, and NQO1. It was speculated that SCP could significantly increase the expression level of Nrf2 compared to CP, thereby activating the expression of downstream ARE genes. The expression levels of p38 in the upstream pathway to regulate Nrf2 content were significantly higher in both the CP and SCP-treated groups, while a higher level of JNK was observed only in the SCP-treated groups. The present study provided insights towards the application of cysteic acid modified peptide in protecting cell from oxidative damage through the JNK/Nrf2 pathway.

Background Identifying changes in serum metabolites before the occurrence of acute myocardial infarction (AMI) is an important approach for finding novel biomarkers of AMI. Methods In this prospective cohort study, serum samples obtained from patients at risk of AMI (n = 112) and non-risk controls (n = 89) were tested using high-resolution metabolomics (HRM). Partial least-squares discriminant analysis (PLS-DA), along with univariate analysis using a false discovery rate (FDR) of q = 0.05 were performed to discriminate metabolic profiles and to determine significantly different metabolites between healthy control and AMI risk groups. Results PLS-DA significantly separated the AMI risk sera from control sera. The metabolites associated with amino acid biosynthesis, 2-oxocarboxylic acid, tryptophan, and amino sugar and nucleotide sugar metabolism pathways were mainly elevated in patients at risk of AMI. Further validation and quantification by MS/MS showed that tryptophan, carnitine, L-homocysteine sulfinic acid (L-HCSA), and cysteic acid (CA) were upregulated, while L-cysteine and L-cysteine sulfinic acid (L-CSA) were downregulated, specifically among AMI risk sera. Additionally, these discriminant metabolic profiles were not related to hypertension, smoking or alcoholism. Conclusion In conclusion, detecting upregulated L-HCSA and CA along with carnitine among patients at risk for AMI could serve as promising non-invasive biomarkers for early AMI detection.

Introduction Our objectives were to evaluate the long-term renal function after radical cystectomy (RC) and ileal conduit diversion (ICD) and to analyze year-by-year the estimated glomerular filtration rate (eGFR) and morphologic upper urinary tract changes. Patients and Methods We retrospectively identified 226 patients who had undergone RC and ICD from 1980 to 2008, with regular postoperative follow-up visits. The eGFR was calculated using the Modification of Diet in Renal Disease equation at baseline and during follow-up. A decrease in renal function was defined as > 1 mL/min/1.73 m 2 annually. Results The median follow-up period after RC was 91 months (range, 61-235 months). The median eGFR decreased from 66 mL/min/1.73 m 2 (range, 17-139 mL/min/1.73 m 2 ) to 59 mL/min/1.73 m 2 (range, 33-102 mL/min/1.73 m 2 ). A rapid decline in renal function occurred during the first 2 postoperative years (−9 mL/min/1.73 m 2 and −4 mL/min/1.73 m 2 in the first and second year, respectively), with a moderate to slight decrease in the subsequent years. Urinary obstruction was diagnosed in 51 patients (23%). Among the patients who underwent prompt surgical treatment, we did not find any association with the eGFR decline ( P = .8). Conclusion Patients with urinary ICD have a lifelong risk of chronic kidney disease. Regular monitoring of renal function and the morphologic upper urinary tract will permit early diagnosis and treatment of modifiable factors, avoiding irreversible kidney damage.

In order to investigate in detail the influence of chemical treatments (reduction, hydrolyzed eggwhite protein (HEWP) treatment, and oxidation) on damaged hair keratin fibers, the structure of cross-sections at various depths of excessively bleached (damaged) black human hair resulting from a permanent waving process was directly analyzed using Raman spectroscopy. It was found that l -cysteine (CYS) largely reacted with the gauche-gauche-gauche (GGG) conformation of disulfide (–SS–) groups (while CYS did not react with the trans-gauche-trans (TGT) conformation). In particular, not only the GGG content, but also the cysteic acid content existing throughout the cortex region of the excessively bleached human hair remarkably decreased by performing the oxidation process after reduction. On the other hand, the GGG content of the excessively bleached black human hair increased, while the TGT content decreased by performing the oxidation process after reduction and then HEWP treatment processes. From these experiments, the authors concluded that some of the keratin associated protein (KAP), which has a rich –SS– content and cysteic acid content was eluted from the cortex region along with the disconnection of –SS– groups, thereby leading to the remarkable reduction in the reconnection of –SS– groups of the excessively bleached black human hair after the permanent waving process (the reduction and oxidation processes). Also, the authors concluded that the HEWP treatment process in the permanent waving process caused the reconstruction of the KAP, thereby contributing to the acceleration of the reconnection of –SS– groups during the oxidation process.

The assessment of quantification results of the alcohol abuse marker ethyl glucuronide (EtG) in hair in comparison to the cut-off values for the drinking behavior may be complicated by cosmetic hair bleaching. Thus, the impact of increasing exposure to hydrogen peroxide on the EtG content of hair was investigated. Simultaneously, the change of absorbance in the range of 1000-1100 cm(-1) indicative for the oxidation of cystine was investigated non-destructively by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) using pulverized portions of the respective hair samples. Hair samples treated with hydrogen peroxide consistently displayed a significantly increased absorbance at 1040 cm(-1) associated with the formation of cysteic acid. The EtG content decreased significantly if the hair was treated with alkaline hydrogen peroxide as during cosmetic bleaching. It could be shown that ATR-FTIR is capable of detecting an exposure to hydrogen peroxide when still no brightening was visible and already before the EtG content deteriorated significantly. Thus, hair samples suspected of having been exposed to oxidative treatment may be checked non-destructively by a readily available technique. This assay is also possible retrospectively after EtG extraction and using archived samples.

