Presence Of Free Thiols Research Articles

Reversible Polymer-Protein Functionalization by Stepwise Introduction of Amine-Reactive, Reductive-Responsive Self-Immolative End Groups onto RAFT-Derived Polymers.

Many promising therapeutic protein or peptide drug candidates are rapidly excreted from an organism due to their small size or their inherent immunogenicity. One way to counteract these effects is PEGylation, in which the biopolymer is shielded by synthetic polymers exploiting their stealth properties. However, these modifications are often accompanied by a reduction in the biological function of the protein. By using responsive moieties that bridge the polymer to the protein, a reversible character is provided to this type of conjugation. In this regard, the reductive-responsive nature of disulfides can be exploited via self-immolative structures for reversible linkage to aminic lysine residues and the N-terminus on the protein surface. They enable a traceless release of the intact protein without any further modification and thus preserve the protein's bioactivity. In this study, we demonstrate how this chemistry can be made broadly accessible to RAFT-derived water-soluble polymers like poly(N,N-dimethylacrylamide) (pDMA) as a relevant PEG alternative. A terminal reactive imidazole carbamate with an adjacent self-immolative motif was generated in a gradual manner onto the trithiocarbonate chain transfer moiety of the polymer by first substituting it with a disulfide-bridged alcohol and subsequently converting it into an amine reactive imidazole carbamate. Successful synthesis and complete characterization were demonstrated by NMR, size exclusion chromatography, and mass spectrometry. Finally, two model proteins, lysozyme and a therapeutically relevant nanobody, were functionalized with the generated polymer, which was found to be fully reversible under reductive conditions in the presence of free thiols. This strategy has the potential to extend the generation of reversible reductive-responsive polymer-protein hybrids to the broad field of available functional RAFT-derived polymers.

Histochemical detection of free thiols in glandular cells and tissues of different marine Polychaeta.

Either through differentiated glands or specialised individual cells, the coating epithelia of soft-bodied marine invertebrates are responsible for the secretion of a broad span of peptidic substances, from protective mucins to biocides. These secretions are characterised by the presence of cysteine-rich proteins and peptides, rendering a distinct histochemical signature of secretory epithelia. Through a histochemical procedure for fluorescence microscopy in paraffin sections, we performed a comparative assessment of the distribution of thiol-rich compounds in multiple epithelia of different species of intertidal Polychaeta, which revealed distinctive patterns of distribution that closely relate to ecology, morphoanatomy and physiology. The presence of free thiols was notorious in mucocytes and enzyme-plus toxin-secreting cells. Consequently, strong signals were recorded in the mucocytes of the parapodia of Nereis splendida, the epidermis and pharynx epithelium of Mysta picta and the venom glands of Glycera alba. The findings show an investment in mucus secretion in foragers such as Nereis and Mysta, especially the latter, which is not a native burrower, as a protective response and as lubricant for locomotion. Additionally, nereidids are believed to secret integumentary toxins for defence. On the other hand, Glycera is an ambush predatorial burrower whose behaviour entirely revolves around the delivery of venom making use of its four jaws. The results showed that the detection of thiol-rich compounds in histological sections can be a tool to identify potential toxin secretion and delivery structures, with important consequences for the bioprospecting of novel bioreactives from marine invertebrates for the purpose of drug discovery.

Investigating Protein-Nanocrystal Interactions for Photodriven Activity.

