Tris 2-carboxyethyl Phosphine Research Articles

Loss of Cationic Peptides with Agarose Gel-Immobilized Tris[2- Carboxyethyl]Phosphine (TCEP)

Loss of Cationic Peptides with Agarose Gel-Immobilized Tris[2- Carboxyethyl]Phosphine (TCEP)

Co‐immobilization of an Enzyme and a Metal into the Compartments of Mesoporous Silica for Cooperative Tandem Catalysis: An Artificial Metalloenzyme

Co‐immobilization of an Enzyme and a Metal into the Compartments of Mesoporous Silica for Cooperative Tandem Catalysis: An Artificial Metalloenzyme

Vitamin C in Infant Formula and Adult/Pediatric Nutritional Formula by Ultra-Performance Liquid Chromatography with Ultraviolet Detection: First Action 2012.22

During the AOAC Annual Meeting held in Las Vegas, NV from September 30 to October 3, 2012, the Stakeholder Panel on Infant Formula and Adult Nutritionals convened to review single-laboratory validation data submitted for the method, Vitamin C in Adult/Pediatric Formula by Ultra-Performance Liquid Chromatography with Ultraviolet Detection. This method is a modified version of the method "HPLC-UV Determination of Total Vitamin C in a Wide Range of Fortified Food Products" previously published in Food Chem., 94, 626-631 (2006). The SLV data from the modified method were reviewed and compared to the standard method performance requirements (SMPR 2012.012), and it was concluded that the method meets the requirements. The method was approved as AOAC Official First Action. The method is based on the acidic extraction of ascorbic acid in the presence of Tris[2-carboxyethyl] phosphine (TCEP) as a reducing agent. Separation was achieved on a C18 column with a sodium acetate eluent (pH 5.4) combined with TCEP and decylamine as an ion-pairing agent. Accuracy rates were between 90 and 100%. Repeatability RSD (RSD,) ranged from 1.4 to 2.5%, and intermediate reproducibility RSD (RSDiR) ranged from 1.3 to 7.5%.

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch regulating metabolic pathways. In this study we discovered that the Omega class glutathione transferase GSTO1-1 plays a significant role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1 in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry and immunoprecipitation identified β-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells. In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation of cellular proteins when the cells are exposed to S-nitrosoglutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.

Polymeric redox-responsive delivery systems bearing ammonium salts cross-linked via disulfides.

A redox-responsive polycationic system was synthesized via copolymerization of N,N-diethylacrylamide (DEAAm) and 2-(dimethylamino)ethyl methacrylate (DMAEMA). N,N’-bis(4-chlorobutanoyl)cystamine was used as disulfide-containing cross-linker to form networks by the quaternization of tertiary amine groups. The insoluble cationic hydrogels become soluble by reduction of disulfide to mercaptanes by use of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) or cysteamine, respectively. The soluble polymeric system can be cross-linked again by using oxygen or hydrogen peroxide under basic conditions. The redox-responsive polymer networks can be used for molecular inclusion and controlled release. As an example, phenolphthalein, methylene blue and reactive orange 16 were included into the network. After treatment with DTT a release of the dye could be recognized. Physical properties of the cross-linked materials, e.g., glass transition temperature (Tg), swelling behavior and cloud points (Tc) were investigated. Redox-responsive behavior was further analyzed by rheological measurements.

Multi-omic Data Integration Links Deleted in Breast Cancer 1 (DBC1) Degradation to Chromatin Remodeling in Inflammatory Response

This study investigated the dynamics of ubiquitinated proteins after the inflammatory stimulation of RAW 264.7 macrophage-like cells with bacterial lipopolysaccharide. Ubiquitination is a common protein post-translational modification that regulates many key cellular functions. We demonstrated that levels of global ubiquitination and K48 and K63 polyubiquitin chains change after lipopolysaccharide stimulation. Quantitative proteomic analysis identified 1199 ubiquitinated proteins, 78 of which exhibited significant changes in ubiquitination levels following stimulation. Integrating the ubiquitinome data with global proteomic and transcriptomic results allowed us to identify a subset of 88 proteins that were targeted for degradation after lipopolysaccharide stimulation. Using cellular assays and Western blot analyses, we biochemically validated DBC1 (a histone deacetylase inhibitor) as a degradation substrate that is targeted via an orchestrated mechanism utilizing caspases and the proteasome. The degradation of DBC1 releases histone deacetylase activity, linking lipopolysaccharide activation to chromatin remodeling in caspase- and proteasome-mediated signaling.

