Reactive Thiol Groups Research Articles

ChemInform Abstract: Far-Red Dibenzo[a,j]perylene-8,16-dione Dyes as Fluorescent Enzyme Substrates and Tags for Biomolecules.

Abstract The synthesis of fluorescent 2,10-derivatives of the title chromophore are described. The substituents contain amino-reactive N-hydroxysuccinimde ester and thiol-reactive iodoacetamide groups for attachment to biomolecules. A phosphate derivative was also prepared as an enzyme substrate.

Mechanistic Study of the Reaction of Thiol‐Containing Enzymes with α,β‐Unsaturated Carbonyl Substrates by Computation and Chemoassays

We investigated the reactions between substituted α,β‐unsaturated carbonyl compounds (Michael systems) and thiols by computations as well as chemoassays. The results give insight into variations in the underlying mechanisms as a function of the substitution pattern. This is of interest for the mechanisms of inhibition of the SARS coronavirus main protease (SARS‐CoV Mpro) by etacrynic acid derivatives as well as for the excess toxicity of substituted α,β‐unsaturated carbonyl compounds. This study compares possible reaction courses including 1,4‐addition followed by a ketonization step, and underscores the importance of a base‐catalyzed step for the reactivity of thiol groups in enzymes. Phenyl and methyl substituents at the Michael system decrease the reactivity of the electrophilic compound, but chlorophenyl substituents partly recover the reactivity. Computations also indicate that electron‐pushing substituents lead to a change in the reaction mechanism. The conformation of the Michael system is also found to significantly influence reactivity: the s‐cis conformation leads to higher reactivity than the s‐trans conformation. The computed data explain the trends in measured inhibition potencies of substituted α,β‐unsaturated carbonyl compounds and of reaction rates in chemical assays. They also indicate that the reversibility of inhibition does not stand in contrast to the formation of a new covalent bond between inhibitor and protease.

Intracellular Delivery of the Reactive Oxygen Species Generating Agent d-Penicillamine upon Conjugation to Poly-l-glutamic Acid

D-penicillamine is an aminothiol that is cytotoxic to cancer cells and generates dose dependent reactive oxygen species (ROS) via copper catalyzed oxidation. However, the delivery of D-pen to cancer cells remains a challenge due to its high hydrophilicity, highly reactive thiol group and impermeability to the cell membrane. To overcome this challenge, we investigated a novel poly-L-glutamic acid (PGA) conjugate of D-pen (PGA-D-pen) where D-pen was conjugated to PGA modified with 2-(2-pyridyldithio)-ethylamine (PDE) via disulfide bonds. Confocal microscopy and cell uptake studies showed that the fluorescently labeled PGA-D-pen was taken up by human leukemia cells (HL-60) in a time dependent manner. Treatment of HL-60, murine leukemia cells (P388) and human breast cancer cells (MDA-MB-468) with PGA-D-pen resulted in dose dependent cytotoxicity and elevation of intracellular ROS levels. PGA-D-pen induced apoptosis in HL-60 cells which was verified by Annexin V binding. The in vivo evaluation of the conjugate in the P388 murine leukemia model (intraperitoneal) resulted in significant enhancement in the survival of CD2F1 mice over vehicle control.

Synthesis of Pyridyl Disulfide-Functionalized Nanoparticles for Conjugating Thiol-Containing Small Molecules, Peptides, and Proteins

Previously we reported emulsion polymerization of propylene sulfide with Pluronic F127 as an emulsifier, yielding nanoparticles (NPs) in the 25 nm size range. Immunologically functional NPs were prepared by adding an antigen-Pluronic conjugate to the polymerization mixture ( Reddy , S. T. , et al. ( 2007 ) Nat. Biotechnol. 25, 1159 ). We sought a more flexible scheme for conjugation of antigens and other biomolecules to the NP surfaces that would allow for milder reaction conditions than achievable during the polymerization step. Here, we present the synthesis of such functionalizable NPs in the form of NPs that carry thiol-reactive groups, to which thiol-containing antigens (peptide or protein) or other biomolecules can be conjugated under mild conditions to yield immunofunctional NPs. The Pluronic-stabilized poly(propylene sulfide) (PPS) NPs with thiol-reactive pyridyl disulfide groups are prepared in two steps by (1) emulsion polymerization of propylene sulfide in the presence of a carboxylate-Pluronic and (2) reaction of the carboxylic acid groups on the NP surface with cysteamine pyridyl disulfide and a water-soluble carbodiimide reagent. We choose pyridyl disulfide groups to have a reduction-sensitive disulfide bond linking the antigen to the NP surface, allowing efficient release of antigen inside the cell in response to the reductive conditions within the endosome. The functionalizable NPs are characterized by proton NMR, dynamic light scattering (DLS), UV/vis spectroscopy, and transmission electron microscopy (TEM). Conjugation of small molecules and protein to the NP surface is presented.