We illustrate how intermolecular interactions facilitate ATP-free electron transfer between either native or engineered MoFe protein (MoFeP) from nitrogenase and a CdS nanorod (NR) by following the reduction of H+ to H2. First, by varying the charge on the surface of the NR, we show the role of electrostatic interactions on MoFeP binding to the particle surface and subsequent H+ reduction. Next, the role of strong, semicovalent thiol-CdS interactions was tested using free cysteines on the MoFeP. By blocking free cysteines, we show that the presence of free thiols on the protein has little to no influence on CdS binding and resultant photocatalytic activity. We next studied methods to covalently bind the protein to CdS by modifying the free cysteines with dibenzocyclooctyne (DBCO) and reacting the CdS NRs capped with a mixture of negatively charged thioglycolic acid and thiol-PEG3-azide ligands. As compared to that of the unmodified proteins, a 32.2 ± 1.5% and 61.7 ± 2.1% increase in H2 production was observed from MoFeP and C-MoFeP, respectively. At last, to test the effect of both charge and covalent tethering, positively charged cysteamine/azide CdS NRs were reacted with DBCO-modified C-MoFeP, which showed little improvement over native C-MoFeP alone under irradiation. These results show the importance of both electrostatic associations between the NR and protein and covalently tethering the protein to the semiconductor surface for enhanced electron transfer and photodriven activity.

Application of a label-free and domain-specific free thiol method in monoclonal antibody characterization

Characterization of free thiol variants in antibody therapeutics is important for biopharmaceutical development, as the presence of free thiols may have an impact on aggregate formation, structural and thermal stability, as well as antigen-binding potency of monoclonal antibodies. Most current methods for free thiol quantification involve labeling of free thiol groups by different tagging molecules followed by UV, fluorescence or mass spectrometry (MS) detection. Here, we optimized a label-free liquid chromatography (LC)-UV/MS method for free thiol quantification at a subunit level and compared this method with two orthogonal and conventional approaches, Ellman's assay and peptide mapping with differential alkylation. This subunit unit approach was demonstrated to be able to provide domain-specific free thiol quantification and comparable results with labeling approaches, using a relatively simple and efficient workflow.

Direct measurement of actual levels of nitric oxide (NO) in cell culture conditions using soluble NO donors

Applying soluble nitric oxide (NO) donors is the most widely used method to expose cells of interest to exogenous NO. Because of the complex equilibria that exist between components in culture media, the donor compound and NO itself, it is very challenging to predict the dose and duration of NO cells actually experience. To determine the actual level of NO experienced by cells exposed to soluble NO donors, we developed the CellNO Trap, a device that allows continuous, real-time monitoring of the level of NO adherent cells produce and/or experience in culture without the need to alter cell culturing procedures. Herein, we directly measured the level of NO that cells grown in the CellNO Trap experienced when soluble NO donors were added to solutions in culture wells and we characterized environmental conditions that effected the level of NO in in vitro culture conditions. Specifically, the dose and duration of NO generated by the soluble donors S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), S-nitrosocysteine (CysNO) and the diazeniumdiolate diethyltriamine (DETA/NO) were investigated in both phosphate buffered saline (PBS) and cell culture media. Other factors that were studied that potentially affect the ultimate NO level achieved with these donors included pH, presence of transition metals (ion species), redox level, presence of free thiol and relative volume of media. Then murine smooth muscle cell (MOVAS) with different NO donors but with the same effective concentration of available NO were examined and it was demonstrated that the cell proliferation ratio observed does not correlate with the half-lives of NO donors characterized in PBS, but does correlate well with the real-time NO profiles measured under the actual culture conditions. This data demonstrates the dynamic characteristic of the NO and NO donor in different biological systems and clearly illustrates the importance of tracking individual NO profiles under the actual biological conditions.

The relevance of tissue thiol histochemistry to diagnostic hematopathology

Expression analyses suggest that alterations of the antioxidant state of some diffuse large B-cell lymphomas can assist prognosis; reversibly oxidized thiols may serve as a surrogate marker for identifying such cases. Little is known about the distribution of free thiols and reversibly oxidized thiols in human tissues. We developed a staining technique that enables visualization of tissue thiols in situ using bright field microscopy and validated it using gastrointestinal tissue specimens. We used our thiol staining technique to assess benign tonsillectomy and diffuse large B-cell lymphoma specimens. The gastrointestinal series revealed the presence of free thiols within epithelial cells and cells of the lamina propria. Staining for reversibly oxidized thiols was robust in gastric foveolar cells, intestinal goblet cells and the mucus they produce. Tonsillectomy specimens exhibited diffuse presence of free thiols. Staining for reversibly oxidized thiols was confined to germinal center macrophages and sinus histiocytes. Among the diffuse large B-cell lymphoma specimens, we observed strong staining for free thiols within malignant cells. By contrast to benign B-cells, the malignant cells demonstrated pronounced and diffuse staining for reversibly oxidized thiols. We demonstrated intrinsic differences between benign and malignant cells.

