Promiscuous Enzymes for Residue-Specific Peptide and Protein Late-Stage Functionalization

The late-stage functionalization of peptides and proteins holds significant promise for drug discovery and facilitates bioorthogonal chemistry. This selective functionalization leads to innovative advances in in vitro and in vivo biological research. However, it is a challenging endeavor to selectively target a certain amino acid or position in the presence of other residues containing reactive groups. Biocatalysis has emerged as a powerful tool for selective, efficient, and economical modifications of molecules. Enzymes that have the ability to modify multiple complex substrates or selectively install nonnative handles have wide applications. Herein, we highlight enzymes with broad substrate tolerance that have been demonstrated to modify a specific amino acid residue in simple or complex peptides and/or proteins at late-stage. The different substrates accepted by these enzymes are mentioned together with the reported downstream bioorthogonal reactions that have benefited from the enzymatic selective modifications.     

Involvement of Acidic Amino Acid Residues in Zn2+ Binding to Respiratory Complex I

Proton transfer across membranes and membrane proteins is a central process in biological systems. Zn²⁺ ions are capable of binding to acidic residues, often found within such specific pathways, thereby leading to a blockage. Here we probed Zn²⁺inhibition of the proton‐pumping NADH:ubiquinone oxidoreductase from Escherichia coli by means of electrochemically induced FTIR difference spectroscopy. Numerous conformational changes were identified including those that arise from the reorganization of the membrane arm upon electron transfer in the peripheral arm of the protein. Signals at very high wavenumbers (1781 and 1756 cm^?1) point to the perturbation of acidic residues in a highly hydrophobic environment upon Zn²⁺ binding. In variant D563N^L, which lacks part of the proton pumping activity (residue located on the horizontal amphipathic helix), the spectral signature of Zn²⁺ binding is changed. Our data support a role for this residue in proton translocation.     

Adsorption and Separation of Mercury: Sorption-Desorption of Hg2+ with Cross-Linked Graft Copolymer of Acrylic Acid and its Application in the Metal Ion Separation Process

A selective and reliable method has been developed for the extraction and separation of mercuric ion with cross-linked graft copolymer of acrylic acid based on sorption-desorption studies. The graft copolymer acts as an ion exchanger. The physico-chemical properties of the exchanger, and optimum pH, time, and temperature for Hg²⁺ adsorption were determined. Metal ion adsorption kinetics, isotherms, and thermodynamics have been studied. A plausible mechanism for mercury ion extraction has been suggested. Mercuric ion has been separated quantitatively from various synthetic mixtures containing metal ions (Ni²⁺, Cd²⁺, Pb²⁺, and Zn²⁺).     

Removal of heavy‐metal ions by magnetic beads containing triazole chelating groups

The aim of this study was to prepare magnetic beads that could be used for the removal of heavy‐metal ions from synthetic solutions. Magnetic poly(ethylene glycol dimethacrylate–1‐vinyl‐1,2,4‐triazole) [m‐poly(EGDMA–VTAZ)] beads were produced by suspension polymerization in the presence of a magnetite Fe₃O₄ nanopowder. The specific surface area of the m‐poly(EGDMA–VTAZ) beads was 74.8 m²/g with a diameter range of 150–200 μm, and the swelling ratio was 84%. The average Fe₃O₄ content of the resulting m‐poly(EGDMA–VTAZ) beads was 14.8%. The maximum binding capacities of the m‐poly(EGDMA–VTAZ) beads from aquous solution were 284.3 mg/g for Hg²⁺, 193.8 mg/g for Pb²⁺, 151.5 mg/g for Cu²⁺, 128.1 mg/g for Cd²⁺, and 99.4 mg/g for Zn²⁺. The affinity order on a mass basis was Hg²⁺ > Pb²⁺ > Cu²⁺ > Cd²⁺> Zn²⁺. The binding capacities from synthetic waste water were 178.1 mg/g for Hg²⁺, 132.4 mg/g for Pb²⁺, 83.5 mg/g for Cu²⁺, 54.1 mg/g for Cd²⁺, and 32.4 mg/g for Zn²⁺. The magnetic beads could be regenerated (up to ca. 97%) by a treatment with 0.1M HNO₃. These features make m‐poly(EGDMA–VTAZ) beads potential supports for heavy‐metal removal under a magnetic field. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Selective and Simultaneous Extractions of Zn and Cu Ions by Hollow Fiber SLM Modules Containing HEH(EHP) and LIX84