Byproducts of protein, lipid and DNA oxidative damage and antioxidant enzyme activities in seizure

Purpose To get more insight into molecular mechanisms underlying oxidative stress and its role in different types of seizure, in this study, oxidative byproducts of proteins, lipids and DNA, as well as, antioxidant enzyme activities were studied in adult patients with epilepsy. Methods Study was performed in 60 patients with epilepsy and in 25 healthy controls. Plasma protein reactive carbonyl derivatives (RCD) and protein thiol groups (P-SH), byproducts of oxidative protein damage, as well as antioxidant enzyme activities, superoxide dismutase (SOD) and glutathione peroxidase (GPX) were studied spectrophotometrically. Urinary 8-epi-prostaglandin F 2α (8-epi-PGF 2α) and 8-hydroxy-2′-deoxyguanosine (8-OHdG), representative byproducts of lipid and DNA oxidative damage, respectively, were determined by enzyme immunoassay. Results RCD levels were significantly increased ( p = 0.001), while P-SH content was decreased in patients with first seizure ( p = 0.052) compared to controls, independently of the seizure type. Urinary 8-epi-PGF 2α and 8-OHdG were significantly increased in patients with epilepsy ( p = 0.001 and p = 0.001). Rise in 8-epi-PGF 2α was more pronounced in patients with generalized tonic–clonic seizure (GTCS) compared to those with partial seizure (PS). Both SOD and GPX activity were significantly increased in epileptic patients compared to controls ( p = 0.001 and p = 0.001), but only SOD activity was significantly higher in patients with GTCS than in those with PS. Conclusions Data on enhanced protein, lipid and DNA oxidation, together with upregulated antioxidant enzyme activities, confirm the existence of systemic oxidative stress in patients with epilepsy. It might be speculated that post-translational modification to existing functional proteins, particularly alterations to ion channels, might be at least partially responsible for acute early changes in neuronal networks.

Allyl sulfur compounds and cellular detoxification system: effects and perspectives in cancer therapy

Natural organosulfur compounds (OSCs) have been shown to have chemopreventive effects and to suppress the proliferation of tumor cells in vitro through the induction of apoptosis. The biochemical mechanisms underlying the antitumorigenic and anti-proliferative effects of garlic-derived OSCs are not fully understood. Several modes of action of these compounds have been proposed, and it seems likely that the rate of clearance of allyl sulfur groups from cells is a determinant of the overall response. The aim of this review is to focus attention on the effects of natural allyl sulfur compounds on the cell detoxification system in normal and tumor cells. It has been already reported that several natural allyl sulfur compounds induce chemopreventive effects by affecting xenobiotic metabolizing enzymes and inducing their down-activation. Moreover, different effects of water- and oil-soluble allyl sulfur compounds on enzymes involved in the detoxification system of rat tissues have been observed. A direct interaction of the garlic allyl sulfur compounds with proteins involved in the detoxification system was studied in order to support the hypothesis that proteins possessing reactive thiol groups and that are involved in the detoxification system and in the cellular redox homeostasis, are likely the preferential targets of these compounds. The biochemical transformation of the OSCs in the cell and their adducts with thiol functional groups of these proteins, could be considered relevant events to uncover the anticancer properties of the allyl sulfur compounds. Although additional studies, using proteomic approaches and transgenic models, are needed to identify the molecular targets and modes of action of these natural compounds, the allyl sulfur compounds can represent potential ideal agents in anticancer therapy, either alone or in association with other antitumor drugs.