Analysis and characterization of aggregation of a therapeutic Fc-fusion protein

Protein aggregation was observed in a purification intermediate of a therapeutic Fc-fusion protein stored at −30°C, even though the protein was stable at 4 and −80°C. The protein was expressed in Escherichia coli as an inclusion body, refolded, and purified using chromatography columns. To study the nature of this aggregation, a series of experiments were conducted to investigate factors that contributed to the protein instability during freezing. We found that the presence of free thiols in the protein is the intrinsic cause. The free thiol cross-linking sites were determined to be at the peptide moiety of the Fc-fusion protein using LC–MS. Partially frozen accompanied by the elevated pH and increased salt and protein concentrations were identified as extrinsic factors that facilitated the aggregation. These results provided important insights into purification process improvement and solution storage of this Fc-fusion protein.

Thrombospondin 1 (TSP-1) Partially Contributes to Shear or Stirring- Dependent TGF-beta1 Activation In Vitro and During Platelet-Rich Thrombi Formation In Vivo.

Thrombospondin 1 (TSP-1) has been shown to activate latent TGF-beta1 in vitro, but its in vivo role is unclear because TSP-1-null mice have a much less severe phenotype than TGF-beta1-null mice. TSP-1 is stored in platelet alpha-granules and released, along with latent TGF-beta1, with activation. Thus, it is strategically located to contribute to TGF-beta1 activation at sites of platelet deposition. We recently demonstrated that stirring and/ or shear could activate latent TGF-beta1 released from platelets, probably in part through thiol-disulfide exchange and we have identified TSP-1 in human platelet releasates by immunoblotting and observed that it labeled with a thiol-reactive biotin-maleimide probe (MPB) by LC/MS/MS analysis, indicating the presence of free thiols. Remarkably, we found that the amount of TSP-1 detected by immunoblotting in human platelet releasates decreased with stirring or shear and that pretreatment of platelet releasates with the thiol-reactive compounds MPB, NEM or BMCC largely prevented the loss of TSP-1 with shear or stirring. To further assess the role of TSP-1 in TGF-beta1 activation, we have now studied the effect of stirring and shear on TGF-beta1 activation in samples from WT and TSP-1-null mice. Unstirred sera or platelet releasates from WT and TSP-1-null mice had similar levels of total TGF-beta1 [serum, 91 ± 15 ng/mL in WT (n=23) and 108 ± 15 ng/ mL in TSP-1-null mice (n=23; p=0.13); platelet releasates, 58 ± 14 ng/mL in WT (n=14) and 53 ± 16 ng/mL in TSP-1-null mice (n=14; p=0.45)]. With either stirring or shear, the TSP-1-null samples demonstrated less activatibility, but the defect was only partial [stirring increased active TGF-beta1 in serum from 0.6 ± 0.2 ng/mL to 2.2 ± 0.8 ng/mL in WT mice and from 0.5 ± 0.2 ng/mL to 1.6 ± 0.5 ng/mL in TSP-1-null mice (n=23) (p=0.05 using ANOVA analysis for the linear model comparing two-way interactions of stirred samples between WT and TSP-1-null mice); stirring increased active TGF-beta1 in platelet releasates from 0.3 ± 0.2 ng/mL to 2.4 ± 1.5 ng/mL in WT mice (n=14) and from 0.2 ± 0.2 ng/mL to 1.1 ± 0.6 ng/mL in TSP-1-null mice (n=14) (p=0.005)]. To assess in vivo TGF-beta1 activation we induced carotid artery thrombi with FeCl3 in WT and TSP-1-null mice. When thrombi were removed 5 min after total occlusion, the thrombus extracts from WT and TSP-1-null mice contained similar amounts of total and active TGF-beta1 [total TGF-beta1, 5.6 ± 2.4 ng/mL in WT and 5.5 ± 1.6 ng/mL in TSP-1-null mice (n=9) (p=0.9) and active TGF-beta1 (expressed as a percentage of total), 1.7 ± 0.7% in WT and 2.5 ± 1.4% in TSP-1-null mice (n=9) (p=0.15)]. When thrombi were removed 120 min after total occlusion, however, the percentage of active TGF-beta1 in the thrombus extracts was higher in WT than TSP-1-null mice [4.5 ± 2.0% in WT mice (n=9) and 3.3 ± 1.5% in TSP- 1-null mice (n=9) (p=0.02 using ANOVA analysis comparing interactions between time and type of mice)]. We conclude that:TSP-1 released from human platelets undergoes a shear-induced, thiol-dependent conformational change,TSP-1 contributes partially to shear or stirring-dependent TGF-beta1 activation in vitro in mice, andTime-dependent activation of TGF-beta1 released from thrombi formed in vivo in mice is also partially dependent on TSP-1.These data suggest a significant, but minor role of TSP-1 in TGF-beta1 activation in vivo and thus provide an explanation for the less severe phenotype of TSP-1-null mice than TGF-beta1-null mice.

