Immobilized metal ion affinity chromatography of proteins on IDA-Fe3+

Several proteins, selected for their varied isoelectric points within pH range ca. 4 to ca. ll, bind to immobilized (chelated to iminodiacetate) ferric ion, IDA-Fe³⁺, when applied in a 50 mM buffer, pH 6.0. These proteins can be displaced from IDA-Fe³⁺ columns by an increase of pH from 6 to 8, an increase of sodium chloride from 0 to 1 Malar and, in particular, by both. Apotransferrins, in contrast to other proteins, are able to scavenge Fe³⁺ ion from IDA-Fe³⁺. Interestingly, two proteins, both quite acidic, behave quite differently on IDA-Fe³⁺: α₁-acid glycoprotein (sialic acid) does not bind, whereas phosvitin (phosphate 1) binds avidly. IDA-Fe³⁺ sorbent, due to its unusual sorptive properties, represents a new addition of particular significance to the family of chromatographic sorbents available for protein purification.     

Determination of iminodiacetic acid in the glyphosate by ion chromatography

In this paper,a simple method based on ion chromatography（IC） with conductivity detection was developed for the determination of iminodiacetic acid（IDA） in the herbicide of glyphosate.Under optimized chromatographic conditions,good linear relationship,sensitivity and reproducibility were obtained.The detection limit（LOD） for IDA obtained by injecting 25 μL of sample was 31.8 μg/L（S/N = 3）.Relative standard deviation（RSD） of repeated analysis for the peak areas was less than 1.53%（n = 6）.A spiking study was performed with satisfactory recoveries between 92.8%and 103.6%.It was confirmed that this method could be applied in glyphosate products.     

基于天冬氨酸的固定化金属离子亲和色谱材料用于磷酸化肽选择性富集

采用点击化学的方法将自然界中的天冬氨酸(aspartic acid)键合到硅球上(命名为Click Asp),并将Fe3+配位到Click Asp上,合成固定金属离子亲和色谱(IMAC)材料(Fe3+-Click Asp);采用红外光谱、X射线电子能谱和扫描电镜等表征证明Fe3+-Click Asp成功合成。将此IMAC材料用于蛋白质酶解液和牛奶中的磷酸化肽的富集,实现了磷酸化肽的高选择性富集。本研究为磷酸化蛋白质组学提供了新材料和新方法。     

螯合金属离子亲和色谱法分离α-氨基酸和肽

 以ＳｅｐｈａｄｅｘＧ１０为基质螯合二价铜离子的亲和色谱法分离α－氨基酸和肽，使之得以完全分离。对分离过程的原理进行了讨论。     

Selective retention of basic compounds by metal aquo‐ion affinity chromatography

A novel metal aquo‐ion affinity chromatography has been developed for the analysis of basic compounds using heat‐treated silica gel containing hydrated metal cations (metal aquo‐ions) as the packing material. The packing materials of the metal aquo‐ion affinity chromatography were prepared by the immobilization of a single metal component such as Fe(III), Al(III), Ag(I), and Ni(II) on silica gel followed by extensive heat treatment. The immobilized metals form aquo‐ions to present cation‐exchange ability for basic analytes and the cation‐exchange ability for basic analytes depends on pK_a of the immobilized metal species. In the present study, to evaluate the retention characteristics of metal aquo‐ion affinity chromatography, the on‐line solid‐phase extraction of drugs was investigated. Obtained data clearly evidence the selective retention capability of metal aquo‐ion affinity chromatography for basic analytes with sufficient capacity.     

Adsorption affinity of anions on metal oxyhydroxides

The dependences of anion (phosphate, carbonate, sulfate, chromate, oxalate, tartrate, and citrate) adsorption affinity anions from geometric characteristics, acid-base properties, and complex forming ability are generalized. It is shown that adsorption depends on the nature of both the anions and the ionic medium and adsorbent. It is established that anions are generally grouped into the following series of adsorption affinity reduction: PO ₄ ^3? , CO ₃ ^2? > C₂O ₄ ^2? , C(OH)(CH₂)₂(COO) ₃ ^3? , (CHOH)₂(COO) ₂ ^2? > CrO ₄ ^2? ? SO ₄ ^2? .     

