Differential interactions of plasmid DNA, RNA and endotoxin with immobilised and free metal ions

Separation of negatively charged molecules, such as plasmid DNA (pDNA), RNA and endotoxin forms a bottleneck for the development of pDNA vaccine production process. The use of affinity interactions of transition metal ions with these molecules may provide an ideal separation methodology. In this study, the binding behaviour of pDNA, RNA and endotoxin to transition metal ions, either in immobilised or free form, was investigated. Transition metal ions: Cu2+, Ni2+, Zn2+, Co2+ and Fe3+, typically employed in the immobilised metal affinity chromatography (IMAC), showed very different binding behaviour depending on the type of metal ions and their existing state, i.e. immobilised or free. In the alkaline cell lysate, pDNA showed no binding to any of the IMAC chemistries tested whereas RNA interacted significantly with Cu2+-iminodiacetic acid (IDA) and Ni2+-IDA but showed no substantial binding to the rest of the IMAC chemistries. pDNA and RNA, however, interacted to varying degrees with free metal ions in the solution. The greatest selectivity in terms of pDNA and RNA separation was achieved with Zn2+ which enabled almost full precipitation of RNA while keeping pDNA soluble. For both immobilised and free metal ions, ionic strength of solution affected the metal ion-nucleic acid interaction significantly. Endotoxin, being more flexible, was able to interact better with the immobilised metal ions than the nucleic acids and showed binding to all the IMAC chemistries. The specific interactions of immobilised and/or free metal ions with pDNA, RNA and endotoxin showed a good potential, by selectively removing RNA and endotoxin at high efficiency, to develop a simplified pDNA purification process with improved process economics.     

Immobilized metal affinity chromatography for the separation of photosystems I and II from the thermophilic cyanobacterium Synechococcus elongatus

Immobilized metal affinity chromatography (IMAC) of solubilized, photosystem II (PS II) enriched particles from the thermophilic cyanobacterium Synechococcus elongatus was studied. A chelating Sepharose Fast Flow column was charged with various metal ions (Mn2+, Fe2+, Fe3+, Ni2+, Co2+, Ca2+, Sr2+, Zn2+ and Cu2+) and their affinity to photosystem I (PS I) and PS II was examined. Among all the metal ions tested, only copper was able to bind the two protein complexes. For elution of the column, a pH gradient, a pH step gradient and gradients of imidazole, amino acids, organic acids and various other eluents were tested; only the pH step gradient, which selectively eluted PS II at a pH between 6 and 5, was useful for the separation of PS I and PS II. All other gradients proved to be inappropriate for the separation of these two photosystems. Mechanisms of protein elution by these compounds are discussed. Alternatively, a separation of PS I and PS II at pH 7.5 could be achieved when an IMAC column was used on which the free coordination positions of the bound copper ions were occupied by imidazole. When solubilized photosystems were loaded on to this column, PS I replaced imidazole and remained bound on the column, whereas PS II was highly enriched in the effluent.     

多齿配体-硅胶色谱介质的制备与评价

以新型环保多齿螯合剂——亚氨基二琥珀酸(IDS)为配体,在优化条件下,合成了IDS-Silica固定相。用电位滴定法测定了固定相上IDS的键合量。考察了IDS-Silica柱的色谱特性以及金属螯合特性。使用制备柱成功地分离了标准蛋白质混合物,该制备柱展现出了典型的阳离子交换特性。用电感耦合等离子体原子发射光谱法考察了金属离子在IDS-Silica固定相上的键合特性。结果表明,金属离子在IDS-Silica固定相上键合量的变化规律与它们同该固定相螯合的强弱顺序一致。通过比较金属Cu~(2+)在4种不同氨羧类配体硅胶柱上的键合量,发现IDS对金属离子具有强的螯合能力。IDS对金属离子的强螯合特性为其今后作为固定金属亲和色谱填料奠定了基础,为缓解亲和柱在使用过程中固定金属离子的流失提供了一种有效的解决方法。     