In order to investigate in detail the internal structure changes in virgin black human hair keratin fibers resulting from bleaching treatments, the structure of cross-sections at various depths of black human hair, which had been impossible due to high melanin grande content, was directly analyzed using Raman spectroscopy. The gauche – gauche – gauche (GGG) content of the SS groups existing from the cuticle region to the center of cortex region of the virgin black human hair remarkably decreased, while the gauche – gauche – trans and trans – gauche – trans contents were not changed by performing the excessive bleaching treatment. In particular, it was found that not only the β-sheet and/or random coil content, but also the α-helix content existing throughout the cortex region of virgin black human hair decreased. In addition, the transmission electron microscope observation shows that the proteins in the cell membrane complex, the cuticle and cortex of the virgin black human hair were remarkably eluted by performing the excessive bleaching treatment. From these experiments, the author concluded that the SS groups, which have a GGG conformation were decomposed and finally converted to cysteic acid, and the α-helix structure of some of the proteins existing in the keratin was changed to the random coil structure, or eluted from the cortex region, thereby leading to the reduction in the protein density of the virgin human hair after the excessive bleaching treatment.

A novel cysteic acid modified carbon paste electrode (cysteic acid/CPE) based on electrochemical oxidation of l -cysteine was developed to simultaneously determine ofloxacin and gatifloxacin in the presence of sodium dodecyl benzene sulfonate (SDBS). Fourier transform infrared spectra (FTIR) indicated that l -cysteine was oxidated to cysteic acid. Electrochemical impedance spectroscopy (EIS) and cyclic voltammograms (CV) indicated that cysteic acid was successfully modified on electrode. The large peak separation (116 mV) between ofloxacin and gatifloxacin was obtained on cysteic acid/CPE while only one oxidation peak was found on bare electrode. And the peak currents increased 5 times compared to bare electrode. Moreover, the current could be further enhanced in the presence of an anionic surfactant, sodium dodecyl benzene sulfonate. The differential pulse voltammograms (DPV) exhibited that the oxidation peak currents were linearly proportional to their concentrations in the range of 0.06–10 μM for ofloxacin and 0.02–200 μM for gatifloxacin, and the detection limits of ofloxacin and gatifloxacin were 0.02 μM and 0.01 μM (S/N = 3), respectively. This proposed method was successfully applied to determine ofloxacin and gatifloxacin in pharmaceutical formulations and human serum samples.

A chiral electrochemical sensor has been prepared via cysteic acid modified glassy carbon (CyA-GC) electrode to discriminate tyrosine (Tyr) enantiomers. Sweep cycles of CyA-GC in modification process and pH of solution were investigated to obtain optimum experimental conditions. Electrochemical impedance spectroscopy was employed to characterize the electrical conductivity of the modified electrode. For the first time, different oxidation peak potentials (Ep) of l- and d-Tyr were observed in the differential pulse voltammograms obtained in the solution containing l- or d-Tyr. Further study showed that only one peak appeared in mixture solution containing l- and d-Tyr and Ep of that peak shifted positively and linearly with an increasing percentage of l-isomer of Tyr racemic mixture. Keywords: Chiral electrochemical recognition, Tyrosine, Cysteic acid, Modified electrode

This manuscript concerns the tissue-specific transcription of mouse and cattle glutamate decarboxylase-like protein 1 (GADL1) and the biochemical activities of human GADL1 recombinant protein. Bioinformatic analysis suggested that GADL1 appears late in evolution, only being found in reptiles, birds, and mammals. RT-PCR determined that GADL1 mRNA is transcribed at high levels in mouse and cattle skeletal muscles and also in mouse kidneys. Substrate screening determined that GADL1, unlike its name implies, has no detectable GAD activity, but it is able to efficiently catalyze decarboxylation of aspartate, cysteine sulfinic acid, and cysteic acid to β-alanine, hypotaurine, and taurine, respectively. Western blot analysis verified the presence of GADL1 in mouse muscles, kidneys, C2C12 myoblasts, and C2C12 myotubes. Incubation of the supernatant of fresh muscle or kidney extracts with cysteine sulfinic acid resulted in the detection of hypotaurine or taurine in the reaction mixtures, suggesting the possible involvement of GADL1 in taurine biosynthesis. However, when the tissue samples were incubated with aspartate, no β-alanine production was observed. We proposed several possibilities that might explain the inactivation of ADC activity of GADL1 in tissue protein extracts. Although β-alanine-producing activity was not detected in the supernatant of tissue protein extracts, its potential role in β-alanine synthesis cannot be excluded. There are several inhibitors of the ADC activity of GADL1 identified. The discovery of GADL1 biochemical activities, in conjunction with its expression and activities in muscles and kidneys, provides some tangible insight toward establishing its physiological function(s).

Redox catalytic process involved in the paired electrosynthesis of l -cysteine and l -cysteic acid from l -cystine is investigated by cyclic voltammetric technique and also confirmed by preparative electrolysis. The cyclic voltammetric behaviour shows that in the catholyte, in situ deposited tin (Sn) surface acts as a redox catalyst for the electro-reduction of l -cystine to l -cysteine whereas in the anolyte, the electro-generated bromine acts as a homogeneous redox mediator to enhance the electro-oxidation of l -cystine. l -Cysteine hydrochloride monohydrate ( l -cysteine) and l -cysteic acid are prepared from l -cystine by preparative electrolysis with high purity and high yield using graphite cathode and DSA anode. At optimum concentration of l -cystine with 1:1 concentration ratio (catholyte:anolyte), the material yield obtained for l -cysteine is above 80% and that for l -cysteic acid is close to 60% in the paired electrosynthesis process in the batch operation. Scope for further experiments in conversion efficiency is also discussed.

The effect on primary, secondary, tertiary and quaternary structure of Pseudozyma (formerly Candida) antarctica lipase B (PalB) on exposure to hydrogen peroxide was investigated using nano-electrospray ionization-mass spectrometry (nano-ESI-MS), liquid chromatography tandem mass spectrometry (LC/MS/MS), circular dichroism (CD), and dynamic light scattering (DLS). Treatment with hydrogen peroxide generated heavier protein variants, with a mass gain that increased with increasing incubation time. Furthermore, elevated concentration of H(2)O(2) was shown to result in partial fragmentation of the protein. Proteolytic digestion of the enzyme gave primary sequence coverage of more than 90%, revealing oxidation of methionine, tryptophan and cystine residues. The active site histidine was not observed in oxidized form in any of the experiments. However, oxidation of cystine to cysteic acid indicated disruption of disulphide bridges, and CD evaluations confirmed that severe changes to the secondary structure towards random coil had occurred. The structural changes could be an effect of the observed amino acid side chain oxidations, and was correlated with deactivation of the lipase. From DLS experiments, it was seen that the lipase exposed to both high temperature and H(2)O(2) formed large and intermediate sized aggregates, not observed for the heat-treated enzyme. The findings reported here could lay the basis for developing enzyme variants with higher oxidative stability.