Abstract

The selective extractions of Zn²⁺ and Cu²⁺ from their mixed solutions of sulfate medium have been studied using hollow fiber supported liquid membranes (HFSLM). The HFSLM contained two kinds of extractants; one contained 2-ethylhexylphosphonic acid mono-2-ethylhexyl ester [HEH(EHP)], the commercial name of which is PC88A, for Zn extraction; the other contained the hydroxy oxime reagent LIX84 for Cu extraction. Individual runs of each HFSLM were made to determine the effect of operational variables on the permeation rates of metal ions and their separation factors. In addition, the simultaneous and selective extractions of both Zn²⁺ and Cu²⁺ from their mixed solutions were demonstrated using the PC88A and LIX84 HFSLMs together. The performance of simultaneous extraction was compared with those of the individual runs.     

New Organic-Inorganic Type Acrylamide Aluminumtungstate: Preparation,Characterization and Analytical Applications as a Cation Exchange Material

Abstract

A crystalline sample of organic-inorganic cation exchanger acrylamide aluminumtungstate has been synthesized. The material behaves as a mono-functional cation-exchanger with an ion–exchange capacity 1.25 meq/g for Na⁺ ions. The material has been characterized on the basis of thermal stability, chemical stability, FTIR, TGA-DTA, X-ray, and SEM studies. The effect of time and temperature on the distribution coefficient of metal ion was studied. It was concluded that 30°C appeared to be the most favorable temperature. Sorption behavior of the metal ions was studied in different solvent systems. On the basis of distribution studies, the material was found to be selective for Pb²⁺ ions. Its selectivity was examined by achieving some important binary separations like Mg²⁺-Pb²⁺, Hg²⁺-Pb²⁺, Ca²⁺-Pb²⁺ Zn²⁺-Pb²⁺, Ni²⁺-Pb²⁺, and Al³⁺-Pb²⁺. The practical applicability of the cation-exchanger was demonstrated in the separation of Pb²⁺ ions from a synthetic mixture.     

Tetraamminezinc complex integrated interpenetrating polymer network nanocomposite membrane for phosphorous recovery

Phosphorous recovery from wastewater by nanofiltration (NF) is a feasible and sustainable approach, but challenges still exist in the development of highly efficient membranes for the selective permeation of phosphorus. Herein an interpenetrating network (IPN) of crosslinked polyvinyl alcohol (PVA) is integrated with Zn²⁺ moieties through coordination with the amino groups of crosslinked polyethyleneimine (PEI) and deposited on borate crosslinked polydopamine-grafted carbon nanotubes (B-PDA-CNT) intermediate layer incorporated into the microfiltration substrate to form the composite membrane. The positively charged membrane in acidic conditions affords good rejection (>85%) of heavy metals ions including Cd²⁺, Cu²⁺, Zn²⁺, Pb²⁺, and Ni²⁺. Meanwhile, a low phosphorus rejection of <15% is realized with the application of a relatively low transmembrane pressure. This presupposes that the developed approach achieves selective removal of heavy metals from a phosphate solution and is attractive for low pressure recovery of phosphorus. © 2018 American Institute of Chemical Engineers AIChE J, 65: 755–765, 2019     

Behavior of acrylic acid–itaconic acid hydrogels in swelling,shrinking, and uptakes of some metal ions from aqueous solution

An experimental research of the absorption properties of metal ions onto synthetic hydrogel obtained by solution polymerization of acrylic acid and itaconic acid in presence of N,N′‐methylenebisacrylamide as crosslinking agent was carried out. The swelling behavior in aqueous salt solutions was studied as a function of divalent cation concentration (Cu²⁺, Zn²⁺, Ni²⁺, Co²⁺, Cd²⁺, Pb²⁺, Hg²⁺) in the external solution ranging from 10^?5 to 1M, at 25°C. The ability of these hydrogels to bind cations was measured at different pH values and metal ion concentrations. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 530–536, 2003     