Separation and evaluation of soybean protein hydrolysates prepared by immobilized metal ion affinity chromatography with different metal ions

Metal ion affinity chromatography is widely used to purify peptides on the basis of the dissimilarities of their amino acids. However, researchers are interested in the separation differences between different metal ions in this method. In our study, four kinds of commonly used metal ions are compared by the amount of immobilized metal ion on iminodiacetic acid-Sepharose and binding amount of soybean peptide to immobilized iminodiacetic acid-Mn(+) adsorbents and evaluated by high-performance liquid chromatography (HPLC) profiles. The results show that due to the different adsorption behaviors of metal ions, the binding ability order of soybean protein peptide on the column should be Fe(3+) > Cu(2+) > Zn(2+) > Ca(2+). The HPLC profiles show that peptides adsorbed by four kinds of metal ions display similar strong hydrophobic characteristics.     

Purification of S-oxynitrilase from Sorghum bicolor by immobilized metal ion affinity chromatography on different carrier materials.

The purification of the hydroxynitrile lyase (EC 4.1.2.11, S-oxynitrilase) from Sorghum bicolor is compared using different strategies. A new procedure is presented, which exploits the affinity of S-oxynitrilase towards metal ions as a key step in purification. The metal ions are immobilized by chelators on different carrier materials, e.g. Sepharose beads, microporous membranes or poly(ethylene glycol). A systematic examination demonstrates the excellent potential of immobilized metal affinity chromatography as a preparative separation method.     

Chelated mercury as a ligand in immobilized metal ion affinity chromatography of proteins

Chelation of mercuric ions by an iminodiacetate-Sepharose gel was evaluated. The retentive properties of iminodiacetate-Sepharose gel column was studied towards proteins varying the composition of eluting systems from 2-mercaptoethanol to NaCl and imidazole, determining also the extent of mercury leaching. It was demonstrated that chelated mercury contained free sites for interaction with proteins such as bromelain and recombinant human granulocyte colony stimulating factor from E. coli. The extraction of the latter by chromatography of its inclusion bodies solution on Hg(II)-loaded Sepharose-iminodiacetate gel was also evaluated.     

Preparation of an iminodiacetic acid-modified capillary and its performance in capillary liquid chromatography and immobilized metal chelate affinity capillary electrophoresis

We prepared iminodiacetic acid (IDA)-modified and Cu(II)-IDA-modified capillaries through polymerization of N-(vinylbenzylimino) diacetic acid. The fundamental performance of these capillaries was examined in capillary liquid chromatography (LC) and immobilized metal chelate affinity capillary electrophoresis (IMACE). Copper(II), cobalt(II), and hematin were detected at different retention times by means of capillary LC with a chemiluminescence detector, during which the IDA-modified capillary was used. The difference in the retention times was attributed to the difference in the interaction between metal ions or complex and IDA moieties on the inner wall of the capillary. In addition, human serum albumin (HSA) and human serum gamma-globulin (HgammaG) were separated and detected using IMACE with an absorption detector, during which the Cu(II)-IDA-modified capillary was used. The separation of HSA and HgammaG was achieved through the interaction between proteins and Cu(II) chelate moieties on the inner wall of this capillary.     

Separation of chitosan oligomers by immobilized metal affinity chromatography

A novel approach for chitosan oligosaccharide (COS) separation by immobilized metal affinity chromatography (IMAC) based on the differences in the interactions of chelated copper (II) ions with various COS (dimers, trimers, tetramers) is described. Polyhydroxylic chromatographic supports (agarose CL-6B and silica) were functionalized with various chelating functions such as iminodiacetate (IDA), carboxymethyl-aspartate (CM-Asp) and tris(carboxymethyl)ethylenediamine (TED). The COS retention capacities of the columns were between 2 and 6 mg/cm(3), depending on the chelating group. The COS were separated and/or enriched up to 95% for dimer and trimer and 90% for the tetramer, with yields of 60-95%.     

Specific capture of uranyl protein targets by metal affinity chromatography

To improve general understanding of biochemical mechanisms in the field of uranium toxicology, the identification of protein targets needs to be intensified. Immobilized metal affinity chromatography (IMAC) has been widely developed as a powerful tool for capturing metal binding proteins from biological extracts. However uranyl cations (UO2(2+)) have particular physico-chemical characteristics which prevent them from being immobilized on classical metal chelating supports. We report here on the first development of an immobilized uranyl affinity chromatography method, based on the cation-exchange properties of aminophosphonate groups for uranyl binding. The cation distribution coefficient and loading capacity on the support were determined. Then the stability of the uranyl-bonded phase under our chromatographic conditions was optimized to promote affinity mechanisms. The successful enrichment of uranyl binding proteins from human serum was then proven using proteomic and mass spectral analysis.     

