RESUMO
Supramolecular metallogels combine the rheological properties of gels with the color, magnetism, and other properties of metal ions. Lanthanide ions such as Eu(III) can be valuable components of metallogels due to their fascinating luminescence. In this work, we combine Eu(III) and iminodiacetic acid (IDA) into luminescent hydrogels. We investigate the tailoring of the rheological properties of these gels by changes in their metal:ligand ratio. Further, we use the highly sensitive Eu(III) luminescence to obtain information about the chemical structure of the materials. In special, we take advantage of computational calculations to employ an indirect method for structural elucidation, in which the simulated luminescent properties of candidate structures are matched to the experimental data. With this strategy, we can propose molecular structures for different EuIDA gels. We also explore the usage of these gels for the loading of bioactive molecules such as OXA, observing that its aldose reductase activity remains present in the gel. We envision that the findings from this work could inspire the development of luminescent hydrogels with tunable rheology for applications such as 3D printing and imaging-guided drug delivery platforms. Finally, Eu(III) emission-based structural elucidation could be a powerful tool in the characterization of advanced materials.
Assuntos
Európio , Hidrogéis , Európio/química , Hidrogéis/química , Luminescência , Iminoácidos/química , Reologia , Substâncias Luminescentes/química , Ligantes , Géis/químicaRESUMO
Iminodiacetic acid (IDA) is one of the chelating ligands most frequently employed in immobilized metal-ion affinity chromatography (IMAC) due to its ability to act as electron-pair donor, forming stable complexes with intermediate and borderline Lewis metal ions (electron acceptor). Thus, IDA can also be employed in ion exchange chromatography to purify positively charged proteins at neutral pH values. This study aimed to evaluate IDA as an ionogenic group (ion exchanger) immobilized on poly (ethylene vinyl alcohol) (PEVA) hollow fiber membranes for immunoglobulin G1 (IgG1) monoclonal antibody (MAb) purification. IDA-PEVA membranes showed considerable promise for MAb purification, since IgG1 was recovered in eluted fractions with traces of contaminants as confirmed by Western blotting and ELISA analysis. Quantification of IgG1 showed that a purity of 94.2% was reached in the elution step. Breakthrough curve and batch adsorption experiments showed that the MAb dynamic binding capacity (DBC) of 3.10 mg g-1 and the maximum adsorption capacity of 70 mg g-1 were of the same order of magnitude as those found in the literature. The results obtained showed that the IDA-PEVA hollow fiber membrane could be a powerful adsorbent for integrating large-scale processes for purification of MAb from cell culture supernatant.
Assuntos
Anticorpos Monoclonais/química , Cromatografia de Afinidade/métodos , Iminoácidos/química , Imunoglobulina G/química , Membranas Artificiais , Adsorção , Animais , Anticorpos Monoclonais/isolamento & purificação , Quelantes/química , Concentração de Íons de Hidrogênio , Imunoglobulina G/isolamento & purificação , Íons , Ligantes , Metais/química , CamundongosRESUMO
Iminodiacetic acid (IDA) and tris(2-aminoethyl)amine (TREN) chelating ligands were immobilized on poly(ethylene vinyl alcohol) (PEVA) hollow-fiber membranes after activation with epichlorohydrin or butanediol diglycidyl ether (bisoxirane). The affinity membranes complexed with Cu(II) were evaluated for adsorption of human immunoglobulin G (IgG). The effects of matrix activation and buffer system on adsorption of IgG were studied. Isotherms of batch IgG adsorption onto finely cut membranes showed that neither of the chelates, IDA-Cu(II) or TREN-Cu(II), had a Langmuirean behavior with negative cooperativity for IgG binding. A comparison of equilibrium and dynamic maximum capacities showed that the dynamic capacity for a mini-cartridge in a cross-flow filtration mode (52.5 and 298.4 mg g(-1) dry weight for PEVA-TREN-Cu(II) and PEVA-IDA-Cu(II), respectively) was somewhat higher than the equilibrium capacity (9.2 and 73.3 mg g(-1) dry weight for PEVA-TREN-Cu(II) and PEVA-IDA-Cu(II), respectively). When mini-cartridges were used, the dynamic adsorption capacity of IDA-Cu(II) was the same for both mini-cartridge and agarose gel.
Assuntos
Cobre/química , Imunoglobulina G/isolamento & purificação , Adsorção , Cátions Bivalentes , Quelantes/química , Cromatografia de Afinidade , Epicloroidrina/química , Compostos de Epóxi/química , Etilenodiaminas/química , Iminoácidos/química , Cinética , Membranas Artificiais , Polivinil/química , Ligação Proteica , Soluções , TermodinâmicaRESUMO
Endotoxins (also known as lipopolysaccharides (LPS)) are undesirable by-products of recombinant proteins, purified from Escherichia coli. LPS can be considered stable under a wide range of temperature and pH, making their removal one of the most difficult tasks in downstream processes during protein purification. The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration. Immobilized metal affinity chromatography (IMAC) enables the affinity interactions between the metal ions (immobilized on the support through the chelating compound) and the target molecules, thus enabling high-efficiency separation of the target molecules from other components present in a mixture. Affinity chromatography is applied with Ca2+ -iminodiacetic acid (IDA) to remove most of the LPS contaminants from the end product (more than 90%). In this study, the adsorption of LPS on an IDA-Ca2+ was investigated. The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal. It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads. The factors such as pH (4.0 or 5.5) and ionic strength (1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than 100 EU/mL and 100 000 EU/mL. This new protocol represents a substantial advantage in time, effort, and production costs.