Recent developments in the technique of combinatorial peptide ligand libraries, for enhancing the visibility of the low-abundance proteome, are reviewed here. Novel en bloc elution systems, allowing essentially complete proteome recovery in a single step, are reported here, particularly, en bloc elution with 3–5% boiling sodium dodecyl sulphate (SDS) or in urea–thiourea–CHAPS added with either 40 mM formic acid or 25 mM cysteic acid. Novel capturing systems are also discussed: in particular, although capturing at pH 7.2 in physiological saline has always been recommended, it is shown that capturing also at acidic (pH 3.8) and alkaline (pH 9.5) values substantially increments the total captured protein population. Some examples of detection of novel proteins by the described methodology are also discussed. In particular, in the case of venom proteins, where essentially all components had been detected and fully described by conventional means, the application of the ligand library technology allowed the discovery of two, previously unreported, trace enzymes necessary for the maintenance of the native structure of venom components, namely peroxiredoxin and glutaminyl cyclase. In the case of the red blood cell (RBC) cytoplasmic proteome, where a grand total of 1570 components of the 2% minority proteomes have been identified, these findings allowed to unravel the genetic defect of a rare RBC disease, called congenital dyserythropoietic anemia type II. The mutations are located in the SEC23B gene coding for the SEC23B protein, detected for the first time in the RBC proteome thanks to the peptide capturing technology.

The electrochemical oxidation of L–cysteine (CySH) in presence of carbon nanotubes (CNTs) formed a composite film at a glassy carbon electrode (GCE) as a novel modifier for directly electroanalytical determination of terbinafine without sample pretreatment in biological fluid. The determination of terbinafine at the modified electrode with strongly accumulation was studied by differential pulse voltammetry (DPV). The peak current obtained at + 1.156 V (vs. SCE) from DPV was linearly dependent on the terbinafine concentration in the range of 8.0 × 10 − 8 –5.0 × 10 − 5 M in a B–R buffer solution (0.04 M, pH 1.81) with a correlation coefficient of 0.998. The detection limit (S/N = 3) was 2.5 × 10 − 8 M. The low-cost modified electrode showed good sensitivity, selectivity, and stability. This developed method had been applied to the direct determination of terbinafine in human serum samples with satisfactory results. It is hopeful that the modified electrode will be applied for the medically clinical test and the pharmacokinetics in future.

Hydrogen peroxide (H2O2) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H2O2 to highly purified human hemoglobin (HbA0) induced structural changes that primarily resided within β subunits followed by the internalization of the heme moiety within α subunits. These modifications were observed when an equal molar concentration of H2O2 was added to HbA0 yet became more abundant with greater concentrations of H2O2. Mass spectrometric and amino acid analysis revealed for the first time that βCys-93 and βCys-112 were oxidized extensively and irreversibly to cysteic acid when HbA0 was treated with H2O2. Oxidation of further amino acids in HbA0 exclusive to the β-globin chain included modification of βTrp-15 to oxyindolyl and kynureninyl products as well as βMet-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the β subunits as a result of the H2O2 attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two α-peptide fragments (α128 - α139) and a heme moiety with the loss of iron, cross-linked between αSer-138 and the porphyrin ring. The novel oxidative pathway of HbA0 modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute.

The effect of H(2)O(2) on smooth muscle heavy meromyosin (HMM) and subfragment 1 (S1) was examined. The number of molecules that retained the ability to bind ATP and the actinactivated rate of P(i) release were measured by single-turnover kinetics. H(2)O(2) treatment caused a decrease in HMM regulation from 800- to 27-fold. For unphosphorylated and phosphorylated heavy meromyosin and for S1, approximately 50% of the molecules lost the ability to bind to ATP. H(2)O(2) treatment in the presence of EDTA protected against ATPase inactivation and against the loss of total ATP binding. Inactivation of S1 versus time correlated to a loss of reactive thiols. Treatment of H(2)O(2)-inactivated phosphorylated HMM or S1 with dithiothreitol partially reactivated the ATPase but had no effect on total ATP binding. H(2)O(2)-inactivated S1 contained a prominent cross-link between the N-terminal 65-kDa and C-terminal 26-kDa heavy chain regions. Mass spectral studies revealed that at least seven thiols in the heavy chain and the essential light chain were oxidized to cysteic acid. In thiophosphorylated porcine tracheal muscle strips at pCa 9 + 2.1 mM ATP, H(2)O(2) caused a approximately 50% decrease in the amplitude but did not alter the rate of force generation, suggesting that H(2)O(2) directly affects the force generating complex. Dithiothreitol treatment reversed the H(2)O(2) inhibition of the maximal force by approximately 50%. These data, when compared with the in vitro kinetic data, are consistent with a H(2)O(2)-induced loss of functional myosin heads in the muscle.

Carbon nanotubes (CNTs) and cysteic acid based on electrochemical oxidation of l -cysteine (CySH) to form a novel composite thin film material at a glassy carbon electrode (GCE) for electroanalytical determination of nimesulide. The determination of nimesulide at the composite modified electrode with strong accumulation of nimesulide was studied by differential pulse voltammetry (DPV). The peak current obtained at +1.251 V (versus SCE) from DPV was linearly dependent on the nimesulide concentration in the range of 1.0 × 10 −7 –1.0 × 10 −5 M in 0.05 M H 2 SO 4 solution with a correlation coefficient of 0.997. The detection limit (S/N = 3) was found to be 5.0 × 10 −8 M. The low-cost modified electrode showed good sensitivity, selectivity, stability and had been applied to the determination of nimesulide in pharmaceutical formulation and human serum samples with satisfactory results.