Competitive electrochemical immunoassay for detection of β-amyloid (1–42) and total β-amyloid peptides using p-aminophenol redox cycling

β-Amyloid (1–42) peptide (Aβ(1–42)) is believed to be important for diagnosis and prognosis of Alzheimer's disease (AD) serving as a reliable molecular biomarker. However, the levels of Aβ(1–42) may differ by gender and age; thus, assay of Aβ(1–42) only might be unable to discriminate between AD and health or other types of dementia. In this work, we reported a sensitive and selective electrochemical method for detection of both Aβ(1–42) and total Aβ using p-aminophenol (p-AP) redox cycling on antibody-modified gold electrodes. Specifically, the conjugates performed between streptavidin-conjugated alkaline phosphatase (SA-ALP) and biotinylated Aβ peptides were captured by the antibody-modified electrodes, which induced the production of electrochemically active p-AP from the p-aminophenyl phosphate (p-APP) substrate. In the presence of tris(2-carboxyethyl)phosphine (TCEP), p-AP could be cycled after its electro-oxidization on the electrode, enabling the increase of the anodic current. Because native Aβ competed with the conjugates to bind the anchored antibody, the signal decreased with the increase of native Aβ concentration. A detection limit of 5pM was achieved. To demonstrate the viability of the method for analysis of Aβ(1–42) and total Aβ in real sample, artificial cerebrospinal fluid (aCSF) containing Aβ(1–40), Aβ(1–42) and Aβ(1–16) was tested. We believe that the simultaneous detection of Aβ(1–42) and total Aβ would be valuable for the early diagnosis of AD.

Comparing the performances of electrochemical sensors using p-aminophenol redox cycling by different reductants on gold electrodes modified with self-assembled monolayers

p-Aminophenol (p-AP) redox cycling using chemical reductants is one strategy for developing sensitive electrochemical sensors. However, most of the reported reductants are only used on indium-tin oxide (ITO) electrodes but not gold electrodes due to the high background current caused by the oxidation reaction of the reductants on the highly electrocatalytic gold electrodes. Therefore, new strategies and/or reductants are in demand for expanding the application of p-AP redox cycling on gold electrodes. In this work, we compared the performances of several reductants in p-AP redox cycling on self-assembled monolayers (SAMs)-modified gold electrodes. Among the tested reagents, nicotinamide adenine dinucleotide (NADH), tris(2-carboxyethyl)phosphine (TCEP) and cysteamine were demonstrated to be suitable for p-AP redox cycling on the alkanethiol-modified gold electrodes because of their low background current. The rate of chemical reaction between reductants and p-quinone imine (QI, the electrochemically oxidized product of p-AP) increases in the order of NADH<TCEP<cysteamine. The system benefits from the low background current, fast chemical reaction and amenability and sensitivity of the sensor with TCEP or cysteamine as the reductant; these performance enhancements were demonstrated by the detection of microRNA. A detection limit of 2pmolL−1 was achieved. We believe that our work will be valuable for the development of electrochemical sensors using p-AP redox cycling on gold electrodes.

TCEP-based rSDS–PAGE AND nLC–ESI-LTQ-MS/MS for oxaliplatin metalloproteomic analysis

