Disulfide-linked Polymer Research Articles

Unveiling the adsorption mechanism of beta-blockers and sulfonamides in aqueous environment using disulfide-linked polymer

Herein, we investigated the environmental application of negatively charged disulfide-linked polymer (DCOP) for selectively removing charged pharmaceuticals, such as beta-blockers and sulfonamides. To compare the effect of surface charge on the adsorption affinity of DCOP for target compounds, atenolol (ATL) and propranolol (PPL) were selected as positively charged beta-blockers, whereas sulfamethazine (SMZ) and sulfasalazine (SFS) were selected as negatively charged sulfonamides. Adsorption experiments, including those considering kinetic, isotherm, and water matrix effects, were performed to study the adsorption behavior of DCOP. Further, density functional theory (DFT) calculations were performed to verify the adsorption mechanism through computational calculation. Accordingly, DCOP exhibited faster and higher adsorption performance for beta-blockers than sulfonamides. The maximum adsorption capacities calculated by the Langmuir model of ATL (Qm,ATL 28.57 mg/g) and PPL (Qm,PPL 20.75 mg/g) were higher than those of SMZ (Qm,SMZ 13.72 mg/g) and SFS (Qm,SFS 13.42 mg/g). Moreover, the relatively hydrophobic compounds, such as PPL and SFS, were removed more efficiently than the hydrophilic compounds such as ATL and SMZ, revealing the importance of hydrophobic interaction. Moreover, DCOP exhibited a similar adsorption performance in the presence of humic acid and tap water. The adsorption energy calculated via DFT modeling corroborated the preferential adsorption of beta-blockers, supporting the experimental results. Consequently, DCOP could be used as a practical adsorbent in terms of one-pot synthesis, structural stability, and anti-interference ability by water matrix factors.

Selective removal of cationic organic pollutants using disulfide-linked polymer

Dyes are widely used in various industries, and the industrial use of dyes has increased steadily over the past few decades. However, it is difficult to remove these dyes from effluents by using conventional wastewater treatment processes owing to their chemical and physical stability. Herein, we propose the possibility of using disulfide-linked polymer (DiS-COP) as an effective adsorbent for selectively removing cationic dyes. The selective adsorption ability of DiS-COP was tested using two cationic dyes, namely methylene blue (MB) and crystal violet (CV), and two anionic dyes, namely methyl orange (MO) and orange G (OG). The adsorption equilibrium is reached in less than 4 h for all dyes with extremely fast removal for CV. The kinetic results were analyzed using the pseudo-first-order, pseudo-second-order, and intraparticle diffusion model. The maximum adsorption capacity for cationic dyes, as obtained using the Langmuir isotherm, was considerably higher than that for anionic dyes. DiS-COP exhibited 10 (Qm,MB 211 mg/g) and 56 times (Qm,CV 1181 mg/g) higher maximum adsorption capacities for MB and CV, respectively, than that for the anionic dye MO (Qm,MO 21 mg/g). Selective adsorption of cationic dyes in the binary system was successfully realized. The results of the pH effect experiment revealed the selective adsorption mechanism. The electrostatic interaction between negatively charged DiS-COP and charged dye molecules induced the selective attraction of cationic dyes. Moreover, the effect of π-π stacking between DiS-COP and the dye molecules, as well as that between the adsorbed dye molecules and the free dye molecules in the solution, facilitated the dye adsorption. The removal mechanism was further elucidated using Fourier transform infrared spectrometry and X-ray photoelectron spectroscopy. In addition, the results of the adsorption experiments involving the selected pharmaceuticals indicated that DiS-COP was able to remove these cationic pharmaceuticals to a significantly greater extent than it was able to remove the those anonic and neutrally charged pharmaceuticals. The DiS-COP used in this study, which can be easily prepared via a one-pot reaction, has the potential for use as an adsorbent to selectively remove cationic organic pollutants.

Stepwise pH-sensitive and biodegradable polypeptide hybrid micelles for enhanced cellular internalization and efficient nuclear drug delivery

The short blood circulation time, reduced cellular uptake, and uncontrollable drug release still hinder the polymer micelle as an efficient drug delivery vehicle in clinical applications. In this study, a series of stepwise pH-sensitive and biodegradable polypeptide hybrid terpolymers, poly (lysine-co-N,N-bis(acryloyl) cystamine-co-dimethylmaleic anhydride) (PLB-DMMA), were designed and synthesized to achieve prolonged circulation time, enhanced cellular uptake and controllable anti-cancer drug release. The synthesized terpolymers can self-assemble into spherical nano-micelles (NMs) with narrow distributions and exhibited stepwise responses to extracellular and intracellular pH condition of the tumor cell. The as prepared NMs showed a negative surface charge under normal physiological conditions exhibiting advantageous stability during blood circulation. By the first-step pH response, the surface charge of the NMs switched from negative to positive to enhance cellular uptake under the slightly acidic tumor extracellular environment. After internalization into tumor cells, the second-step pH response resulted in an endosome escape of the NMs via the “proton-sponge” effect in the acidic endo/lysosome environment. Additionally, a rapid drug release was triggered in response to the intracellular reductive environment of tumor cells via the destruction of disulfide-linked polymer chains to enhance the nucleus delivery of DOX. in vitro cell assays showed that the blank NMs showed negligible systemic toxicity against normal cells while the DOX-loaded NMs significantly inhibited growth of the tumor cells. In general, it was suggested that the as developed stepwise pH-sensitive and biodegradable PLB-DMMA based NMs would be a smart and promising drug delivery candidate for anti-cancer chemotherapy.