Speciation of tin in lemon juice: An example of trace metal speciation in food

A scheme is presented for the separation and identification of different tin species in lemon juice. Soluble tin species are separated from other constituents by means of column-switching liquid chromatography and detected by electrothermal atomic absorption spectrometry (AAS). Other metals (copper, zinc, iron, calcium and magnesium) are detected in the same fraction by on-line flame-AAS. Gas chromatography/mass spectrometry of the organic ligands after derivatization confirmed that citric acid is the important ligand for metal binding in lemon juice. Tin is also partly bound to particulate matter, which is investigated by infrared spectroscopy and model experiments. It is shown that tin is most probably bound to carboxylic groups of pectin (analogously to calcium). Investigations of different lemon juices showed large variations of total tin, but few differences between tin species. Only one sample contained organotin compounds at a detectable level.     

Immobilized Metal Affinity Chromatography of Phosphorylated Proteins Using High Performance Sorbents

The interactions of two model phosphoproteins (porcine pepsin and ovalbumin) with two different immobilized metal affinity chromatography (IMAC) sorbents containing immobilized Fe³⁺, Ga³⁺, and UO₂ ²⁺ ions have been investigated under various conditions. Both proteins were adsorbed on immobilized uranyl ions under acidic conditions similar to those on immobilized Fe³⁺ and Ga³⁺ ions. The retained proteins could be released either by the presence of phosphate ions in the elution buffer (immobilized Ga³⁺ and Fe³⁺ ions) or by an increased pH (all tested immobilized ions). The IMAC sorbents employed could be used under the conditions of high-performance chromatography and are suitable for the separation and analysis of intact phosphoproteins.     

Adsorption and selectivity characteristics of several human serum proteins with immobilised hard Lewis metal ion-chelate adsorbents

In this investigation, human serum has been used as an example of a crude protein mixture to define the protein binding characteristics and selectivity of several immobilised hard Lewis metal ion affinity chromatographic (IMAC) adsorbents. Specifically, the binding properties of immobilised O-phosphoserine (im-OPS) and 8-hydroxyquinoline (im-8-HQ), with immobilised iminodiacetic acid as a control system, have been investigated in combination with the hard Lewis metal ions, Al3+, Ca2+, Fe3+, Yb3+, and the borderline metal ion, Cu2+, over the pH range pH 5.5 to pH 8.0 with buffers of 0.5 M ionic strength. The same IMAC adsorbents were also investigated for their protein binding capabilities with buffers of an ionic strength of 0.06 M at pH 5.5 and pH 8.0. The binding behaviour of four "marker" proteins, namely transferrin, alpha2-macroglobulin, gammaglobulin and human serum albumin have furthermore been employed to monitor the differences in protein selectivity exhibited by these IMAC systems. The experimental findings confirm that these hard Lewis metal ion IMAC adsorbents function in a "mixed" binding mode with both coordination and electrostatic characteristics evident, depending on the ionic strength and pH of the equilibration or elution buffers. Based on a screening protocol, several members of the im-Mn+-8-HQ and im-Mn+-OPS adsorbent series have been identified with high selectivity for transferrin and alpha2-macroglobulin. These hard Lewis metal ion IMAC adsorbents thus provide attractive alternatives for selective fractionation of human serum proteins.     

固定金属离子亲和色谱--蛋白质分离的方法、原理、特性和应用

介绍了固定金属离子亲和色谱法(IMAC)的方法原理、金属螯合柱的制备、固定金属离子与蛋白质的相互作用以及影响这些作用的因素、不同色谱条件下各种作用力对蛋白质保留值的贡献、蛋白质的洗脱原理和IMAC在蛋白质分离纯化中的应用,论述了IMAC的特点、不足、克服的方法和今后应解决的问题。     

Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry

On-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization-mass spectrometry (IMAC/CE/ESI-MS) offers selective preconcentration of phosphorylated peptides with identification of the phosphorylated amino acid(s). The preconcentration provides low concentration limits of detection and capillary electrophoresis separates the peptides. Recently, we reported a fast, simple, and sensitive on-line IMAC/CE/ESI-MS/MS method for the determination of phosphopeptides at low-pmole levels. That work is expanded here by use of multiple stage tandem mass spectrometry (MS(n), n = 2,3) to isolate and fragment target ions to provide more reliable assignments of phosphorylated residues. The application of IMAC/CE/ESI-MS(n) is demonstrated by the analysis of tryptic digests of alpha- and beta-casein and in-gel tryptic digests of beta-casein.     