The methyl-accepting chemotaxis proteins are a family of receptors in bacteria that mediate chemotaxis to diverse signals. To explore the plasticity of these proteins, we have developed a simple method for selecting cells that swim to target attractants. The procedure is based on establishing a diffusive gradient in semi-soft agar plates and does not require that the attractant be metabolized or degraded. We have applied this method to select for variants of the Escherichia coli aspartate receptor, Tar, that have a new or improved response to different amino acids. We found that Tar can be readily mutated to respond to new chemical signals. However, the overall change in specificity depended on the target compound. A Tar variant that could detect cysteic acid still showed a strong sensitivity to aspartate, indicating that the new receptor had a broadened specificity relative to wild-type Tar. Tar variants that responded to phenylalanine or N-methyl aspartate, or that had an increased sensitivity to glutamate showed a strong decrease in their response to aspartate. In at least some of the cases, the maximal level of sensitivity that was obtained could not be attributed solely to substitutions within the binding pocket. The new tar alleles and the techniques described here provide a new approach for exploring the relationship between ligand binding and signal transduction by chemoreceptors and for engineering new receptors for applications in biotechnology.

We used a proteomic approach to identify proteins that associate with keratins 8 or 18 (K8/K18) in a pervanadate-dependent manner. Pervanadate triggers Ran-K8/K18 binding and a gel-migration-shift of Ran from 25 to 27 kDa, which does not occur upon exposure to H2O2 or vanadate or if pervanadate is excluded during cell solubilization. Generation of 27-kDa Ran is not related to hyperphosphorylation, is heat-insensitive, but occurs upon conversion of Ran cysteines to cysteic acid. The pervanadate-mediated Ran cysteine --> cysteic acid oxidation and its related gel migration shift affects other proteins including actin. Mutation of the three Ran cysteines (Cys-85, -112, and -120) showed that Ran Cys-112 oxidation generates 27-kDa Ran and accounts for its keratin binding. Proteasome inhibition accentuates Ran-keratin binding after cell exposure to pervanadate. Therefore, cell-free exposure to pervanadate causes cysteine to cysteic acid oxidation of Ran and several other proteins and Ran-K8/K18 association. In cells, stabilization of oxidized Ran by proteasome inhibition promotes Ran-keratin interaction. Keratin sequestration of oxidized Ran may provide a back-up protective mechanism in some cases of oxidative injury.

It was found out, the condition that polyaniline (PANI) formed selectively on wool. PANI was selectively formed on wool in the case of polymerization of aniline, using potassium iodate (KIO 3 ), as an oxidizing agent in the presence of wool. IR spectrum of PANI–wool composite formed, using KIO 3 , showed the presence of cysteic acid units (Cy SO 3 H) resulted from the oxidation of cystine bonds (Cy S S Cy) in wool. Selective formation of PANI on wool was considered to occur by a concentration of aniline on wool bearing cysteic acid units. It became apparent by IR analysis of oxidized wool that the oxidized layer of the wool textiles was thicker and cystine bonds in wool were converted into more cysteic acids by oxidation, using KIO 3 than using (NH 4 ) 2 S 2 O 8 and K 2 Cr 2 O 7 . Therefore, formation of PANI on wool occurred remarkably in the case of polymerizing aniline, using KIO 3 .

Site-specific chemical modification is a useful technology in characterisation of proteins, but the number of chemical probes of the protein structure reacting with proteins under mild conditions in aqueous solutions is rather limited. Here we studied the reaction of osmium tetroxide, 2,2′-bipyridine (Os,bipy) with several peptides using capillary zone electrophoresis (CZE) and matrix-assisted laser desorption-ionisation–time-of-flight mass spectrometry (MALDI–TOF MS). Both techniques showed formation of a stable complex of Os,bipy with tryptophan residues. In CZE peaks with different migration times and UV-Vis spectra were observed. MALDI–TOF MS showed the formation of a product with characteristic isotopic pattern corresponding to the presence of osmium atom. Oxidation of cysteine and methionine side chains to cysteic acid and methionine sulfone by Os,bipy was detected by CZE and confirmed by MALDI–TOF and post-source decay (PSD) mass spectra. PSD showed specific shifts of molecular weights of the peptides and their fragments after the derivatisation. We believe that Os,bipy may become a useful agent in the characterisation of proteins.

The proteomics analysis reported here shows that a major cellular response to oxidative stress is the modification of several peroxiredoxins. An acidic form of the peroxiredoxins appeared to be systematically increased under oxidative stress conditions. Peroxiredoxins are enzymes catalyzing the destruction of peroxides. In doing so, a reactive cysteine in the peroxiredoxin active site is weakly oxidized (disulfide or sulfenic acid) by the destroyed peroxides. Cellular thiols (e.g. thioredoxin) are used to regenerate the peroxiredoxins to their active state. Tandem mass spectrometry was carried out to characterize the modified form of the protein produced in vivo by oxidative stress. The cysteine present in the active site was shown to be oxidized into cysteic acid, leading to an inactivated form of peroxiredoxin. This strongly suggested that peroxiredoxins behave as a dam upon oxidative stress, being both important peroxide-destroying enzymes and peroxide targets. Results obtained in a primary culture of Leydig cells challenged with tumor necrosis factor α suggested that this oxidized/native balance of peroxiredoxin 2 may play an active role in resistance or susceptibility to tumor necrosis factor α-induced apoptosis.