In this work, the reactivity of the citostatic drugs such as oxaliplatin, cisplatin and carboplatin towards proteins and the stability of Pt–protein complexes along their storage were evaluated. Neither native-PAGE nor nrSDS–PAGE seems to be suitable for the separation of carboplatin-binding proteins. A reducing electrophoretic separation procedure able to maintain the integrity of oxaliplatin–protein complexes has been developed. The method is based on SDS–PAGE under conditions provided by the thiol-free reducing agent tris (2-carboxyethyl) phosphine (TCEP), which allowed the separation of oxaliplatin-binding proteins in narrow bands with almost quantitative recoveries. Different amounts of platinum-bound protein bands covering the range 0.3–2.0μg were excised and mineralised for platinum determination, showing good linearity. Limits of detection for a mixture of five standard proteins (transferrin, albumin, carbonic anhydrase, myoglobin and cytochrome c) incubated with oxaliplatin were within the range 11.0–44.0pg of platinum, which were satisfactory for their application to biological samples. The suitability of the TCEP-based SDS–PAGE for the separation of platinum-enriched protein fractions of a kidney cytosol from a rat treated with oxaliplatin was demonstrated. The identification of high Pt to protein ratio cytosolic fractions was carried out by separating the cytosolic platinum-binding proteins by SEC–ICP-MS. Several cytosolic renal proteins were identified in those gel bands containing platinum-enriched protein fractions using nLC–ESI-LTQ-MS/MS after in-gel digested with trypsin. In addition, fractions containing platinum-enriched proteins with lower theorical molecular weight were directly analysed by nLC–ESI-LTQ-MS/MS after in-solution tryptic digestion allowing protein identification.

Direct measurement of active thiol metabolite levels of clopidogrel in human plasma using tris(2-carboxyethyl)phosphine as a reducing agent by LC-MS/MS

A simple, robust, and rapid LC-MS/MS method has been developed and validated for the simultaneous quantitation of clopidogrel and its active metabolite (AM) in human plasma. Tris(2-carboxyethyl)phosphine (TCEP) was used as a reducing agent to detect the AM as a disulfide-bonded complex with plasma proteins. Mixtures of TCEP and human plasma were deproteinized with acetonitrile containing 10 ng/mL of clopidogrel-d4 as an internal standard (IS). The mixtures were separated on a C18 RP column with an isocratic mobile phase consisting of 0.1% formic acid in acetonitrile and water (90:10, v/v) at a flow rate of 0.3 mL/min. Detection and quantification were performed using ESI-MS. The detector was operated in selected reaction-monitoring mode at m/z 322.0→211.9 for clopidogrel, m/z 356.1→155.2 for the AM, and m/z 326.0→216.0 for the IS. The linear dynamic range for clopidogrel and its AM were 0.05-20 and 0.5-200 ng/mL, respectively, with correlation coefficients (r) greater than 0.9976. Precision, both intra- and interday, was less than 8.26% with an accuracy of 87.6-106%. The validated method was successfully applied to simultaneously analyze clinical samples for clopidogrel and its AM.

Trisulfide Modification Impacts the Reduction Step in Antibody–Drug Conjugation Process

Antibody-drug conjugates (ADCs) utilizing cysteine-directed linker chemistry have cytotoxic drugs covalently bound to native heavy-heavy and heavy-light interchain disulfide bonds. The manufacture of these ADCs involves a reduction step followed by a conjugation step. When tris(2-carboxyethyl)phosphine (TCEP) is used as the reductant, the reaction stoichiometry predicts that for each molecule of TCEP added, one interchain disulfide should be reduced, generating two free thiols for drug linkage. In practice, the amount of TCEP required to achieve the desired drug-to-antibody ratio often exceeds the predicted, and is variable for different lots of monoclonal antibody starting material. We have identified the cause of this variability to be inconsistent levels of interchain trisulfide bonds in the monoclonal antibody. We propose that TCEP reacts with each trisulfide bond to form a thiophosphine and a disulfide bond, yielding no net antibody free thiols for conjugation. Antibodies with higher levels of trisulfide bonds require a greater TCEP:antibody molar ratio to achieve the targeted drug-to-antibody ratio.

Vortex-Induced Amyloid Superstructures of Insulin and Its Component A and B Chains

Insulin is an amyloid-forming polypeptide built of two disulfide-linked chains (A and B), both themselves amyloidogenic. An interesting property of insulin is that agitation strongly influences the course of its aggregation, resulting in characteristic chiral superstructures of amyloid fibrils. Here, we investigate the self-assembly of these superstructures by comparing the quiescent and vortex-assisted aggregation of insulin and its individual A and B chains in the presence or absence of reducing agent tris(2-carboxyethyl)phosphine (TCEP). Our study shows that only the B chain in the presence of TCEP is converted into aggregates with morphology (according to atomic force microscopy) and optical activity (manifested as an extrinsic Cotton effect induced in bound thioflavin T) characteristic of amyloid superstructures that are normally formed by insulin in the absence of TCEP. In contrast to more rigid B-peptide fibrils, elongated aggregates of the A peptide become amorphous upon agitation. Moreover, the aggregation of equimolar mixture of both peptides does not produce highly ordered entities. Our results suggest that the dynamics of the B chain are the driving force for the assembly of superstructures, with the A chain being complicit as long as its own dynamics are controlled by the firm attachment to the B chain provided by the intact covalent structure of insulin.