Effect of metal ion hydration on the interaction between sodium carboxylates and aluminum(III) or chromium(III) ions in aqueous solution

The interaction between sodium octanoate, decanoate, and dodecanoate and aluminum(III) and chromium(III) has been studied in water at natural pH values, starting well below the surfactant critical micelle concentration, using electrical conductivity, turbidity, and potentiometric measurements. With decanoate or dodecanoate, maximum interaction occurs at 3:1 stoichiometry, corresponding to charge neutralization. Although the solutions become turbid with both metal ions, indicating phase separation, differences are observed and attributed to the fact that aluminum(III) is relatively labile to substitution and rapidly replaces its water ligands, whereas chromium(III) is substitution inert. This shows up in well-defined floc formation with Al(3+), whereas Cr(3+) suspensions do not precipitate, probably because that replacement of coordinated water by carboxylate ligands is impeded. This can be overcome by increasing temperature, and differences in the thermal behavior with Al(3+) and Cr(3+) are suggested to be due to increased involvement of substitution reactions in the latter case. The effect of octanoate on the trivalent metal ions is less clear, and with Cr(3+) interaction only occurs when the carboxylate is in excess. Hydrophobic interactions between alkyl chains play a major role in driving phase separation. At high surfactant concentrations, the solid phases do not dissolve, in contrast to what is observed with the corresponding alkylsulfates. This has implications for use of these systems in metal separation through froth flotation. The concentration of metal ions in supernatant solution has been determined for sodium dodecanoate and sodium dodecylsulfate with Al(3+) and Cr(3+) over the whole surfactant concentration range by inductively coupled plasma-mass spectrometry (ICP-MS). From this, association constants have been determined and are found to be larger for the carboxylate than the alkylsulfate, in agreement with the greater Lewis basicity of the -CO(2)(-) group.     

A comparison of the binding of metal complexes to duplex and quadruplex DNA

Electrospray ionisation mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy were used to compare the binding of mononuclear nickel, ruthenium and platinum complexes to double stranded DNA (dsDNA) and quadruplex DNA (qDNA). CD studies provided evidence for the binding of intact complexes of all three metal ions to qDNA. ESI mass spectra of solutions containing platinum or ruthenium complexes and qDNA showed evidence for the formation of non-covalent complexes consisting of intact metal molecules bound to DNA. However, the corresponding spectra of solutions containing nickel complexes principally contained ions consisting of fragments of the initial nickel molecule bound to qDNA. In contrast ESI mass spectra of solutions containing nickel, ruthenium or platinum complexes and dsDNA only showed the presence of ions attributable to intact metal molecules bound to DNA. The fragmentation observed in mass spectral studies of solutions containing nickel complexes and qDNA is attributable to the lower thermodynamic stability of the former metal complexes relative to those containing platinum or ruthenium, as well as the slightly harsher instrumental conditions required to obtain spectra of qDNA. This conclusion is supported by the results of tandem mass spectral studies, which showed that ions consisting of intact nickel complexes bound to qDNA readily undergo fragmentation by loss of one of the ligands initially bound to the metal. The ESI-MS results also demonstrate that the binding affinity of each of the platinum and ruthenium complexes towards qDNA is significantly less than that towards dsDNA.     

Investigation of the binding properties of heavy-metal-peptide complexes in plant cell cultures using HPLC-ICP-MS

Metal-induced, sulfhydryl-rich peptides (phytochelatins) found in plants, algae, yeasts and fungi have been described as sequesters and detoxifiers of heavy metal ions. High-performance liquid chromatography (HPLC) coupled on-line with inductively coupled plasma mass spectrometry (ICP-MS) has been used for the determination of heavy metal binding properties of phytochelatins in Silene vulgaris cell cultures. The induction of phytochelatins and the binding of heavy metals to these complexes were investigated by exposure of cell cultures with different concentrations of Cd, Cu, Pb and Zn. An in vitro heavy metal saturation assay and in vivo stress experiments with those elements able to bind to phytochelatins were carried out in order to characterize the binding affinity and binding stability of these compounds. It is shown that of the metals investigated, Cu binds most stably to phytochelatins under in vitro and in vivo conditions.     