Glial cell hyperpolarization previously has been reported to be induced by high frequency stimulation or glutamate. We now report that it also is produced by the glutamate-containing dipeptide N-acetylaspartylglutamate (NAAG), by its non-hydrolyzable analog beta-NAAG, and by NAAG in the presence of 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), a potent inhibitor of the NAAG degradative enzyme glutamate carboxypeptidase II. The results indicate that NAAG mimics the effect of nerve fiber stimulation on the glia. Although glutamate has a similar effect, the other presumed product of NAAG hydrolysis, N-acetylaspartate, is without effect on glial cell membrane potential, as is aspartylglutamate (in the presence of 2-PMPA). The hyperpolarization induced by stimulation, glutamate, NAAG, beta-NAAG, or NAAG plus 2-PMPA is completely blocked by the Group II metabotropic glutamate receptor antagonist (S)-alpha-ethylglutamate but is not altered by antagonists of Group I or III metabotropic glutamate receptors. The N-methyl-D-aspartate receptor antagonist MK801 reduces but does not eliminate the hyperpolarization generated by glutamate, NAAG or stimulation. These results, in combination with those of the preceding paper, are consistent with the premise that NAAG could be the primary axon-to-glia signaling agent. When the unstimulated nerve fiber is treated with cysteate, a glutamate reuptake blocker, there is a small hyperpolarization of the glial cell that can be substantially reduced by pretreatment with 2-PMPA before addition of cysteate. A similar effect of cysteate is seen during a 50 Hz/5 s stimulation. From these results we suggest that glutamate derived from NAAG hydrolysis appears in the periaxonal space under the conditions of these experiments and may contribute to the glial hyperpolarization.

Four acidic, isoelectric buffers, for peptide and protein separations, have been recently described and adopted in capillary zone electrophoresis: cysteic acid [Cys-A, isoelectric point (pI) 1.85], iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). These four buffers allow to explore an acidic portion of the titration curves of macroions, covering about 1.6 pH units (from pH 1.85 to ca. 3.45), thus permitting resolution of compounds having coincident titration curves at a given pH value. Given the rather acidic pI values of these buffers, their long-term stability has been investigated, by monitoring pH and conductivity changes upon increasing storage times. When dissolved in plain water, all four buffers appear to give constant pH and conductivity readings up to 15 days; after that, the conductivity keeps steadily increasing in a similar fashion. The same parameters, when the same buffers are dissolved in 6 M urea, appear to be stable for only one week, with the conductivity progressively augmenting after this period. A similar behaviour is exhibited by histidine (pI 7.70), a neutral, isoelectric buffer adopted for separation of DNA fragments. By mass spectrometry, Cys-A shows minute amounts (ca. 1%) of a degradation product after ageing for 3 weeks; in the same time period, Glu is extensively degraded (20%). No degradation species could be detected in IDA and Asp solutions. It is additionally shown that the acidic buffers are not quite stationary in the electric field, but can be transported at progressively higher rates (according to the pI value) from the cathodic to the anodic vessel. This is due to the fact that, at their respective pI values, a fraction of the amphotere has to be negatively charged in order to provide counterions to the excess of protons due to bulk water dissociation. Guidelines are given for the proper use and storage of such buffers.

The flavoenzyme d -aspartate oxidase from beef kidney (DASPO, EC 1.4.3.1) has been overexpressed in Escherichia coli. A purification procedure, faster than the one used for the enzyme from the natural source (bDASPO), has been set up yielding about 2 mg of pure recombinant protein (rDASPO) per each gram of wet E. coli paste. rDASPO has been shown to possess the same general biochemical properties of bDASPO, except that the former contains only FAD, while the latter is a mixture of two forms, one active containing FAD and one inactive containing 6-OH-FAD (9–20% depending on the preparation). This results in a slightly higher specific activity (about 15%) for rDASPO compared to bDASPO and in facilitated procedures for apoprotein preparation and reconstitution. Redox potentials of −97 mV and −157 mV were determined for free and l -(+)-tartrate complexed DASPO, respectively, in 0.1 M KPi, pH 7.0, 25°C. The large positive shift in the redox potential of the coenzyme compared to free FAD (−207 mV) is in agreement with similar results obtained with other flavooxidases. rDASPO has been used to assess a possible oxidative activity of the enzyme towards a number of compounds used as agonists or antagonists of neurotransmitters, including d -aspartatic acid, d -glutamic acid, N-methyl- d -aspartic acid, d,l -cysteic acid, d -homocysteic acid, d,l -2-amino-3-phosphonopropanoic acid, d -α-aminoadipic acid, d -aspartic acid-β-hydroxamate, glycyl- d -aspartic acid and cis-2,3-piperidine dicarboxylic acid. Kinetic parameters for each substrate in 50 mM KPi, pH 7.4, 25°C are reported.

We propose a system of protein sequence analysis with dl differentiation using capillary electrophoresis (CE). This system consists of a protein sequencer and a CE. After fractionation of phenylthiohydantoin (PTH)-amino acids from the protein sequencer, optical resolution for each PTH-amino acid is performed by CE using some chiral selectors such as digitonin, o -trimethyl-β-cyclodextrin (TM-β-CD) and others. In addition, optical resolution of all standard PTH- dl -amino acids including PTH- dl -carboxymethyl-Cys (CM-Cys) and cysteic acid (CYA) except for PTH- dl -Lys was successfully developed. The resolution of PTH- dl -Lys could not be reconfirmed due to low reproducibility and the impurities. As a model peptide, [ d -Ala 2 ]-methionine enkephalin ( l -Tyr– d -Ala–Gly– l -Phe– l -Met), was used and the sequence with dl differentiation was completely determined.

After reviewing previous attempts to use the extent of amino acid racemisation (AAR) for the determination of the age of archaeological samples containing proteins, the authors describe their own approach. Following an optimised protein hydrolysis with low racemisation the d - and l -amino acid content in fossil bone samples of known age (radiocarbon method) was determined by HPLC after precolumn derivatisation. Based on the obtained half-lives of racemisation and plotting the d / l ratio as a function of time for various amino acids, calibration curves are obtained which can be used for the age determination of fossil bone samples in the range of 2000–500 000 years. Another method is presented for the determination of age of textiles in the range of 100–1800 years. This method is based on the determination by an amino acid analyser of the age-dependent alteration of amino acid composition of proteins. Cystine, methionine and tyrosine content decreases, while cysteic acid content increases with age. Prediction equations were developed as linear regressions of the age of wool based on cysteic acid, cystine and tyrosine contents.