Coupling of Proton Binding in Extracellular Domain to Channel Gating in Acid-Sensing Ion Channel

Protonation of several amino acid residues in the extracellular domain (ECD) of acid-sensing ion channel (ASIC) causes conformational changes that lead to opening of the channel. It is not clear how conformational changes in ECD are coupled to channel gating. Here, we show that the loop connecting β9 and α4 at the base of the thumb region of ECD interacts with post-TM1 to stabilize the channel in the closed state. Flexibility of these two regions is important for optimum gating of the channel. In ASIC1a, when Y71 (post-TM1) and W287 (β9-α4 loop) were mutated to cysteine, they formed disulfide bond in the closed state. Breaking of the disulfide bond by reducing agent dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) potentiated the current significantly. Engineered cysteine G288C reacted with sulfhydryl-specific methanethiosulfonate ethyltrimethylammonium (MTSET) in the open state but not in closed/steady desensitized state, suggesting gating-associated conformational movement of this loop. We also identified a salt bridge between highly conserved R64 at TM1 and D432 at TM2 that is important for optimum gating. Based on our results and other published work, we propose that proton binding in ECD is followed by the displacement of the β9-α4 loop of the thumb, leading to the rotation of TM1. Conformational movement propagates to TM2 and the channel gate opens by the concomitant movement of TM2 and breaking of the salt bridge between R64 and D432.

Microwave-Mediated Reduction of Disulfide Bridges with Supported (Tris(2-carboxyethyl)phosphine) as Resin-Bound Reducing Agent

We report on the synthesis and use of a new supported reagent consisting in tris(2-carboxyethyl)phosphine (TCEP) immobilized on hydrophilic PEG based resin beads. Used in conjunction with a 5 min microwave (MW) irradiation, "supported TCEP" reduced disulfide bridges in free thiols in peptides having two or more cysteine residues. Separation of reaction products from reducing agent was easily performed by simple filtration.

Identification of a Disulfide Bridge Important for Transport Function of SNAT4 Neutral Amino Acid Transporter

SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine (TCEP), indicating the possible involvement of disulfide bridge(s). Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H2O2. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.

Half Antibody Fragments Improve Biosensor Sensitivity without Loss of Selectivity

We show for the first time that half antibody fragments obtained by reduction via tris(2-carboxyethyl)phosphine (TCEP) gave a larger response with shear-mode resonating mass sensors than physisorbed whole antibody or antibody immobilized via Protein G. The reduced antibody is shown to preserve the antigen-binding region and was determined via the antigen binding response. Reduction exposed the native thiol group in the antibody that readily chemisorbed onto the gold-coated sensor surfaces with the right orientation for antigen binding. Comparing responses obtained on a quartz crystal microbalance for the detection of pathogenic Escherichia coli O157:H7 using TCEP-reduced antibody with native antibody showed that the proposed method enhances device sensitivity. Examining the half antibody fragments for detection of the pathogen in the presence of the nonpathogenic wild strain showed that the antibody fragments retained their specific antigen binding capability without loss of selectivity.

Automated high performance liquid chromatography with on-line reduction of disulfides and chemiluminescence detection for determination of thiols and disulfides in biological fluids