Rapid synthesis of a silsesquioxane-based disulfide-linked polymer for selective removal of cationic dyes from aqueous solutions

Developing a facile and rapid method to prepare adsorbents for an efficient and selective removal of dyes is of great importance for human health and environment. In this work, a silsesquioxane-based disulfide-linked polymer (DLP) was rapidly synthesized by a simple oxidative coupling reaction of octa(3-mercaptopropyl)silsesquioxane within 2 min at room temperature. The synthesized DLP demonstrated selective and efficient adsorption of cationic dyes over anionic dyes with the maximum capacities of 12.94 mg g−1 for methylene blue, 18.47 mg g−1 for rhodamine B and 26.69 mg g−1 for crystal violet. The high uptake and selectivity for cationic dyes was attributed to the electrostatic interactions between the anionic charges from sulfur and oxygen anions formed by the deprotonation of thiols and hydroxyls in the DLP and the cationic charges from the dyes, and the swelling behavior of the DLP. To investigate the adsorption process, the effect of various factors such as contact time, initial concentration, temperature, and pH on the adsorption activity of cationic dyes was studied. The adsorption equilibrium data showed a better fitting to the Langmuir isotherm model than the Freundlich model and the adsorption kinetics fitted well with the pseudo-second-order kinetic model, indicating that the adsorption process is mostly governed by the chemisorption behavior. Additionally, the absorbent was recyclable with the adsorption capacity being >95% after five times recycle adsorption. These results indicated that the present disulfide-linked polymer could be promisingly applied as an efficient adsorbent for the selective removal of cationic dyes from industrial wastewater.

Polymer ligand-assisted fabrication of multifunctional and redox-responsive self-assembled magnetic nanoclusters for bimodal imaging and cancer treatment.

Multifunctional nanoplatforms that can combine diagnostic functions and therapeutic capabilities are highly required for the construction of novel cancer diagnosis-therapy integrative systems. In this work, we developed multifunctional and tumor redox-responsive magnetic nanoclusters (RMNs) by the self-assembly of iron oxide nanoparticles with a chlorin e6 (Ce6)-disulfide-linked polymer ligand. The core of RMNs could enhance the T2-weighted MR contrast capability, and the surface ligand could improve the system stability and load Ce6. In addition, the obtained RMNs also exhibited excellent biocompatibility and higher cytotoxicity upon near infrared (NIR)-irradiation. These RMNs with a size of ∼90 nm can effectively facilitate their accumulation in tumor sites through the enhanced permeability and retention (EPR) effect and subsequently are endocytosed into a highly reductive intracellular environment that activates fluorescence and photodynamic therapeutic antitumor activity. Furthermore, the in vivo imaging and therapy results not only confirmed that RMNs can as bimodal imaging probes for the early-stage diagnosis of cancer but also demonstrated the superior photodynamic therapeutic efficiency of RMNs in suppressing tumor growth. These results indicated that RMNs show great potential for simultaneous diagnostic imaging and photodynamic therapy.

Triggered Cargo Release by Encapsulated Enzymatic Catalysis in Capsosomes

We report the coencapsulation of glutathione reductase and disulfide-linked polymer-oligopeptide conjugates into capsosomes, polymer carrier capsules containing liposomal subcompartments. The architecture of the capsosomes enables a temperature-triggered conversion of oxidized glutathione to its reduced sulfhydryl form by the encapsulated glutathione reductase. The reduced glutathione subsequently induces the release of the encapsulated oligopeptides from the capsosomes by reducing the disulfide linkages of the conjugates. This study highlights the potential of capsosomes to continuously generate a potent antioxidant while simultaneously releasing small molecule therapeutics.