Assuntos
Cromatografia de Afinidade/métodos , Endotoxinas/isolamento & purificação , Proteínas Recombinantes/química , Adsorção , Cálcio/química , Escherichia coli , Iminoácidos/química , Íons/química , Metais/química , Proteínas/químicaRESUMO
Monoclonal antibodies (MAbs) have been used for therapies and some analytical procedures as highly purified molecules. Many techniques have been applied and studied, focusing on monoclonal antibodies purification. In this study, an immobilized metal affinity chromatography membrane was developed and evaluated for the purification of anti-TNP IgG(1) mouse MAbs from cell culture supernatant after precipitation with a 50% saturated ammonium sulfate solution. The chelating ligands iminodiacetic acid, carboxymethylated aspartic acid (CM-Asp), nitrilotriacetic acid, and tris (carboxymethyl) ethylenediamine in agarose gels with immobilized Ni(II) and Zn(II) ions were compared for the adsorption and desorption of MAbs. The most promising chelating ligand--CM-Asp--was then coupled to poly(ethylene vinyl alcohol) (PEVA) hollow fiber membranes. According to SDS-PAGE and ELISA analyses, a higher selectivity and a purification factor of 85.9 (fraction eluted at 500 mM Tris) were obtained for IgG(1) using PEVA-CM-Asp-Zn(II). The anti-TNP MAb could be eluted under mild pH conditions causing no loss of antigen binding capacity.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Quelantes/química , Cromatografia de Afinidade/métodos , Imunoglobulina G/isolamento & purificação , Metais/química , Adsorção , Anticorpos Monoclonais/química , Ácido Aspártico/química , Cromatografia de Afinidade/instrumentação , Etilenodiaminas/química , Iminoácidos/química , Imunoglobulina G/química , Ligantes , Membranas Artificiais , Níquel/química , Ácido Nitrilotriacético/química , Propriedades de Superfície , Zinco/químicaRESUMO
The presented study focuses on the feasibility of immobilized metal affinity chromatography for purification of Madin Darby canine kidney cell culture-derived influenza virus particles. Therefore, influenza virus A/Puerto Rico/8/34 was screened for adsorption to different transition metal ions attached to iminodiacetic acid. Subsequently, capturing of the same virus strain using zinc-modified iminodiacetic acid membrane adsorbers was characterized regarding viral recoveries, host cell nucleic acid and total protein depletion as well as zinc-ion-leaching. In addition, the effect of the imidazole proton pump on virus stability was studied based on the hemagglutination activity. During adsorption in the presence of 1M sodium chloride the majority of virus particles were recovered in the product (64% hemagglutination activity). Host cell nucleic acid and total protein content were reduced to approximately 7 and 26%, respectively. This inexpensive and rapid method was applied reproducibly for influenza virus A/Puerto Rico/8/34 preparations on the laboratory scale. However, preliminary results with other virus strains indicated clearly a strong strain dependency for viral adsorption.
Assuntos
Vírus da Influenza A/isolamento & purificação , Adsorção , Animais , Técnicas de Cultura de Células , Linhagem Celular , Cromatografia de Afinidade/métodos , Cães , Iminoácidos/química , Vírus da Influenza A/crescimento & desenvolvimento , Membranas Artificiais , Reprodutibilidade dos Testes , Cultura de Vírus , Zinco/química , Zinco/farmacocinéticaRESUMO
The purification of IgG from human plasma was studied by comparing two affinity membranes complexed with Ni(II), prepared by coupling iminodiacetic acid (IDA) and Tris(2-aminoethyl)amine (TREN) to poly(ethylenevinyl alcohol), PEVA, hollow fiber membranes. The Ni(II)-TREN-PEVA hollow fiber membrane had lower capacity for human IgG than the complex Ni(II)-IDA-PEVA, but with similar selectivity. The IgG in peak fractions eluted from the Ni(II)-IDA-PEVA with a stepwise concentration gradient of Tris-HCl pH 7.0 (100-700 mM) reached a purity of 98% (based on IgG, IgM, IgA, albumin, and transferrin nephelometric analysis). Adsorption IgG data at different temperatures (4-37 degrees C) were analyzed using Langmuir model resulting in a calculated maximum capacity at 25 degrees C of 204.6 mg of IgG/g of dry membrane. Decrease in Kd with increasing temperature (1.7x10(-5) to 5.3x10(-6) M) indicated an increase in affinity with increased temperature. The positive value of enthalpy change (26.2 kJ/mol) indicated that the adsorption of IgG in affinity membrane is endothermic. Therefore, lower temperature induces adsorption as verified experimentally.