New alpha-amino phenylalanine tetrazole ligand for immobilized metal affinity chromatography of proteins

A new chelating compound has been developed for use in the immobilized metal affinity chromatographic (IMAC) separation of proteins. The bidentate ligand, alpha-amino phenylalanine tetrazole, 4, was synthesized via a five-step synthesis from N-fluorenylmethoxycarbonyl phenylalanine and then immobilized onto silica through the epoxide coupling procedure. The binding behavior of the resulting IMAC sorbent, following chelation with Zn2+ to a density of 183 micromol Zn2+ ions/g silica, was characterized by the retention of proteins in the pH range of 5.0-8.0, and by the adsorption behavior of lysozyme with frontal chromatography at pH 6.0 and 8.0. The prepared column showed the separation ability to four test proteins and the retention time of these proteins increased with an increase in pH. From the derived isotherms, the adsorption capacity, qm, for the binding of lysozyme to immobilized Zn2+-alpha-amino phenylalanine tetrazole-silica was found to be 1.21 micromol/g at pH 6.0 and 1.20 micromol/g sorbent at pH 8.0, respectively, whilst the dissociation constants KD at these pH values were 5.22x10(-6) and 3.49x10(-6) M, respectively, indicating that the lysozyme was retained more stable under alkaline conditions, although the binding capacity in terms of micromole protein per gram sorbent remained essentially unchanged.     

Electrospray ionization mass spectrometry of phosphopeptides isolated by on-line immobilized metal-ion affinity chromatography

Electrospray ionization mass spectrometry (ESI/MS) affords a rapid and sensitive technique for determining peptides produced by the enzymatic digestion of phosphoroteins. When coupled with on-line immobilized metal-ion affinity chromatography (IMAC), the combmation allows separation and mass spectrometric identification of phosphorylated and nonphosphorylated peptides. In this study, the feasibility and general applicability of on-line IMAC/ESI/MS is investigated by using immobilized ferric ions for selective chelation of several phosphotyrosine and phosphoserine peptides. The sensitivity and practicality of the technique for phosphoproteins are demonstrated via the analysis of 30 pmol (～0.7 μg) of bovine β-casein purified by sodium dodecylsulfate-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, and digested in situ with trypsin. It is observed that on-line IMAC/ESI/MS suffers less from sample losses than experiments performed off-line, suggesting that the limiting factors in sensitivity for this technique are the purification procedures and sample handling rather than the IMAC and mass spectrometry. Thus, the ability to inject the tryptic digest of an electroblotted protein directly onto the column without buffer exchange and to analyze the eluent directly via on-line coupling of the IMAC column to the mass spectrometer greatly reduces sample losses incurred through sample handling and provides a convenient method for analyzing phosphopeptides at low levels.     

Speciation of M+3-hydroxypyrone chelation complexes by electrospray ionization ion trap and Fourier transform ion cyclotron resonance mass spectrometry

Kojic acid (5-hydroxy-2-hydroxymethyl-4-pyrone) is known to have a high affinity for transition metals, and it and its derivatized cogeners are used both analytically and clinically. The interactions between kojic acid (KA) and eleven +3 metals (Al(+3), As(+3), Cr(+3), Ga(+3), Fe(+3), In(+3), Yb(+3), Y(+3), Gd(+3), Nd(+3), La(+3)) were examined by electrospray ionization mass spectrometry (ESI-MS) using an ion trap in an aqueous medium. For a subset of five ions, Fourier transform ion cyclotron resonance (FTICR)-MS was conducted to provide accurate mass confirmation of peak assignments for metals having clustering of abundant isotopes. KA readily formed complexes with all the metal ions tested. The most common complexes observed were ML(3)H(+) and M(2)L(5). Different behavior was seen for small and large ionic radius ions, with a relative cut-off between In(+3) ( approximately 80 pm) and Yb(+3) ( approximately 87 pm); a striking trend in % collision energy vs. cluster complexity was revealed. The KA-Cr(+3)complex shows a high affinity for H(2)O molecules in the gas phase, whilst In(+3) shows a preference for dimetal complexes and Y(+3) a deviant behavior when complexed to two neutrals.     