Plasma amino acids were analysed in patients with migraine with (9) and without (80) aura, in patients with tension headache (14) and in controls (62). The neuroexcitatory amino acids glutamic acid, glutamine, glycine, cysteic acid and homocysteic acid were elevated in migraine patients while total thiols (cysteine/cystine) were reduced. Patients with tension headache had values which were similar to those of controls. Tryptophan was elevated in migraine patients without aura only. Studies on two patients showed that the raised resting excitatory amino acid levels became still further elevated during a migraine attack. These results show that high concentrations of neurotransmitter amino acids occur normally in migraine patients and suggest that this profile may be a contributory factor in migraine attacks. Tension headache, however, has different biochemical parameters.

A possible mechanism for the formation of cysteine, cysteic acid and partially oxidized cystine species in keratin following exposure to UVC radiation at 254 nm is proposed. The primary photolysis products are the radical ions RSSR'' and RSSR'-, and it is suggested that partially oxidized cystine species are formed from reactions of the radical cation only. The mechanisms m is supported by Fourier transform IR (FT-IR) and optical reflectance spectroscopy of UVC-treated wool exposed to air, oxygen and nitrogen atmospheres, and by previous electron spin resonance (ESR) studies on disulphides exposed to both UV and ionizing radiation. The mechanism supports the Symons assignment for the sulphur radical “species X” as [RS(SR)SR]', rather than RSS'. Exposure of wool to less energetic UV radiation produces different products via a quenching mechanism involving the formation of the radical anion RSSR'- only, which precludes the formation of oxidized cystine intermediates. The formation of S-sulphonate residues at longer wavelengths suggests that a competing singlet oxygen process may also occur.

Recent studies have demonstrated that interactions between cysteic acid and arginine residues have a marked effect on the low energy decompositions of protonated peptides in the gas phase. This work extends these findings to show that analogous interactions operate in the presence of other acidic amino acid residues, although the effects are less pronounced. Thus, the presence of aspartic or glutamic acid residues (X) in the sequence, RLXIFSXFR, attenuates the proton-sequestering properties of the arginine residues in the [M+2H]2+ ion, thereby promoting charge-proximal fragmentation of the peptide backbone. Significant differences in fragmentation behavior are observed for the doubly protonated peptide incorporating two aspartic acid residues, in comparison with the glutamic acid-containing analogue. Low energy collisional activation of [M+2H]2+ ions of RLDIFSDFR, but not RLEIFSEFR, yields significant d-type ions associated with cleavage at the acidic residues. The proposed mechanism invokes arginine/aspartic acid side-chain interaction and is blocked by the additional methylene group in the glutamic acid side-chain. b-Type fragmentation is promoted C-terminal to the aspartic acid residues; this is attributed to nucleophilic attack of the side-chain carboxyl group on the carbonyl carbon of the adjacent peptide bond and is promoted by an interaction between the acidic side chain and the guanidino group of the N-terminal arginine residue. This effect is not observed for the glutamic acid-containing analogue. Molecular mechanics calculations indicate lowest energy conformations consistent with the mass spectrometric data; specifically, the additional methylene group in the glutamic acid side-chain markedly increases the distance between the carboxyl group and the adjacent peptide bond.

Hydrolyzing a protein in acid for a single hydrolysis interval, normally 24 h, will lead to inaccurate estimates of the amino acid composition of that protein due to an effect of the time of hydrolysis on peptide bond cleavage and amino acid degradation. The simultaneous yield and decay of amino acids during the hydrolysis of a protein can be described by a compartmental model with parameters for the hydrolysis and loss rates specific to each amino acid in a protein. The amino acid composition of the protein prior to hydrolysis can be determined by nonlinear regression of data derived from multiple hydrolysis intervals. In the present study egg-white lysozyme was hydrolyzed in 6 M HCl using 18 hydrolysis intervals (range, 2-141 h) using the conventional duplicate hydrolyses/interval system. Hydrolysis and loss rates were determined for each amino acid. Increasing the number of hydrolysis intervals prior to the maximum point on the hydrolysis curve, and including an hydrolysis interval greater than 100 h increased the accuracy with which the hydrolysis and loss rates were estimated. Most of the amino acids underwent some degree of loss during hydrolysis. Of particular note was the loss rate for cysteic acid, which was greater than that found for serine which is commonly regarded as an acid-labile amino acid. The determined amino acid composition of the protein, based on the nonlinear regression of the data from four different series of hydrolysis intervals, was compared with the known amino acid composition (sequencing). Using the routine duplicate sampling system, a nonlinear regression including 10 hydrolysis intervals (2, 6, 10, 14, 18, 22, 26, 30, 60, and 141 h) resulted in a mean amino acid recovery of 100% (range, 94-110%) and provided an acceptable compromise between accuracy and the cost of analysis.

Cysteine sulfinate decarboxylase (CSD) which decarboxylates cysteine sulfinic acid (CSA) to form hypotaurine is thought to be involved in the biosynthesis of taurine. It was recently localized in astrocytes in the cerebellum and hippocampus by immunocytochemistry. Another sulfur-containing aminoacid (SCAA), homocysteic acid (HCA), was also found in astrocytes in these regions. We therefore investigated the specificity of CSD vs CSA and HCA as well as the related analogs homocysteine sulfinic acid (HCSA) and cysteic acid (CA). CSD was immunotrapped from brain and liver tissue supernatant using a specific CSD antiserum and Protein-A Sepharose. It was then incubated with the l -form of the various SCAA. Reaction products were identified and quantified by pre-column o -phthalaldehyde derivatization HPLC. CA and HCA from 2.5 to 25 mM inhibited the formation of hypotaurine from CSA (0.25 mM). Moreover, the inhibition curves were parallel for liver and brain CSD. CA or HCA (25 mM) elicited a near-total inhibition. HCSA did not produce a significant inhibition up to 25 mM. Incubation with 25 mM CSA or CA led to the formation of hypotaurine and taurine, respectively. The ratio of formation of taurine to that of hypotaurine was similar for CSD from liver and brain. In contrast no homotaurine, the decarboxylated reaction product of HCA, could be detected following incubation with 25 mM HCA. According to the sensitivity of the HPLC analysis this indicates that the decarboxylation of HCA, if any, was 130-fold and 50-fold less than that of CSA by CSD from liver and brain, respectively, in our experimental conditions. Similarly, following incubation with HCSA, no new peak appeared on the chromatogram when compared to a blank sample. These results show that CSD from either brain or liver has a high specificity for CSA and CA, which are the SCAA involved in the biosynthesis of taurine. HCA is an inhibitor of CSD but does not appear to be a substrate for CSD in vitro . HCSA is neither a substrate nor an inhibitor of CSD in vitro . Accordingly, CSD is unlikely to play a role in the metabolism of HCA or HCSA in vivo . Copyright © 1996 Elsevier Science Ltd.