Calcium Regulates S100A12 Zinc Sequestration by Limiting Structural Variations

Antimicrobial proteins such as S100A12 and S100A8/A9 are highly expressed and secreted by neutrophils during infection and participate in human immune response by sequestering transition metals. At neutral pH, S100A12 sequesters Zn²⁺ with nanomolar affinity, which is further enhanced upon calcium binding. We investigated the pH dependence of human S100A12 zinc sequestration by using Co²⁺ as a surrogate. Apo-S100A12 exhibits strong Co²⁺ binding between pH 7.0 and 10.0 that progressively diminishes as the pH is decreased to 5.3. Ca²⁺-S100A12 can retain nanomolar Co²⁺ binding up to pH 5.7. NMR spectroscopic measurements revealed that calcium binding does not alter the side-chain protonation of the Co²⁺/Zn²⁺ binding histidine residues. Instead, the calcium-mediated modulation is achieved by restraining pH-dependent conformational changes to EF loop 1, which contains Co²⁺/Zn²⁺ binding Asp25. This calcium-induced enhancement of Co²⁺/Zn²⁺ binding might assist in the promotion of antimicrobial activities in humans by S100 proteins during neutrophil activation under subneutral pH conditions.     

Zn<Superscript>2+</Superscript>-exchange kinetics and antimicrobial properties of synthetic zirconium umbite (K<Subscript>2</Subscript>ZrSi<Subscript>3</Subscript>O<Subscript>9</Subscript>·H<Subscript>2</Subscript>O)

Zirconium umbite, K₂ZrSi₃O₉·H₂O, is a microporous framework ion exchanger whose potential as a carrier for Zn²⁺ ions in antimicrobial formulations has not yet been investigated. Accordingly, batch Zn²⁺-exchange kinetics of synthetic zirconium umbite (K-UM) and the subsequent antimicrobial action of the zinc-bearing phase (Zn-UM) against Staphylococcus aureus and Escherichia coli are reported. Nonstoicheiometric over-exchange of Zn²⁺ for K⁺ was observed and attributed to hydrolysis and complexation reactions of Zn²⁺ within the umbite framework. The exchange process, which was described by a simple pseudo-first-order model (k ₁ = 2.69 × 10⁻⁴ min⁻¹, R ² = 0.992), did not achieve equilibrium within 120 h at 25 °C, by which time the uptake of zinc was found to be 1.04 mmol g⁻¹. The minimal bactericidal concentrations of Zn-UM for E. coli and S. aureus were found to be >10 g cm³ and <1.0 g cm³, respectively.     

Protective Role of Glutathione in the Hippocampus after Brain Ischemia

Stroke is a major cause of death worldwide, leading to serious disability. Post-ischemic injury, especially in the cerebral ischemia-prone hippocampus, is a serious problem, as it contributes to vascular dementia. Many studies have shown that in the hippocampus, ischemia/reperfusion induces neuronal death through oxidative stress and neuronal zinc (Zn²⁺) dyshomeostasis. Glutathione (GSH) plays an important role in protecting neurons against oxidative stress as a major intracellular antioxidant. In addition, the thiol group of GSH can function as a principal Zn²⁺ chelator for the maintenance of Zn²⁺ homeostasis in neurons. These lines of evidence suggest that neuronal GSH levels could be a key factor in post-stroke neuronal survival. In neurons, excitatory amino acid carrier 1 (EAAC1) is involved in the influx of cysteine, and intracellular cysteine is the rate-limiting substrate for the synthesis of GSH. Recently, several studies have indicated that cysteine uptake through EAAC1 suppresses ischemia-induced neuronal death via the promotion of hippocampal GSH synthesis in ischemic animal models. In this article, we aimed to review and describe the role of GSH in hippocampal neuroprotection after ischemia/reperfusion, focusing on EAAC1.     

Poly(2‐acrylamido glycolic acid‐co‐2‐acrylamido‐2‐methyl‐1‐propane sulfonic acid): Synthesis,characterization, and retention properties for environmentally impacting metal ions

Poly(2‐acrylamido glycolic acid‐co‐2‐acrylamido‐2‐methyl‐1‐propane sulfonic acid) [P(AGA‐co‐APSA)] was synthesized by radical polymerization in an aqueous solution. The water‐soluble polymer, containing secondary amide, hydroxyl, carboxylic, and sulfonic acid groups, was investigated, in view of their metal‐ion‐binding properties, as a polychelatogen with the liquid‐phase polymer‐based retention technique under different experimental conditions. The investigated metal ions were Ag⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Pb²⁺, and Cr³⁺, and these were studied at pHs 3, 5, and 7. P(AGA‐co‐APSA) showed efficient retention of all metal ions at the pHs studied, with a minimum of 60% for Co(II) at pH 3 and a maximum close to 100% at pH 7 for all metal ions. The maximum retention capacity (n metal ion/n polymer) ranged from 0.22 for Cd²⁺ to 0.34 for Ag⁺. The antibacterial activity of Ag⁺, Cu²⁺, Zn²⁺, and Cd²⁺ polymer–metal complexes was studied, and P(AGA‐co‐APSA)–Cd²⁺ presented selective antibacterial activity for Staphylococcus aureus with a minimum inhibitory concentration of 2 μg/mL. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Synthesis,characterization, and properties for Hg2+ of new chelating resin containing sulfoxide and 3‐aminopyridine