Tracking of antibody reduction fragments by capillary gel electrophoresis during the coupling to microparticles surface

Due to their high specificity and efficiency, antibodies are ideal ligands for target-specific ultrasound contrast agents. The present study focuses on the chemical stability of antibodies during functionalisation with sulfosuccinimidyl-pyridyldithiopropionamidohexanoate (SPDP), a heterobifunctional linker, which exposes free thiol groups upon treatment with a reducing agent. Thiolated antibodies can then react with thiol-reactive group, such as maleimide present on the microbubble surface to form stable covalent complexes. The immunoglobulin structure relies on several intra- and inter-chain disulfide bridges which might be affected by reducing agents. A capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method with UV detection was applied to address the effect of the functionalisation process on the structural integrity of the antibodies and revealed that antibody disulfide bonds are prone to reduction as function of the reducing agents. Depending on the coupling conditions, various IgG fragments were identified reflecting different combinations between the light and heavy chains. Furthermore, two commonly used reducing agents, namely triscarboxyethylphosphine (TCEP) and 1,4-dithiothreitol (DTT) were compared under various preparation conditions. Results showed that reduction conditions based on DTT as a reducing agent under acidic pH were more appropriate to preserve intra- and inter-disulfide bridges of SPDP-modified antibodies.

Red‐Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy

Fluorescent markers emitting in the red are extremely valuable in biological microscopy since they minimize cellular autofluorescence and increase flexibility in multicolor experiments. Novel rhodamine dyes excitable with 630 nm laser light and emitting at around 660 nm have been developed. The new rhodamines are very photostable and have high fluorescence quantum yields of up to 80 %, long excited state lifetimes of 3.4 ns, and comparatively low intersystem-crossing rates. They perform very well both in conventional and in subdiffraction-resolution microscopy such as STED (stimulated emission depletion) and GSDIM (ground-state depletion with individual molecular return), as well as in single-molecule-based experiments such as fluorescence correlation spectroscopy (FCS). Syntheses of lipophilic and hydrophilic derivatives starting from the same chromophore-containing scaffold are described. Introduction of two sulfo groups provides high solubility in water and a considerable rise in fluorescence quantum yield. The attachment of amino or thiol reactive groups allows the dyes to be used as fluorescent markers in biology. Dyes deuterated at certain positions have narrow and symmetrical molecular mass distribution patterns, and are proposed as new tags in MS or LC-MS for identification and quantification of various substance classes (e.g., amines and thiols) in complex mixtures. High-resolution GSDIM images and live-cell STED-FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy.

The Role of Thiols and Disulfides on Protein Stability

There has been a tremendous increase in the number of approved drugs derived from recombinant proteins; however, their development as potential drugs has been hampered by their instability that causes difficulty to formulate them as therapeutic agents. It has been shown that the reactivity of thiol and disulfide functional groups could catalyze chemical (i.e., oxidation and beta-elimination reactions) and physical (i.e., aggregation and precipitation) degradations of proteins. Because most proteins contain a free Cys residue or/and a disulfide bond, this review is focused on their roles in the physical and chemical stability of proteins. The effect of introducing a disulfide bond to improve physical stability of proteins and the mechanisms of degradation of disulfide bond were discussed. The qualitative/quantitative methods to determine the presence of thiol in the Cys residue and various methods to derivatize thiol group for improving protein stability were also illustrated.

On the Regioselective Intramolecular Nucleophilic Addition of Thiols to C60

AbstractThe first intramolecular nucleophilic addition of a thiol to a fullerene double bond is reported. Whereas the reaction of cysteine (4) or cystine (11) with formaldehyde and [60]fullerene in chlorobenzene at reflux afforded the new compound 10 bearing a thiazolidine moiety fused to the fullerene sphere, the reaction of commercially available N‐methylglycine (12; sarcosine) with 1,4‐dithiane‐2,5‐diol (13) and C60 in chlorobenzene at reflux led to the desired cyclized compound 3 in poor yield. The favorable geometrical approach of the reactive thiol group towards the fullerene double bond is followed by a 5‐exo‐trig intramolecular nucleophilic addition of the S–H group to the adjacent double bond of the fullerene to form compound 3 in which a tetrahydrothiophene ring is simultaneously fused to the pyrrolidine and the fullerene moieties. Compound 3 was spectroscopically and electrochemically characterized and CV revealed that saturation of a second double bond of the fullerene sphere (cis‐1 regioisomer) is compensated by the electronegative character of the sulfur atom, resulting in reduction potentials similar to those observed for fullerene monoadducts. The two‐fold cyclization reaction occurs in a totally regioselective stepwise process leading exclusively to the cis‐1 isomer. Theoretical calculations (DFT and the ONIOM approach) predict that the formation of the sulfur pentagonal ring is highly favored and does not present structural constraints. Furthermore, the activation barrier for the nucleophilic attack of the thiol group on the fullerene surface was found to be substantially higher in comparison with the analogous previously reportedhydroxy group (activation barrier about 8 kcal mol–1 larger for SH than for OH), thus accounting for the poor yields of tetrahydrothiophene 3. The formation of 3 competes with the formation of 1,2‐dimethylpyrrolidino[3,4:1,2][60]fullerene (15), which is formed as the main reaction product by loss of the SH group. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Metabolic and hematological profiles in mature horses supplemented with different selenium sources and doses1,2