Preconcentration of the herbicide glyphosate and its metabolite AMPA by immobilised metal ion affinity chromatography (IMAC)

A method based on Immobilised Metal Ion Affinity Chromatography (IMAC) using a chelating resin (Spheron Oxine 1000) loaded with Pd(II) is evaluated for the extraction and preconcentration of glyphosate and AMPA from natural water samples. The efficiency of the metal-loaded resin in retaining glyphosate and AMPA and the ability of different reagents to recover them is investigated. The most effective elution solution for the recovery of both analytes from the resin is found to be a mixture of 0.1 M HCI and 1 M NaCl. The effect of flow rate, analyte concentration, and sample volume is evaluated. The optimised experimental conditions are then used in the extraction of the analytes from spiked natural waters. The use of the Pd loaded resin led to recoveries ranging from 80-90% for glyphosate and 60-80% for AMPA.     

Metallo supramolecular assemblies of bis-squaraines by allosteric Ca2+ ion binding

Alkyl chain tethered bis-squaraines bind to Ca2+ ions through the participation of the negatively charged oxygen of the central cyclobutene moiety to form folded H-type aggregates. The initially formed Ca2+ complex is preorganized to facilitate cooperative allosteric binding of Ca2+, resulting in the formation of extended supramolecular arrays. The electronic absorption, IR, and ESI-MS studies support the formation of metallo supramolecular architectures of the folded H-type dimers of the bis-squaraines.     

Preparation of core–shell structure Fe3O4@SiO2 superparamagnetic microspheres immoblized with iminodiacetic acid as immobilized metal ion affinity adsorbents for His‐tag protein purification

The core–shell structure Fe₃O₄/SiO₂ magnetic microspheres were prepared by a sol–gel method, and immobiled with iminodiacetic acid (IDA) as metal ion affinity ligands for protein adsorption. The size, morphology, magnetic properties and surface modification of magnetic silica nanospheres were characterized by various modern analytical instruments. It was shown that the magnetic silica nanospheres exhibited superparamagnetism with saturation magnetization values of up to 58.1 emu/g. Three divalent metal ions, Cu²⁺, Ni²⁺ and Zn²⁺, were chelated on the Fe₃O₄@SiO₂–IDA magnetic microspheres to adsorb lysozyme. The results indicated that Ni²⁺‐chelating magnetic microspheres had the maximum adsorption capacity for lysozyme of 51.0 mg/g, adsorption equilibrium could be achieved within 60 min and the adsorbed protein could be easily eluted. Furthermore, the synthesized Fe₃O₄@SiO₂–IDA–Ni²⁺ magnetic microspheres were successfully applied for selective enrichment lysozyme from egg white and His‐tag recombinant Homer 1a from the inclusion extraction expressed in Escherichia coli. The result indicated that the magnetic microspheres showed unique characteristics of high selective separation behavior of protein mixture, low nonspecific adsorption, and easy handling. This demonstrates that the magnetic silica microspheres can be used efficiently in protein separation or purification and show great potential in the pretreatment of the biological sample. Copyright © 2015 John Wiley & Sons, Ltd.     

Immobilized metal ion affinity chromatography of synthetic peptides. Binding via the alpha-amino group.

Peptides synthesized by the solid-phase method can be efficiently purified in a single immobilized metal affinity chromatography step based on interaction with the alpha-amino group if, after coupling of each amino acid residue, unreacted amino groups are irreversibly blocked by acetylation and if no strongly metal-binding amino acids (His, Trp, Cys) are present in the sequence. A difference in basicity for alpha- and epsilon-amino functions of ca. 2 pH units is sufficiently large to allow selective binding of peptides to immobilized metal ions via the unprotonated alpha-amino group. The binding is pH-dependent: on Cu(2+)- and Ni(2+)-loaded supports most peptides are maximally retarded at pH values around 7.5 and 8.5, respectively. The decreased binding strength at lower pH values is due to protonation of the alpha-amino function, whereas the reduced affinity at higher pH is caused by metal ion transfer from the matrix to the peptide. The metal ion is captured in a multidentate chelate where, in addition to the alpha-amino group, up to three adjacent deprotonated amide nitrogens are coordinated to the metal. If the pH is raised further, additional metal ions may be bound in biuret-like structures. Immobilized Ni2+, owing to its higher selectivity and affinity, is the preferred chromatographic support if slightly basic conditions can be tolerated.     

Trace analysis of heavy metal ions by ion chromatography

Summary An ion chromatographic separation technique for heavy metal ions is described. Using pressure-stable, silica-based, ion-exchange supports and standard HPLC equipment with post-column reaction detector high resolution is achieved as well as extremely high sensitivity in the parts per trillion (ppt)-range.