Designing new metal affinity peptides by random mutagenesis of a natural metal-binding site

The metal-binding site of a Helicobacter pylori ATPase 439 (heli(WT)-tag) was successfully used as a new fusion peptide for immobilized metal ion affinity chromatography (IMAC). It produced higher yields than the frequently used his6-tag. Due to stronger binding of the peptide to metal ions, harsher elution conditions were, however, necessary. This undesired side-effect was overcome by modifying the heli(WT)-tag by polymerase chain reaction-directed mutagenesis. The modified tags were screened by an automated high-throughput IMAC system, leading to a heliM14-tag peptide that could be eluted under conditions similar to those of the his6-tag but at the same time produced 20% higher yields of the desired protein.     

Adsorption of endotoxins on Ca2+ iminodiacetic acid by metal ion affinity chromatography

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 100000 EU/mL. This new protocol represents a substantial advantage in time, effort, and production costs.     

Coordination chemistry of a terpyridine-tris(pyrazolyl) ditopic ligand

A new ditopic ligand, 4'-(4-(2,2,2-tris(1H-pyrazol-1-ido)ethoxymethyl)phenyl)-2,2':6',2'-terpyridine (pzt), has been prepared and its coordination chemistry studied. Metal ions with a preference for octahedral geometry form ML(2) complexes that are readily isolated and characterised, with the metal ion being bound to the terpyridine sites of both ligands. Other metal ions bind to the terpyridine site of just one ligand. In the case of silver(i), a dinuclear M(2)L(2) complex has been isolated in which each silver ion is coordinated to the terpyridine site of one ligand and to a single pyrazolyl donor group from the second ligand. Evidence for binding of metal ions to the tris(pyrazolyl) binding site was obtained by electrospray mass spectrometry and NMR techniques. The free ligand and three metal complexes, including the disilver complex, have been characterised by X-ray crystallographic techniques.     

Fractionation and identification of metalloproteins from a marine cyanobacterium

Trace metals are essential for the growth of marine cyanobacteria, being required for key cellular processes such as photosynthesis and respiration. Despite this, the metalloproteomes of marine cyanobacteria are at present only poorly defined. In this study, we have probed the major cobalt, iron, manganese, and nickel-binding proteins in the marine cyanobacterium Synechococcus sp. WH8102 by using two different fractionation approaches combined with peptide mass fingerprinting. For the identification of intact metalloproteins, multidimensional native chromatography was used to fractionate the proteome, followed by inorganic mass spectrometry to identify metal-enriched fractions. This approach led to the detection of nickel superoxide dismutase together with its predicted cofactor. We also explored the utility of immobilized metal affinity chromatography (IMAC) to isolate subpopulations of proteins that display affinity for a particular metal ion. We conclude that low-resolution 2D liquid chromatography is a viable fractionation technique to correlate relatively low-abundance metal ions with their few cellular destinations (e.g. Ni), but challenges remain for more abundant metals with multiple destinations such as iron. IMAC has been shown as a useful pre-fractionation technique to screen for proteins with metal-binding capacity, and may become a particularly valuable tool for the identification of metal-trafficking proteins.     

Current and prospective applications of metal ion-protein binding

Since immobilized metal ion affinity chromatography (IMAC) was first introduced, several variants of this method and many other metal affinity-based techniques have been devised. IMAC quickly established itself as a highly reliable purification procedure, showing rapid expansion in the number of preparative and analytical applications while not remaining confined to protein separation. It was soon applied to protein refolding (matrix-assisted refolding), evaluation of protein folding status, protein surface topography studies and biosensor development. In this review, applications in protein processing are described of IMAC as well as other metal affinity-based technologies.