This study was conducted to determine the effects of dietary Se source and dose on metabolic and hematological profiles, and their relationships with oxidative status in horses. Twenty-five mature horses were blocked by BW and randomly allocated to 1 of 5 dietary treatments: negative control (CTRL: 0.085 mg of Se/kg of DM), 3 different dietary concentrations of organic Se provided by Se yeast (SY02, SY03, and SY04 containing 0.2, 0.3, and 0.4 mg of total Se/kg of DM, respectively), and 1 positive control provided by sodium selenite (SS03 containing 0.3 mg of total Se/kg of DM). Horses were fed the same basal diet (6 kg of grass hay and 3 kg of concentrate per horse daily) and received their respective treatments for a continuous period of 112 d. Jugular venous blood samples were collected before the morning feed on d 0, 28, 56, 84, and 112. Whole blood was analyzed for hematological profile, and plasma was analyzed for metabolites of energy, protein, and mineral metabolism; enzymatic activities and metabolites related to liver and muscle damage; and markers of inflammatory and oxidative status. Plasma metabolites related to energy, protein, and mineral metabolism, acute phase proteins, and enzyme activities related to hepatocellular, hepatobiliary, and muscle damage were not affected by Se source or dose. There were no differences among treatments in either reactive oxygen metabolites or thiol group concentrations in plasma. However, a linear decrease (P < 0. 01) in plasma total antioxidants was observed with increasing Se yeast supplementation. Furthermore, total antioxidant concentrations were less in SY03 than SS03 horses (P < 0.05), and were less in SY03 and SY04 than CTRL horses (P < 0.05). These results could be interpreted as an improvement in the preventive antioxidant systems of horses fed Se yeast. Total white blood cell count was not affected by treatment. There was a tendency for horses receiving greater concentrations of Se yeast to have greater lymphocyte counts (P = 0.09), with greater lymphocyte counts in blood of SY03 vs. SS03 horses (P < 0.05). Despite the lack of effect of Se source and dose on markers of inflammatory and liver status, the hematological profile seems to indicate an immunomodulatory action, as shown by mild changes in the white blood cell populations in response to Se yeast inclusion.

Chitosan-graft-6-mercaptonicotinic Acid: Synthesis, Characterization, and Biocompatibility

Thiolated chitosans are relatively new thiolated biopolymers exhibiting mucoadhesive, enzyme inhibitory, and permeation enhancing properties. A drawback, however, is their pH dependent reactivity. The aim of this study was therefore to develop a novel thiolated chitosan showing a non pH dependent reactivity of its thiol groups. For this purpose, 6-mercaptonicotinic acid (6-MNA) was covalently attached to chitosan by a carbodiimide mediated reaction. The obtained conjugate was characterized in vitro by quantification of immobilized thiol groups, cytotoxicity, in situ gelling properties, and disulfide bond formation at different pH. The synthesized conjugate displayed up to 973.80 micromol thiol groups per gram of polymer in reduced form. The polymer was nontoxic and showed in situ gelling properties. Furthermore, disulfide bond formation and therefore gelling properties occurred at various pH ranges. The reactivity of thiol groups was in the same range at pH 3 and pH 6.8. According to these results, chitosan-6 mercaptonicotinic acid seems to have some promising features to be used particularly for mucoadhesive formulations.