Covalent regulation of ULVWF string formation and elongation on endothelial cells under flow conditions.

The adhesion ligand von Willebrand factor (VWF) is a multimeric glycoprotein that mediates platelet adhesion to exposed subendothelium. On endothelial cells, freshly released ultra-large (UL) VWF multimers form long string-like structures to which platelets adhere. The formation and elongation of ULVWF strings were studied in the presence of the thiol-blocking N-ethylmaleimide (NEM). The presence of thiols in ULVWF and plasma VWF multimers was determined by maleimide-PEO(2)-Biotin labeling and thiol-chromatography. Finally, covalent re-multimerization of ULVWF was examined in a cell- and enzyme-free system. We found that purified plasma VWF multimers adhere to and elongate ULVWF strings under flow conditions. The formation and propagation of ULVWF strings were dose-dependently reduced by blocking thiols on VWF with NEM, indicating that ULVWF strings are formed by the covalent association of perfused VWF to ULVWF anchored to endothelial cells. The association is made possible by the presence of free thiols in VWF multimers and by the ability of (UL) VWF to covalently re-multimerize. The data provide a mechanism by which the thrombogenic ULVWF strings are formed and elongated on endothelial cells. This mechanism suggests that the thiol-disulfide state of ULVWF regulates the adhesion properties of strings on endothelial cells.

Organoditelluride-tethered polymers that spontaneously generate nitric oxide when in contact with fresh blood

An organoditelluride (RTeTeR) species (5,5′-ditelluro-2,2′-dithiophenecarboxylic acid, DTDTCA) that catalytically decomposes endogenous S-nitrosothiols (RSNOs) to nitric oxide (NO) in the presence of free thiols (RSHs) is used to prepare new types of NO generating polymers (NOGPs). In this work, DTDTCA is covalently linked to poly(allylamine hydrochloride) (PAH) to form a water soluble NOGP (polymer 2). This polymer is further crosslinked within a cellulose membrane to yield an interpenetrating polymer network (IPN) (polymer 3). The catalytic NO generation ability of these NOPGs is clearly observed viachemiluminescence detection of liberated NO. The observed NO fluxes of the IPNs in the presence of given concentrations of RSNOs and reducing thiols can be tuned by adjusting the DTDTCA loading of polymer 2 crosslinked within the cellulose support. Polymer 3 is shown to spontaneously generate NO when in contact with fresh blood via amperometric sensor measurements. Hence, these new types of NOGPs are potentially useful as catalytic layers to create new amperometric RSNO sensors and possibly blood contacting medical devices that exhibit improved hemocompatiblity.

Gamma-Glutamyltransferase from the outer cell envelope of Treponema denticola ATCC 35405.