Continuous Molecular Evolution of Protein-Domain Structures by Single Amino Acid Changes

Protein structures cluster into families of folds that can result from extremely different amino acid sequences [1]. Because the enormous amount of genetic information generates a limited number of protein folds [2], a particular domain structure often assumes numerous functions. How new protein structures and new functions evolve under these limitations remains elusive. Molecular evolution may be driven by the ability of biomacromolecules to adopt multiple conformations as a bridge between different folds [3-6]. This could allow proteins to explore new structures and new tasks while part of the structural ensemble retains the initial conformation and function as a safeguard [7]. Here we show that a global structural switch can arise from single amino acid changes in cysteine-rich domains (CRD) of cnidarian nematocyst proteins. The ability of these CRDs to form two structures with different disulfide patterns from an identical cysteine pattern is distinctive [8]. By applying a structure-based mutagenesis approach, we demonstrate that a cysteine-rich domain can interconvert between two natively occurring domain structures via a bridge state containing both structures. Comparing cnidarian CRD sequences leads us to believe that the mutations we introduced to stabilize each structure reflect the birth of new protein folds in evolution.

The Glycoprotein NOWA and Minicollagens Are Part of a Disulfidelinked Polymer That Forms the Cnidarian Nematocyst Wall

The nematocyst is a unique extrusive organelle involved in the defense and capture of prey in cnidarians. Minicollagens and the glycoprotein NOWA are major components of the nematocyst capsule wall, which resists osmotic pressure of 15 MPa. Here we present the recombinant expression of NOWA, which spontaneously assembles to globular macromolecular particles that are sensitive to reduction as the native wall structure. Ultra-structural analysis showed that the Hydra nematocyst wall is composed of several layers of globular particles, which are interconnected via radiating rodlike protrusions. Evidence is presented that native wall particles contain NOWA and minicollagen, supposed to be linked via disulfide bonds between their homologous cysteine-rich domains. Our data suggest a continuous suprastructure of the nematocyst wall, assembled from wall proteins that share a common oligomerization motif.

Mutations in α-Chain of C4BP That Selectively Affect Its Factor I Cofactor Function

C4b-binding protein (C4BP) inhibits all pathways of complement activation, acting as a cofactor to the serine protease factor I (FI) in the degradation of activated complement factors C4b and C3b. C4BP is a disulfide-linked polymer of seven alpha-chains and a unique beta-chain, the alpha- and beta-chains being composed of eight and three complement control protein (CCP) domains, respectively. In previous studies we have localized cofactor activity and binding of C4b to alpha-chain CCP1-3 of C4BP, whereas the binding of C3b required additionally CCP4. Likewise, introduced point mutations that decreased binding of C4b/C3b caused a decrease in cofactor activity. In the present study, we describe two mutants of C4BP, K126Q/K128Q and F144S/F149S, clustered on alpha-chain CCP3, which selectively lost their ability to act as cofactors in the cleavage of both C4b and C3b. Both mutants show the same binding affinity for C4b/C3b as measured by surface plasmon resonance and have the same inhibitory effect on formation and decay of the classical pathway C3-convertase as the wild type C4BP. It appears that C4b and C3b do not undergo the same conformational changes upon binding to the C4BP mutants as during the interaction with the wild type C4BP, which then results in the observed loss of the cofactor activity.

Structural Requirements for the Complement Regulatory Activities of C4BP

C4b-binding protein (C4BP) is a regulator of the classical complement pathway C3 convertase (C4bC2a complex). It is a disulfide-linked polymer of seven alpha-chains and a unique beta-chain; the alpha- and beta-chains are composed of eight and three complement control protein (CCP) domains, respectively. To elucidate the importance of the polymeric nature of C4BP and the structural requirements for the interaction between C4b and the alpha-chain, 19 recombinant C4BP variants were created. Six truncated monomeric variants, nine polymeric variants in which individual CCPs were deleted, and finally, four variants in which double alanine residues were introduced between CCPs were functionally characterized. The smallest truncated C4BP variant still active in regulating fluid phase C4b comprised CCP1-3. The monomeric variants were less efficient than polymeric C4BP in degrading C4b on cell surfaces. All three N-terminal CCP domains contributed to the binding of C4b and were important for full functional activity; CCP2 and CCP3 were the most important. The spatial arrangements of the first CCPs were found to be important, as introduction of alanine residues between CCPs 1 and 2, CCPs 2 and 3, and CCPs 3 and 4 resulted in functional impairment. The results presented here elucidate the structural requirements of individual CCPs of C4BP, as well as their spatial arrangements within and between subunits for expression of full functional activity.

The human MUC2 intestinal mucin has cysteine-rich subdomains located both upstream and downstream of its central repetitive region.