Free Thiol Group of MD-2 as the Target for Inhibition of the Lipopolysaccharide-induced Cell Activation

MD-2 is a part of the Toll-like 4 signaling complex with an indispensable role in activation of the lipopolysaccharide (LPS) signaling pathway and thus a suitable target for the therapeutic inhibition of TLR4 signaling. Elucidation of MD-2 structure provides a foundation for rational design of inhibitors that bind to MD-2 and inhibit LPS signaling. Since the hydrophobic binding pocket of MD-2 provides little specificity for inhibitors, we have investigated targeting the solvent-accessible cysteine residue within the hydrophobic binding pocket of MD-2. Compounds with affinity for the hydrophobic pocket that contain a thiol-reactive group, which mediates covalent bond formation with the free cysteine residue of MD-2, were tested. Fluorescent compounds 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid and N-pyrene maleimide formed a covalent bond with MD-2 through Cys(133) and inhibited LPS signaling. Cell activation was also inhibited by thiol-reactive compounds JTT-705 originally targeted against cholesterol ester transfer protein and antirheumatic compound auranofin. Oral intake of JTT-705 significantly inhibited endotoxin-triggered tumor necrosis factor alpha production in mice. The thiol group of MD-2 also represents the target of environmental or endogenous thiol-reactive compounds that are produced in inflammation.

Effect of whey protein enzymatic hydrolysis on the rheological properties of acid-induced gels

A protein dispersion blend of β-lactoglobulin and α-lactalbumin was heat-denatured at pH 7.5, hydrolyzed by α-chymotrypsin and then acidified with glucono-δ-lactone to form gels at room temperature. Heat treatment induced the formation of whey protein polymers with high concentration of reactive thiol groups (∼37 μmol/g). The reactive thiol group concentration was reduced by half after 40 min enzymatic hydrolysis. It was further reduced after enzyme thermal deactivation. During acidification, the first sign of aggregation for hydrolyzed polymers occurred earlier than for non hydrolyzed polymers. Increasing the hydrolysis duration up to 30 min resulted in more turbid gels characterized by an open microstructure. Elastic and viscous moduli were both reduced, while the relaxation coefficient and the stress decay rate constants were increased by increasing the hydrolysis duration. After one week storage at 5 °C, the hardness of gels made from hydrolyzed polymers increased by more than 50%. The effect of polymer hydrolysis on acid-induced gelation is discussed in relation to the availability and reactivity of thiol groups during gel formation and storage.

Liver apolipoprotein B 100 expression and secretion are down-regulated early postpartum in dairy cows

Fatty liver is a typical condition early postpartum in dairy cows. The aim of the present study was to evaluate gene expression, synthesis and secretion of liver apolipoprotein B 100 (Apo-B 100) in periparturient dairy cows. The relationships between Apo-B 100 with liver lipid accumulation, and metabolic and oxidative status were also investigated. The trial was carried out in a commercial dairy herd. Sixteen multiparous Holstein cows due to calve in April were monitored from 30 d prepartum to 35 d postpartum. Milk yield was recorded, and body condition (BCS) of cows was scored. Blood samples were taken at − 30 ± 3, − 18 ± 2, and − 4 ± 1 d prepartum (before expected calving) and at 3, 17, and 35 d postpartum. Plasma and erythrocyte samples were obtained and analyzed. Glucose, NEFA, BHBA, cholesterol, albumin, Apo-B 100, reactive oxygen metabolites, thiol groups, and glutathione peroxidase were determined in plasma. Reactive oxygen metabolites, thiol groups, glutathione peroxidase and superoxide dismutase were determined in erythrocytes. Liver biopsies were performed on d − 30, 3, and 35 relative to day of calving. Liver samples were analyzed to determine fat accumulation, Apo-B 100 mRNA and Apo-B 100 protein. Milk yield, BCS, and plasma indices of metabolic status showed expected changes for that physiological phase. Oxidative status indices indicated an oxidative stress condition early postpartum. Fat accumulation in liver increased after calving and no significant difference was found between values observed at 3 and 35 DIM. Gene expression as well as cellular and plasma concentration of Apo-B 100 were higher prepartum compared with postpartum. Apo-B 100 mRNA was not different between 3 and 35 DIM, whereas both cellular and plasma Apo-B 100 increased from 3 to 35 DIM. The study demonstrates that Apo-B 100 is regulated at the level of transcription and post-transcription. Changes of gene and protein Apo-B 100 expression may contribute to the accumulation of triglyceride in liver taking place early postpartum in dairy cows.