A new chelating resin (PVESO‐AP) containing sulfoxide and 3‐aminopyridine (AP) was synthesized by using chloromethylated polystyrene‐co‐divinylbenzene (PS‐Cl) as material. Its structure was characterized by infrared spectra and elemental analysis. The adsorption capacities of the newly formed resin for Hg²⁺, Ag⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Ni²⁺ were determined at pH 2 and 5. In addition, the adsorption selective coefficients for Hg²⁺ in several binary mixture systems were investigated by batch experiment method. The results showed that this resin had high adsorption selectivity for Hg²⁺. The adsorption kinetics and isothermal of PVESO‐AP for Hg²⁺ at different temperatures were also investigated. The adsorption kinetics showed that the apparent activation energy E_a was 13.71 kJ/mol. The results also showed that the Langmuir model was better than the Frundlich model to describe the isothermal process of PVESO‐AP resin for Hg²⁺. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Poly[poly(oxypropylene) phosphate] macroionophores for transport and separation of cations in a hybrid: Cation‐exchange polymer and liquid membrane system

Poly[poly(oxypropylene) phosphate]s (PPOPP, M_n = 5800, 8100, 10,400), with different POP units (400, 1200, 2000), were synthesized and applied as cation‐selective macroionophores in a multimembrane hybrid system (MHS). The solution of PPOPP in dichloroethane formed the flowing liquid membrane (FLM) circulating between two polymer cation‐exchange membranes, and subsequently, between two polymer‐made pervaporation (PV) membranes. It was found that the PPOPP macroionophores activate the preferential transport of Zn²⁺ cations from aqueous solutions containing competing Cu²⁺, Ca²⁺, Mg²⁺, K⁺, and Na⁺ cations. The following separation orders were observed for PPOPPs with POP‐400 and POP‐1200: Zn²⁺ > Cu²⁺ ? Ca²⁺, Mg²⁺, K⁺, Na⁺, and for PPOPP with POP‐2000: Zn²⁺ > Cu²⁺,Ca²⁺ ? Mg²⁺, K⁺, Na⁺. Always, the particular cations are separated as: Zn²⁺ > Cu²⁺, Ca²⁺ > Mg²⁺, and K⁺ > Na⁺. The properties of PPOPPs were compared to respective transport and separation characteristics corresponding to those of respective poly(propylene glycol)s and poly(oxypropylene) bisphosphates. The results of investigation indicate that the bifunctional character of PPOPPs is caused by the presence of ionizable groups and probably pseudocyclic POP structures. By comparing the separation of cations in the simple MHS[FLM] system and the system supported by pervaporation unit [MHS[FLM‐PV] it was found that continuous dehydration of an organic FLM improves the system overall performance. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1436–1445, 2004     

Profiling Protein S-Sulfination with Maleimide-Linked Probes

Cysteine residues are susceptible to oxidation to form S-sulfinyl (R-SO₂H) and S-sulfonyl (R-SO₃H) post-translational modifications. Here we present a simple bioconjugation strategy to label S-sulfinated proteins by using reporter-linked maleimides. After alkylation of free thiols with iodoacetamide, S-sulfinated cysteines react with maleimide to form a sulfone Michael adduct that remains stable under acidic conditions. Using this sequential alkylation strategy, we demonstrate differential S-sulfination across mouse tissue homogenates, as well as enhanced S-sulfination following pharmacological induction of endoplasmic reticulum stress, lipopolysaccharide stimulation, and inhibitors of the electron transport chain. Overall, this study reveals a broadened profile of maleimide reactivity across cysteine modifications, and outlines a simple method for profiling the physiological role of cysteine S-sulfination in disease.     