The human oral spirochete Treponema denticola ATCC 35405 was shown to exhibit relatively high enzyme activity toward the gamma-glutamyl amide bond present in N-gamma-L-glutamyl-4-nitroaniline. The enzyme responsible for this catalysis (gamma-glutamyltransferase [GGT]; EC 2.3.2.2) was purified by means of fast protein liquid chromatography to two sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-pure forms from a mild (0.1%) Triton X-100 extract of washed cells. The GGT was studied primarily with regard to its hydrolytic activity by using N-gamma-L-glutamyl-4-nitroaniline as a substrate, although the GGT was shown to catalyze transpeptidation reactions. The high-molecular-mass form of the GGT gave a value of about 213 kDa by SDS-PAGE when heat treatment was omitted and one of 26 kDa after heat treatment; mass spectrometry gave a value of 26.877. The larger form may represent an aggregate with nonprotein structures (possibly of a carbohydrate nature). The preliminary N-terminal sequence of the GGT is MKKPLIGITGSXLYETSQXXF. The enzyme was highly active on glutathione, transferring its Glu residue either to a water molecule or to the Gly-L-Leu dipeptide. The GGT stability was absolutely dependent on the presence of free thiol(s), while no evidence of metalloenzyme nature was obtained. The proposed location of the GGT in the outer cell envelope and its high activity on glutathione, a major nonprotein thiol present in virtually all cells, suggest that the GGT may play a role in the propagation of T. denticola within inflamed periodontal tissues.

The disulfide folding pathway of hirudin elucidated by stop/go folding experiments.

The folding pathway of hirudin was analyzed by structural characterization and stop/go folding experiments of acid-trapped intermediates. The results show that the folding is initiated by a near-random packing, followed by the reorganization and fine adjustment of partially compact intermediates to attain the active molecule. The process of packing is observed as the unfolded hirudin flows sequentially via three groups of equilibrated intermediates, namely one-disulfide, two-disulfide, and three-disulfide (scrambled species) isomers. Nearly all possible disulfide species were found to exist along the pathway. Specific tertiary interactions then take effect at the final stage, refining and consolidating the loosely packed intermediates, in the presence of free thiols, to form the active hirudin. The rate of packing and reorganization can be selectively regulated by a number of external factors, and conditions can be chosen to allow the completion of folding process within 10 min or 10 h.

Ribonuclease inhibitor from human placenta. Purification and properties.

A soluble ribonuclease inhibitor from the human placenta has been purified 4000-fold by a combination of ion exchange and affinity chromatography. The inhibitor has been isolated in 45% yield (about 2 mg/placenta) as a protein that is homogeneous by sodium dodecyl sulfate-gel electrophoresis. In common with the inhibitors of pancreatic ribonuclease from other tissues that have been studied earlier, the placental inhibitor is an acidic protein of molecular weight near 50,000; it forms a 1:1 complex with bovine pancreatic RNase A and is a noncompetitive inhibitor of the pancreatic enzyme, with a Ki of 3 X 10(-10) M. The amino acid composition of the protein has been determined. The protein contains 30 half-cystine plus cysteine residues determined as cysteic acid after performic acid oxidation. At pH 8.6 the nondenatured protein alkylated with iodoacetic acid in the presence of free thiol has 8 free sulfhydryl groups. The inhibitor is irreversibly inactivated by sulfhydryl reagents and also by removal of free thiol from solutions of the protein. Inactivation by sulfhydryl reagents causes the dissociation of the RNase - inhibitor complex into active RNase and inactive inhibitor.

The thermolability of tissue thromboplastins.

1. The thermolability of tissue thromboplastins is markedly increased in the presence of cysteine, glutathione sulphite and hydrosulphite, but not in the presence of cysteine, ascorbate, thiosulphate or thiocyanate. 2. Thermostability is increased by fluoride (and ageing), but is overcome by cysteine. 3. These effects were demonstrated with tissue thromboplastins from brain, chorion and a thyroid carcinoma, and were found equally with conventional water-soluble and lipoid-type substances. 4. Thermolability appears to be dependent upon the presence of free thiols or their equivalent in an enzyme catalysed reaction.