Synthesis and application of novel phenylboronate affinity materials based on organic polymer particles for selective trapping of glycoproteins

We report on synthesis concepts for the fabrication of various novel phenylboronate affinity materials based on polymethacrylate epoxy beads (Fractogel EMD Epoxy (M) 40-90 microm) and the testing of these functionalized polymer particles for selective trapping of a glycoprotein from a standard mixture containing a glycosylated and a nonglycosylated protein. Two inherently different approaches for the functionalization of the bare beads with boronate groups have been elucidated. In the first, the epoxy residues of the polymer particles were converted into reactive thiol groups which were subsequently used as anchor moieties for the immobilization of 4-vinylphenylboronic acid by radical addition or radical polymerization reaction. Three different ways for the generation of sulfhydryl groups have been examined leading to materials with distinct linker chemistries. In the second and more straightforward approach, the epoxy groups were reacted with 4-mercaptophenylboronic acid. The novel materials were thoroughly characterized by (i) quantitation of the sulfur content by elemental analysis, (ii) reactive sulfhydryls were determined in a photospectrometric assay, (iii) boron content was measured by inductively coupled plasma-atomic emission spectrometry, and (iv) the amount of reactive boronate groups was evaluated in a fast binding assay employing adenosine as test compound. A maximum concentration of 1.2 mmol boronate groups per gram dry beads could be achieved by the presented synthesis routes. Employing the novel phenylboronate affinity materials in capture and release experiments in the batch mode, a standard glycoprotein, viz. transferrin (Tf) from human serum was separated from a nonglycosylated protein, BSA. A commercial boronate affinity material based on 3-aminophenylboronic acid modified agarose gel was employed as reference material and was found to perform significantly worse compared to the herein presented novel polymethacrylate particles.

Tumor-targeted PE38KDEL delivery via PEGylated anti-HER2 immunoliposomes

We previously reported the development of PE38KDEL-loaded anti-HER2 poly(lactic-co-glycolic acid) (PLGA) nanoparticles that bind and internalize in HER2-overexpressing breast cancer cells, enabling potent anti-tumor activity. To overcome the problems associated with the short half-lives of this drug delivery system, we have constructed PE38KDEL-loaded anti-HER2 PEGylated liposomes (PE-HER-liposomes). PE-HER-liposomes were constructed with Fab′ of recombinant humanized anti-HER2 monoclonal antibody (anti-HER2 Fab′) covalently linked to PEGylated liposomes containing PE38KDEL (PE-liposomes). We attached anti-HER2 Fab′ to the terminus of PEG (polyethylene glycol) on PEGylated liposomes. Incorporation of pyridylthiopropionoylamino-PEG-distearoylphosphatidylethanolamine (PDP-PEG-DSPE) into PEGylated liposomes followed by mild thiolysis of the PDP groups resulted in the formation of reactive thiol groups at the periphery of the liposomes. Efficient attachment of maleimide-derivatized anti-HER2 Fab′ took place under mild conditions. The characterization of PE-HER-liposomes, such as particle size, was evaluated by dynamic light-scattering detector. The Micro BCA method was used to determine the encapsulation efficiency of PE38KDEL and the quantity of conjugated Fab′. Flow cytometry and confocal microscopy showed that PE-HER-liposomes possessed receptor-specific binding and internalization for HER2-overexpressing SK-BR3 cells. Remarkably, PE-HER-liposomes were more cytotoxic than non-targeted PE-liposomes in HER2-overexpressing breast cancer cells. In conclusion, PE-HER-liposomes could serve as a promising therapeutic candidate for the treatment of HER2-overexpressing breast cancers.