Synthetic Antibody Mimics Based on Cancer-Targeting Immunostimulatory Peptides

De novo cancer-targeting immunostimulatory peptides have been designed and developed as synthetic antibody mimics. A series of bifunctional peptides incorporating NKp30-binding and NK-cell-activating domains were synthesized as linear dimers and then extended into branching trimeric peptides by the incorporation of GRP78-targeting and tumor-cell-binding sequences. A selected trimeric peptide from this small set of peptides displayed binding capabilities on GRP78⁺ HepG2 and A549 target cells. Cell binding diminished in the presence of an anti-GRP78 peptide blocker, thus suggesting GRP78-binding dependence. Similarly, the selected trimeric peptide was also found to exhibit NK cell binding in an NKp30-dependent manner, which translated into NK cell activation as indicated by cytokine secretion. In co-culture, fluorescence microscopy revealed that the target GFP-expressing A549 cells were visibly associated with the effector NK cells when pre-activated with lead trimeric peptide. Accordingly, A549 cells were found to be compromised, as evidenced by the loss of GFP signal and notable detection of early-/late-stage apoptosis. Investigation of the immunological markers related to toxicity revealed detectable secretion of pro-inflammatory cytokines and chemokines, including IFN-γ, TNF-α, and IL-8. Furthermore, administration of peptide-activated NK cells into A549-tumor-bearing mice resulted in a consistent decrease in tumor growth when compared to the untreated control group. Taken together, the identification of a lead trimeric peptide capable of targeting and activating NK cells’ immunotoxicity directly towards GRP78⁺/B7H6^- tumors provides a novel proof-of-concept for the development of cancer-targeting immunostimulatory peptide ligands that mimic antibody-targeting and -activating functions related to cancer immunotherapy applications.     

Morin functionalized Merrifield’s resin: A new material for enrichment and sensing heavy metals

In this paper, we report the synthesis of morin covalently attached to Merrifield’s resin to be used in metal sorption and recognition. The potential in sensing and sorbent properties of these materials for Cu(II), Pb(II), Zn(II) and Cd(II) in batch and using a flow-through optosensing approach is outlined. The sorption capacity was evaluated in a batch approach using atomic absorption spectroscopy. It was observed that morin modified Merrifield’s resin retained all the studied metals with high efficiency (90-95%). The influence of pH on the ion binding capacity of the resin was investigated by batch method as a function of their fluorescence quenching/enhancement efficiency. The sensing properties of the morin modified beads were also evaluated by packing the resins into a flow-through cell in a FIA (flow injection analysis) format. Results demonstrated that fluorescence emission increased upon Zn²⁺ and Cd²⁺ interaction with a higher sensitivity for Zn²⁺. In contrast, fluorescence quenching was observed when Cu²⁺ and Pb²⁺ were injected and reacted with the resin. The potential applicability of the sensing approach for the selective determination of Cu²⁺ and Zn²⁺ ions is discussed.     

Use of Masking Agents in Promoting Selective Transport of Zn2+ through Nafion Membrane

ABSTRACT

The effect of H⁺ ion concentration on the ion-exchange selectivity of Nafion 117 cation-exchange membrane toward Zn²⁺, Pb²⁺, Cu²⁺, Al³⁺, and Fe³⁺ have been studied. A selective transport of any particular metal ion was not possible by controlling the pH alone. However, selective permeation of Zn²⁺ across the membrane could be accomplished by selectively masking the metal ion as the cationic 1,10-phenanthroline complex while the permeation of other interfering ions could be suppressed by masking them as their anionic EDTA complex. About 20% of Zn²⁺ selectively permeated from the mixture in 6 hours and the quantity increased to 67% after 60 hours of permeation.     

HIF-1α Dependent Upregulation of ZIP8, ZIP14, and TRPA1 Modify Intracellular Zn2+ Accumulation in Inflammatory Synoviocytes

Intracellular free zinc ([Zn²⁺]_i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn²⁺]_i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn²⁺ have been identified, proteins that are involved in Zn²⁺ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn²⁺ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn²⁺]_i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn²⁺]_i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca²⁺ oscillations. Assays with fluorescent Zn²⁺ indicators revealed that the basal [Zn²⁺]_i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn²⁺]_i level in the presence of 1 μM Zn²⁺ in inflammatory FLSs. Among the 17 out of 24 known Zn²⁺ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn²⁺]_i level. Taken together, Zn²⁺ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn²⁺]_i in inflammatory FLSs.