Conflicting reports in the literature of appropriate assay procedures for measurement of cysteine dioxygenase activity led us to evaluate the procedure for assay of cysteine dioxygenase activity in rat liver preparations. Cysteine dioxygenase activity was largely in the soluble fraction of liver and was stimulated by addition of NAD+ and Fe2+. The pH optimum of the enzyme was 6.1. Addition of an inhibitor of pyridoxal 5-phosphate-dependent enzymes was necessary to prevent rapid removal of the reaction product cysteine sulfinate. Cysteine sulfinate and cysteic acid were separated by anion-exchange HPLC on a polymer-based column with trimethylamino active groups, and the reaction products were quantitated by measurement of 35S radioactivity or by formation and measurement of fluorescent derivatives. This assay of cysteine dioxygenase under optimal conditions provides a physiologically relevant measure of cysteine dioxygenase activity in liver and hepatocytes based on the observation that this activity was highly correlated with the capacity for cysteine catabolism and taurine production by isolated hepatocytes.

The biosynthesis of the amino acid taurine has been studied in the locust Schistocerca americana gregaria . Tissue concentrations of putative precursors of taurine, and taurine itself were estimated in haemolymph, nervous tissue and muscle. Following this locusts were injected with 20 μCi of [ 35 S]cysteine and tissue samples were taken at times thereafter, and taurine and its precursors were extracted and separated by thin-layer chromatography (TLC). Taurine, cysteine sulphinic acid (CSA) were quantified by scintillation counting. Cysteic acid and hypotaurine were qualitatively detected by autoradiography of TLC plates. Results indicate that in haemolymph and muscle the biosynthetic pathway for taurine is cysteine then CSA, cysteic acid and thereafter taurine. In nervous tissue both cysteic acid and hypotaurine were detected suggesting two possible pathways for taurine since both are immediate precursors of this amino acid. Taurine biosynthesis was markedly greater in juvenile compared with mature adult locusts. It was also observed that picrotoxin treatment and prolonged flying, which have both previously been found to cause a redistribution of taurine in the locust, increased the biosynthesis of this amino acid.

A method for the evaluation of the age of wool carpets and textiles was developed based on the age dependent alteration of amino acid composition of proteins. Samples of 23 wool carpets and textiles of known age, obtained from the Hungarian Museum of Industrial Arts and the Hungarian National Museum were analysed for amino acid content. Results were compared with data obtained for contemporary, untreated wool and wool carpet. The cysteic acid content of wool increases with age. The contemporary wool carpet contained 0.31 g of cysteic acid in 100 g of protein. Comparable figures were 1.87 g for a 550-year old carpet and 4.01–4.39 g for the 1600–1750 year old wool carpets. The cystine content decreased with age, the corresponding figures being 7.88, 3.12 and 1.19-0.97 g/100 g, respectively. Corresponding contents of methionine were 0.43, 0.21 and 0.20-0.00 g/ 100 g and for tyrosine 3.07, 2.11 and 0.20-0.00 g/100 g. Prediction equations were developed as linear regressions of the age of wool on cysteic acid, cystine and tyrosine contents. The 95% confidence intervals of estimates for two samples of unknown age were estimates plus or minus 30 and 38 years.

Folate, cobalamin and pyridoxine deficiency are associated with psychiatric or neurological symptomatology. Disturbances in sulfur amino acid metabolism leading to accumulation of homocysteine occurs in all three conditions as the metabolism of homocysteine depends on enzymes requiring these vitamins as cofactors. Oxidation products of homocysteine (homocysteine sulfinic acid and homocysteic acid) and cysteine (cysteine sulfinic acid and cysteic acid) are excitatory sulfur amino acids and may act as excitatory neurotransmitters, whereas taurine and hypotaurine (decarboxylation products of cysteic acid and cysteine sulfinic acid) may act as inhibitory transmitters. Homocysteic acid and cysteine sulfinic acid have been considered as endogenous ligands for the N-methyl-D-aspartate (NMDA) type of glutamate receptors. The profile of these sulfur amino acid neurotransmitters could be altered in a similar fashion in states of decreased availability of folate, cobalamin or pyridoxine. It is proposed that the mechanism of neuropsychiatric manifestations in all three conditions result from a combination of two insults to homocysteine catabolism in the brain.

Poly(cysteic acid) was synthesized and found to inhibit the cytopathic effect of HIV-1 in CEM cell cultures at concentrations that were not cytotoxic to uninfected CEM cells.

The presence of a cysteine residue(s) near the active site of acylpeptide hydrolase was suggested by inactivation of the enzyme with sulfhydryl-modifying agents and by the substantial protection against inactivation afforded by the competitive inhibitor acetylmethionine. 5,5'-dithiobis-(2-nitrobenzoate) titrations of the native and the denatured enzyme together with analysis for cysteic acid after performic acid oxidation showed that the enzyme contained 12 free SH groups and three disulfide bonds/monomer. Chemical modification with radiolabeled iodoacetamide led to the labeling of Cys-30 and Cys-64 suggesting that one or both of these Cys residues are close to the active site. Modification of one or both of them probably inhibits the enzyme either because of a distortion of the active site or because the adducts present a barrier to the efficient diffusion of substrates into and products out of the active site. Studies on the mechanism of action of acylpeptide hydrolase have employed p-nitrophenyl-N-propyl carbamate as a potent active site-directed inhibitor. Enzyme inactivation, which follows pseudo first-order kinetics, is diminished by the competitive inhibitor acetylmethionine. The inhibited enzyme slowly regains activity at a rate that is increased in the presence of the nucleophile hydroxylamine. A general mechanism involving an acyl-enzyme intermediate is supported by evidence for the formation of acetyl-alanyl hydroxamate during hydrolysis of acetyl-alanine p-nitroanilide in the presence of hydroxylamine. The effect on Vmax and Km during this reaction indicate that hydrolysis of the acyl-enzyme intermediate is rate-limiting.