The human MUC2 mucin is a large secretory glycoconjugate that coats the epithelia of the intestines, airways, and other mucus membrane-containing organs. Previous work has shown that this mucin contains an extended tandem repeat-containing domain rich in Thr and Pro. In the present work we describe two additional regions of this mucin located both upstream and downstream of the tandem repeat array. The carboxyl-terminal domain contains 984 residues and can be divided into mucin-like (139 residues) and cysteine-rich (845 residues) subdomains. This latter subdomain exhibits varying degrees of sequence similarity to a wide range of mucins and mucin-like proteins including those isolated from rats, pigs, cows, and frogs. We also report here the sequence of 1270 residues lying immediately upstream of the tandem repeats. This region contains a repetitive, mucin-like subdomain and a second cysteine-rich stretch of more than 700 residues. Both cysteine-rich subdomains of this mucin have sequence similarity with von Willebrand factor, a serum protein that exists as a disulfide-linked polymer. This suggests that these cysteine-rich subdomains are important in the catenation of mucin monomers into oligomers, the structures that confer viscoelasticity upon mucus.

Substances that polymerize or depolymerize cytoskeletal proteins affect platelet spreading and thrombus-formation on surfaces coated with human collagen isotypes I, IV, and V

The effect of substances that affect platelet cytoskeleton on the interaction of gel-filtered platelets with surfaces coated with human monomeric type I, IV, and V collagen was studied. The sulfhydryl group oxidizing agent azodicarboxylic acid-bis-dimethylamide (diamide) which causes disulfide-linked polymer formation of certain cytoskeletal proteins, the actin-polymerization inhibitor, cytochalasin B, and 2-mercaptopropionylglycine (2-MPG), a cell-permeable SH-reagent, completely abolish adhesion-induced platelet spreading and mural platelet aggregate formation on collagen-coated surfaces. Extrusion of pseudopods was inhibited by cytochalasin B and 2-MPG as well as by diamide, but only the latter caused spherulation of platelets, whereas cytochalasin 8 and 2-MPG left the discoid shape of resting platelets intact. These effects are dose-dependend and are not accounted for by a chemical modification of the collagenous substrates by the cytoskeletal perturbing substances. The present data indicate that (i) cytoskeletal rearrangements are essential in adhesion-induced platelet spreading and aggregate formation on surfaces coated with collagen, but not in supporting the initial attachment of native platelets to the substrate; (ii) both, polymerization and depolymerization of actin filaments affect platelet activation; (iii) the sulfhydryl-disulfide status of the platelet seems to be a possible target for anti-platelet drugs, since chemical modification of platelets by the GSH-GSSG-active substances, diamide and 2-MPG, leads to a reversible inhibition of adhesion-induced platelet activation.

Chemical modification of cytoskeletal proteins of human blood platelets by diamide

Incubation of human blood platelets with diamide (azo-dicarboxylic acid-bis-dimethylamide, DIA) influences the aggregation behaviour considerably. Depending on concentration and incubation time DIA induces a reversible aggregation or brings about complete inhibition. The effect is reversible and may be due to the regeneration of reduced glutathione (GSH) which will be oxidized to GSSG by DIA. DIA causes disulfide-linked polymer formation of certain cytoskeletal proteins. At least three polymer families (P a, P b, P c) with different molecular weights are formed depending on dosage and incubation time of DIA. The appearance of a double band in P a correlates with reversible aggregation, the formation of P c is always accompanied by a complete inhibition of aggregation. A disturbance of cytoskeleton-membrane interaction by polymer formation can be assumed. GSH serves as a reductant of disulfide-linked polymers whereby a direct link between the maintenance of SH/SS status of platelets and GSH can be established.

Purification and characterization of a macromolecular weight cofactor for C3b-inactivator, C4bC3bINA-cofactor, of human plasma

The macromolecular weight cofactor for C3b-inactivator, C4bC3bINA-cofactor, was purified by an improved method and its protein nature was investigated. The purification procedures were composed of the removal of cold insoluble globulin with gelatin-Sepharose, polyethylene glycol fractionation, gel filtration on Bio-Gel A-15m, heparin-Sepharose chromatography, and the removal of IgM with anti-IgM-Sepharose. The molecular weight of C4bC3bINA-cofactor was estimated to be 450,000 by SDS-polyacrylamide gel electrophoresis. ∗ The reduced cofactor gave a single band of 75,000 daltons upon SDS-polyacrylamide gel electrophoresis, suggesting that the cofactor is a disulfide-linked polymer (probably a hexamer) of similar or identical polypeptide chains, each with a molecular weight of 75,000. The cofactor is a glycoprotein and the isoelectric point of the cofactor was estimated to be 6.7. No N-terminal amino acid was detected by the dansylation technique, suggesting that the N-terminal of the cofactor would be blocked. Upon immunoelectrophoresis, the cofactor migrates as a slow β-globulin, and its mobility was shifted towards the anodal side when C4b was added to the cofactor. This result suggested that the cofactor was functionally the same as the C4-binding protein.

Immunoglobulin M heavy chain disease: intracellular origin of the mu chain fragment.

Cells obtained from a patient with mu heavy chain disease synthesize a mu heavy chain fragment with a molecular weight of 55,000. The fragment is detected intracellularly after short labeling times and then is assembled inside the cell and secreted as a disulfide-linked polymer.