S-Nitrosoglutathione Inactivation of the Mitochondrial and Cytosolic BCAT Proteins: S-Nitrosation and S-Thiolation

Specific proteins with reactive thiol(ate) groups are susceptible to nitric oxide (NO) modification, which can result in S-nitrosation, S-thiolation, or disulfide bond formation. In the present study the effect of NO modification on the functionality of human mitochondrial and cytosolic branched-chain aminotransferases (hBCATm and hBCATc, respectively) was investigated. Here, the NO reactive agents, S-nitrosoglutathione (GSNO), S-nitroso-N-acetyl-dl-penacillamine, and sodium nitroprusside, inactivated both isoforms in a dose-dependent manner. Furthermore, low concentrations of GSNO caused a time-dependent loss in BCAT activity (50 +/- 3% and 77 +/- 2% for hBCATc and hBCATm, respectively) correlating with the loss of four and one to two thiol groups, respectively, confirming the thiols as targets for NO modification. Analysis of GSNO-modified hBCATc by quadrupole time-of-flight mass spectrometry identified a major peak containing three NO adducts and a minor peak equivalent to two NO adducts and one glutathione (GSH) molecule, the latter confirmed by Western blot analysis. Moreover, prolonged exposure or increased levels of GSNO caused increased S-glutathionylation and partial dimerization of hBCATc, suggesting a possible shift from regulation by NO to one of adaptation during nitrosated stress. Although GSNO inactivated hBCATm, neither S-nitrosation, S-glutathionylation, nor dimerization could be detected, suggesting differential mechanisms of regulation through NO between isoforms in the mitochondria and cytosol. Reversal of GSNO-modified hBCAT using GSH alone was only partial, and complete reactivation was only possible using the glutaredoxin/GSH system (97 +/- 4% and 91 +/- 3% for hBCATc and hBCATm, respectively), implicating the importance of a full physiological redox system for activation/inactivation. To conclude, these results clearly demonstrate distinct functional/mechanistic responses to GSNO modification between BCAT isoforms and offer intriguing comparisons between the BCAT proteins and the respective cytosolic and mitochondrial hTrx and hGrx proteins.

Catechol estrogens mediated activation of Nrf2 through covalent modification of its quinone metabolite to Keap1

The catechol metabolites (2-OHE and 4-OHE) of estrogen enter a redox cycle, thereby generating not only reactive oxygen species (ROS) but also electrophilic quinones. It is well recognized that chemicals causing oxidative stress or electrophiles activate a transcription factor Nrf2 that is negatively regulated by Keap1, leading to up-regulation of downstream proteins responsible for detoxification of electrophiles in cells. The purpose of the present study is to explore the roles of oxidative and electrophilic stress in Nrf2 activation caused by redox-active catechol estrogens. Exposure of RAW264.7 cells to 2- and 4-OHE activated Nrf2, resulting in induction of heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit (GCLC). Under these conditions, intracellular oxidants were generated; however, subsequent examinations revealed that quinoid metabolites derived from 2- and 4-OHE mainly participate in the Nrf2 activation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis revealed Keap1 undergoes modification by such quinoid species through multiple reactive thiol groups. These results suggest that Nrf2 activation during redox cycling of catechol estrogens is dominantly attributable to formation of their ortho-quinones that covalently bind to Keap1.

Catalytically Important Amino Acid Residues in Endoxylanases from Aspergillus Awamori

ABSTRACTTwo endoxylanases, Xyl-II (23 kDa) andXyl III (27 kDa), from Aspergillus awamori K-1 were modified with several specific reagents. The catalytic role of the carboxyl groups was estimated through chemical modification studies. Water-soluble carbodiimide (CMC) inactivated the xylanases rapidly and completely in a pseudo-first-order process indicating involvement of carboxyl groups in the catalysis. Treatment of the enzymes with diethyl pyrocarbonate (DEPC) resulted in modification of 1.56 histidine residues in the Xyl III (100% retention of the original enzyme activity in the presence of substrate - 0.4% xylan). Titration of the enzymes with 5.5' -dithiobis-(2-nitrobenzoic acid) (DTNB) did not result in the release of 5-mercapto-2- nitrobenzoic acid, suggesting that no free thiol groups exist in Xyl II and Xyl III. The lack of reactive thiol groups in the intact enzymes was confirmed using the specific cysteine-residue modifiers—iodoacetimide and p-chloromercuribenzoate, neither of which had any noticeable effect on xylanase activities after 60 min of incubation. N-Bromosuccinimide (NBS) showed a strong inhibitory effect on both endoxylanases, suggesting that trypthophan residues are essential for the activity, since the presence of substrate protected the enzymes against inactivation.