In general, the reduction of disulfide bonds with tris(2-carboxyethyl)phosphine (TCEP) is performed using off-line operation, which is not only time-consuming but also vulnerable to the spontaneous re-oxidation of thiols during sample preparation and subsequent analysis procedures. To the best of our knowledge, there has been not any case on the on-line reduction for biological disulfides coupled with high performance liquid chromatography (HPLC). In this study, these obstacles are overcome by packing Zn(II)-TCEP complexes into a home-made column. The as-synthesized Zn(II)-TCEP complexes enable efficient reduction of disulfide bonds at pH 3.0. This acidic pH value was compatible with that of the mobile phase for HPLC separation of thiols and disulfides. Therefore, using fluorosurfactant-prepared triangular gold nanoparticles as HPLC postcolumn specific chemiluminescence (CL) reagents for thiols, the feasibility of the established on-line reduction column has been confirmed for the direct identification of both thiols and disulfides by incorporating this reduction column into a single chromatographic separation. Detection limits for these analytes range from 8.3 to 25.4nM and the linear range in a log–log plot can comprise three orders of magnitude. Finally, the utility of this automated on-line reduction of disulfides-HPLC-CL system has been demonstrated for the reliable determination of thiols and disulfides in human urine and plasma samples.

Phosphine Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy

We report that the cyanine dye Cy5 and several of its structural relatives are reversibly quenched by the phosphine tris(2-carboxyethyl)phosphine (TCEP). Using Cy5 as a model, we show that the quenching reaction occurs by 1,4-addition of the phosphine to the polymethine bridge of Cy5 to form a covalent adduct. Illumination with UV light dissociates the adduct and returns the dye to the fluorescent state. We demonstrate that TCEP quenching can be used for super-resolution imaging as well as for other applications, such as differentiating between molecules inside and outside the cell.

Optimising the synthesis, polymer membrane encapsulation and photoreduction performance of Ru(ii)- and Ir(iii)-bis(terpyridine) cytochrome c bioconjugates

Ruthenium(II) and iridium(III) bis(terpyridine) complexes were prepared with maleimide functionalities in order to site-specifically modify yeast iso-1 cytochrome c possessing a single cysteine residue available for modification (CYS102). Single X-ray crystal structures were solved for aniline and maleimide Ru(II) 3 and Ru(II) 4, respectively, providing detailed structural detail of the complexes. Light-activated bioconjugates prepared from Ru(II) 4 in the presence of tris(2-carboxyethyl)-phosphine (TCEP) significantly improved yields from 6% to 27%. Photoinduced electron transfer studies of Ru(II)-cyt c in bulk solution and polymer membrane encapsulated specimens were performed using EDTA as a sacrificial electron donor. It was found that membrane encapsulation of Ru(II)-cyt c in PS140-b-PAA48 resulted in a quantum efficiency of 1.1 ± 0.3 × 10(-3), which was a two-fold increase relative to the bulk. Moreover, Ir(III)-cyt c bioconjugates showed a quantum efficiency of 3.8 ± 1.9 × 10(-1), equivalent to a ∼640-fold increase relative to bulk Ru(II)-cyt c.

Sulfhydryl-dependent dimerization of soluble guanylyl cyclase modulates the relaxation of porcine pulmonary arteries to nitric oxide

The dimeric status of nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC) is obligatory for its catalyzing activity to synthesis the second messenger cyclic guanosine monophosphate (cGMP), which leads to vasodilatation. The present study was conducted to determine whether or not the dimerization of sGC is modulated by thiol-reducing agents and its influences on relaxation of pulmonary arteries caused by NO. The dimers and monomers of sGC and cGMP-dependent protein kinase (PKG) were analyzed by Western blotting. The intracellular cGMP content was measured by enzyme-linked immunosorbent assay. Relaxations of isolated porcine pulmonary arteries were determined by organ chamber technique. Protein levels of sGC dimers were decreased by thiol reductants dithiothreitol (DTT), reduced L-glutathione, L-cysteine, and tris(2-carboxyethyl)phosphine (TCEP), associated with decreased cGMP elevation, attenuated relaxations to NO. DTT at concentrations that affected sGC dimerization and activity showed no effect on PKG dimerization nor relaxation to 8-Br-cGMP. Hypoxia decreased the dimerization and activity of sGC of the arteries. The suppression of DTT and TCEP on sGC dimerization and activity was augmented by hypoxia. In the presence of DTT and TCEP, relaxations of porcine pulmonary artery caused by NO were significantly less under hypoxia compared with those under normoxia. These results suggest that the dimerization and activity of sGC along with NO-induced vasodilatation can be modulated in a thiol-dependent manner. Such a mechanism may be involved in the diminished response of pulmonary arteries to NO under hypoxia.