Capacity-potential data for the adsorption of cysteine and other organosulfur compounds on polycrystalline gold are reported. Cysteine, N-acetylcysteamine and cysteinesulfinic acid form monolayers on gold through the thiol group and significantly lower the interfacial capacity in the double-layer region. However, cysteic acid forms multilayers under the same conditions. The surface excess of the compounds at full monolayer coverage has been estimated and compared with the data for the adsorption of simple n-alkylthiols. It is argued that the packing of the cysteine and related compounds is more compact because of strong electrostatic interactions.

The mechanism electro-oxidation of cysteine, cysteinesulfinic acid and cysteic acid in perchloric acid solutions has been studied using cyclic voltammetry. All the compounds investigated have been found to be chemisorbed on a polycrystalline gold electrode, and oxidized with four, two or one electron respectively. The water molecule coadsorbed with cysteine participates in the cysteine one-electron oxidation with a lower energy requirement, whereas bulk water is involved in multiple electron transfer reactions at the completely covered electrode surface. A strong inhibiting effect of chloride anions on the cysteine oxidation has been observed at the partially covered electrode surface. The effect of the adsorbate surface coverage on the reaction mechanism in anodically catalysed reactions involving oxygen transfer is discussed.

A high-performance liquid chromatographic procedure was developed for the fully automated precolumn derivatization of amino acids by derivatization with o -phthaldialdehyde (OPA) together with the chiral thiols N-isobutyryl- l -cysteine (IBLC) and N-isobutyryl- d -cysteine (IBDC) [(CH 3 ) 2 CHCONHCH(CH 2 SH)COOH]. The derivatization is completed within 2 min at room temperature and the resulting diastereomeric isoindol derivatives exhibit linearity with respect to fluorescence in the range 25–1000 pmol. The derivatives are completely separated within 75 min on an octadecylsilyl stationary phase using a linear gradient generated by sodium acetate buffer (pH 5.95) and methanol-acetonitrile. Fluorescence detection at an excitation wavelength of 230 nm and an emission wavelength of 445 nm makes possible the detection of 1–2 pmol of an amino acid enantiomer. Derivatization with OPA-IBLC permits the separation of a 41-component standard containing seventeen protein l -amino acids and, in addition, glycine, dl -Cysteic acid, the internal standard l - homo -arginine and the non-protein amino acids α-aminoisobutyric acid and dl -isovaline. Replacement of OPA-IBLC with OPA-IBDC (or vice versa ) leads to a reversal in the elution of the derivatives of d - and l -amino acids. The applicability of the method to various fields of biosciences is demonstrated with the detection and determination of d -amino acids in bacteria, microfungi, higher plants, invertebrates and vertebrates, including man, and in the amino acid-containing Murchison meteorite.

The stimulatory effect of excitatory sulphur-containing amino acids on inositol phosphate formation was investigated in primary cultures of cerebellar granule cells. L-Cysteine sulphinate (CSA), L-cysteate (CA), L-homocysteine sulphinate (HCSA), L-homocysteate (HCA) and S-sulpho-L-cysteine (SSC) dose-dependently stimulated the formation of [3H]inositol phosphates exhibiting EC50 values in the range 60-200 microM and maximal effects of six- to 17-fold that of basal [3H]inositol phosphate levels. Endogenous L-glutamate spontaneously released into the extracellular medium or following exposure of cells to HCSA, HCA or SSC did not contribute significantly to formation of [3H]inositol phosphates, whereas 10% of the total [3H]inositol phosphates accumulated following exposure to CSA and CA was due to released L-glutamate. The selective N-methyl-D-aspartate receptor antagonist, D,L-2-amino-5-phosphonopentanoic acid (APV, 500 microM) attenuated by 20% (HCSA) to between 80 and 100% (CSA, CA, SSC, HCA) the formation of [3H]inositol phosphates induced by 1 mM sulphur-containing amino acids. When, however, HCSA was used at 100 microM (a concentration near to its EC50 for phosphoinositide hydrolysis), APV inhibited induced responses by 70%. Sulphur-containing amino acid-stimulated [3H]inositol phosphate formation was unaffected by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM). Inhibition of sulphur-containing amino acid-stimulated [3H]inositol phosphate formation by co-administration of APV and CNQX was similar to that obtained in the presence of APV alone. CSA-, CA-, SSC- and HCA-stimulated [3H]inositol phosphate formation was markedly reduced by removal of Ca2+ from the extracellular medium whereas that stimulated by HCSA was less affected. A similar inhibitory profile was observed when the levels of sulphur-containing amino acid-induced increases in intracellular free calcium ([Ca2+]i) were measured in the presence of 500 microM APV; 1 mM HCSA-induced responses being inhibited by only 30% whereas responses to the remaining sulphur-containing amino acid (also at 1 mM) were inhibited by > 45%. When the sulphur-containing amino acids were used at concentrations approximating their EC50 values for phosphoinositide hydrolysis, APV inhibited the induced increases in [Ca2+]i by 70-100%. HCA and SSC co-administered with the less efficacious but selective metabotropic glutamate receptor agonist, (+-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) at maximally effective concentrations (1 mM) of each agonist stimulated [3H]inositol phosphate formation in an additive manner.(ABSTRACT TRUNCATED AT 400 WORDS)

According to the Fmoc strategy, highly potent endothelin antagonistic cyclic pentapeptides possessing cysteic acid residue(s) were prepared in solid phase peptide synthesis. Use of a tetrabutylammonium salt overcame low solubility of N-(9-fluorenylmethoxycarbonyl)cysteic acid in a conventional solvent such as DMF.