Assuntos
Cromatografia de Afinidade/métodos , Etilenodiaminas/química , Iminoácidos/química , Imunoglobulina G/isolamento & purificação , Níquel/química , Adsorção , Soluções Tampão , Quelantes/química , Cromatografia de Afinidade/instrumentação , Humanos , TemperaturaRESUMO
Redox properties of copper complexes are important for their catalytic functions in vitro and in biological systems, and can contribute to their reactivity toward selected targets. In order to evaluate the influence of different ligands on the reactivity of copper ions, comparative studies were carried out with some copper(II) complexes containing a tridentate imine, or a tetradentate di-Schiff base ligand with a mixed pyridine, pyrazine, or imidazole donor set, acting as catalysts in the oxidation of 2-deoxy-D-ribose. Addition of the reducing agent glutathione (gamma-glutamylcysteinylglycine; GSH), which can also act as a good ligand for copper(I), mediated the oxidation of the substrate. For some of these compounds, a reductive activation followed by competition for the metal ion was verified, with formation of copper(I)-glutathione complex monitored by fluorescence measurements. For others, however, the reduction of the metal by the glutathione seems to not occur. In the presence of hydrogen peroxide, the oxidative damage is significantly enhanced for all the complexes tested. Redox potential measurements by cyclic voltammetry corroborated partially these results, indicating that the most reactive complexes are those with more positive redox potential. Evidence for site-specific attack to 2-deoxy-D-ribose was also observed, consistent with the intermediary formation of a copper-hydroxyl species, [LCu(II)(*OH)], rather than 'free' hydroxyl radical.
Assuntos
Cobre/farmacologia , Iminoácidos/química , Compostos Organometálicos/farmacologia , Ribose/química , Cobre/química , Relação Dose-Resposta a Droga , Eletroquímica , Espectroscopia de Ressonância de Spin Eletrônica , Glutationa/química , Glutationa/farmacologia , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Oxirredução/efeitos dos fármacos , Bases de Schiff/química , Espectrometria de Fluorescência , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/química , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
An on-line affinity selection method using a polymeric monolithic support is proposed for the retention of histidine-containing peptides and their subsequent separation by capillary zone electrophoresis (CZE). Monolithic capillary columns were prepared in fused-silica capillaries of 150 mum inner diameter (ID) by ionizing radiation-initiated in situ polymerization and cross-linking of diethylene glycol dimethacrylate and glycidyl methacrylate, and chemically modified with iminodiacetic acid (IDA) and copper ion. Monolithic microextractors were coupled on-line near the inlet of the separation capillary (fused-silica capillary, 75 mum ID x 28 cm from the microextractor to the detector). Model peptide mixtures of histidine-containing and histidine-noncontaining peptides were assessed. Peptides were released from the sorbent by a 5 mM imidazole solution and then separated by CZE with ultraviolet detection. Relative standard deviation values for migration times and corrected peak areas were found to be lower than 5.8 and 10.5%, respectively. IDA-Cu(II) ion modified monolithic microextractors showed a chromatographic behavior and could be reused at least 25 times. The use of monolithic supports proved to be an advantageous alternative to packed particles for the preparation of microextractors.
Assuntos
Histidina/química , Metacrilatos/química , Peptídeos/análise , Cobre/química , Eletroforese Capilar , Iminoácidos/química , Microscopia Eletrônica de Varredura , Dióxido de Silício/químicaRESUMO
The influence of the morphology of ethylene glycol dimethacrylate-hydroxyethyl methacrylate copolymer [poly(EGDMA-co-HEMA)] base support to obtain different Fe(3+)-containing sorbents and their properties in retention of O-phosphothreonine [Thre(P)] is examined in this paper. Three base supports poly(EGDMA-co-HEMA) (I-III) were obtained using different quantities of initiator in suspension polymerization reactions. These products were submitted to chemical modifications using 1,4-butanediol diglycidyl ether (BDGE) in activation reactions and different chelating agents (iminodiacetic acid, IDA; disodium ethylenediamine tetraacetate, EDTA; and hexamethylenediamine tetrapropanoic acid, HMDTP) in coupling reactions to attain Fe(3+)-containing sorbents. Properties such as specific surface area (S(s)), specific pore volume (V(p)), scanning electron microscopy (SEM), IR spectroscopy, quantity of functional groups (oxirane and carboxyl), amount of chelated metal ion, ligand occupation (L), swelling studies as well as quantity of O-phosphoamino acid retained were used as comparative parameters for matrices. In general, the derivatization reactions proved to be more efficient when higher S(s) of macropores (50-400,000 nm) were available in the matrix. In our case, it was observed when highest percentage of initiator was used. On the other hand, the effect of accessibility of surface area on the yield of coupling reactions was noticed when comparing the different chelating agents since the number of carboxyl groups present in products was higher when the molecular size of the chelating agent was lower. Although all Fe(3+)-containing sorbents resulted efficient to retain Thre(P), the values of retention of the amino acid were slightly higher when IDA-containing matrices were used irrespective of the quantity of metal chelated. This could be probably due to the fact that the IDA ligand could be bounded to the matrix in sites that though accessible for the center of adsorption were hard for Thre(P) to access.