Anion exchange liquid chromatography--inductively coupled plasma-mass spectrometry detection of the Co2+, Cu2+, Fe3+ and Ni2+ complexes of mugineic and deoxymugineic acid

Phytosiderophores, such as mugineic and deoxymugineic acid, are produced by graminaceous plant species in response to Fe deficiency conditions normally experienced in calcareous and alkaline non-calcareous soils. As these phytosiderophores have the ability to form thermodynamically stable complexes with other metal cations present in the growing medium, they have also been implicated in the transport and bioavailability of these metals in the environment. However, routine analytical methodology to detect the various metal complexes formed by these phytosiderophores is lacking. Therefore, as these complexes are negatively charged over a wide range of pH values, anion exchange liquid chromatography (AE LC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) was investigated as a means to separate and quantify these complexes. The metal-phytosiderophore complexes were prepared at pH 7 and separated by NaOH or NH4NO3 gradient elution on a Dionex AS11 anion exchange column. Of the metals tested only the Co2+ and Ni2+ complexes of mugineic and deoxymugineic acid were detected when using a 0-20mM NaOH gradient elution profile. However, the phytosiderophore complexes of Cu2+ and Fe3+ were also detected when using NH4NO3 as the mobile phase at pH 7. Base-assisted hydrolysis of the latter two complexes is proposed to explain their apparent 'instability' in the high pH NaOH mobile phase. The absolute detection limits of the developed methodologies for these metal complexes ranged from 0.1 to 2.8pmol. As phytosiderophore complexes with Cd2+ and Zn2+ were not detected, it was concluded that the dissociation kinetics of these metal-phytosiderophore complexes were too rapid for these complexes to be observed in the present chromatographic conditions.     

Alpha-benzoinomixe: Extracting agent for uranium(VI), tungsten(VI), molybdenum(VI) and some transition metals of the iron family

The effect of pH on the percent extraction of vanadium(V), iron(II), cobalt(II), nickel(II), copper(II), molybdenum(VI), tungsten(VI) and uranium(VI) by -benzoinoxime in different solvents has been studied. The maximum recovery is not appreciably affected by the nature of the solvent, but occurs at different pH values for different metals. The pH corresponding to maximum extraction increases with increasing hydrolysis pK of the species in aqueous solution, and decreases with increasing stability constant of the complexes formed. Alpha-benzoinoxime allows the separation of these metal ions into three groups: V(V), Mo(VI) and W(VI) are extracted at pH=2, U(VI) at pH=5, Fe(II), Cu(II), Co(II) and Ni(II) at around pH=10.     

Investigation on C2-ceramide complexes with transition metal ions using electrospray ionization tandem mass spectrometry

Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is applied for the investigation of C(2)-ceramide complexes with transition metal ions. Ceramide plays an important role in the regulation of various signaling pathways leading to proliferation, differentiation or apoptotic cell death. The formation and fragmentation of doubly charged cluster ions as well as singly charged cluster ions of C(2)-ceramide with transition metal ions (Mn(2+), Fe(2+), Co(2+) and Ni(2+)) are studied by ESI-MS/MS in the positive mode. Tube lens offset voltage and concentrations of C(2)-ceramide and transition metals are optimized to determine the best conditions for generating doubly charged cluster ions. The fragmentation pathways of metal ion complexes with C(2)-ceramide and the compositions of these complexes are determined by collision induced dissociation (CID). All transition metal ions (Mn(2+), Fe(2+), Co(2+) and Ni(2+) except Cu(2+)) shows similar complexation with C(2) ceramide. The unique complexation behavior of copper(II) is responsible for the different geometry of the complexes and relatively lower affinity of ceramide to copper(II) than those to other transition metals.     

Speciation of metal-EDTA complexes by flow injection analysis with electrospray ionization mass spectrometry and ion chromatography with inductively coupled plasma mass spectrometry

Flow injection analysis (FIA) with ESI-MS and ion chromatography (IC) with inductively coupled plasma-MS (ICP-MS) as the complementary technique have been explored for the determination of metal ions as their metal-EDTA complexes. ESI-MS enabled the identification of metal-EDTA complexes such as [Mn(EDTA)](2-), [Co(EDTA)](2-), [Ni(EDTA)](2-), [Cu(EDTA)](2-), [Zn(EDTA)](2-), [Pb(EDTA)](2-), and [Fe(EDTA)](1-) and their MS spectral showed that these metal-EDTA complexes were present in solution. Based on the ESI-MS, ion chromatographic separation and ICP-MS detection of these complexes are possible because IC-ICP-MS requires stable metal-EDTA complex during the chromatographic separation. The separation of these metal-EDTA complexes was achieved on an anion-exchange column with a mobile phase containing 30 mM NH(4)(HPO(4))(2) at pH 7.5 within 7 min with ICP-MS providing element specific detection. The ICP-MS LODs for the metal-EDTA were in the range of 0.1-0.5 microg/L with the exception of Fe (15 microg/L). The proposed method was a simple procedure for sample processing, using direct injection of sample without removal of sample matrix and was successfully applied to the determination of metal-EDTA complexes in real samples.     

Extraction and exchange behavior of metal species in therapeutically applied peat

The binding and availability of metals (Al, Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Ni, Pb, Zn) in therapeutically applied peat (Grosses Gifhorner Moor, Sassenburg/North Germany) was characterized by means of a versatile extraction approach. Aqueous extracts of peat were obtained by a standardized batch equilibrium procedure using high-purity water (pH 4.5 and 5.0), 0.01 mol l(-1) calcium chloride solution, 0.01 mol l(-1) ethylenediaminetetraacetic acid (EDTA) and 0.01 mol l(-1) diethylenetriamine pentaacetic acid (DTPA) solution as metal extractants. In addition, the availability of peat-bound metal species was kinetically studied by collecting aliquots of extracts after different periods of extraction time (5, 10, 15, 30, 60 and 120 min). Metal determinations were performed by atomic spectrometry methods (AAS, ICP-OES) and dissolved organic matter (DOM) was characterized by UV/Vis measurements at 254 and 436 nm, respectively. Of the extractants studied Ca, Mg and Mn were the most available metals, in contrast to peat-bound Fe and Al. The relative standard deviation s(r) of the developed extraction procedures was mostly in the range of 4 to 20%, depending on the metal and its concentration in peat. A pH increase favored the extraction of metals and DOM from peat revealing complex extraction kinetics. Moreover, a competitive exchange between peat-bound metal species and added Cu(II) ions showed that >100 mg of Cu(II) per 50 g wet peat was necessary to exchange the maximum of bound metals (e.g. 21.8% of Al, 3.9% of Fe, 79.0% of Mn, 81.9% of Sr, related to their total content).     

Kinetics and Mechanism of Ni(II) Chelation in Model and Real Solutions of Xylem Sap of Quercus ilex

The kinetics of formation and dissociation of Ni(II) complexes with oxalic and citric acids was studied by cyclic voltammetry in model solutions of xylem sap of Q. ilex (the dominant tree growing on serpentine soils of Northeast Portugal) using representative concentrations, pH and ionic strength. The role of magnesium on complex formation was analyzed from solutions where Mg is present at concentration levels found in the xylem sap of Q. ilex growing on both nonserpentine and serpentine soils. Kinetics studies were also done in diluted solutions of real xylem sap samples, spiked with increasing amounts of magnesium. The values obtained for the apparent rate constants were those anticipated by the proposed model. To test the validity of the methodology and mechanisms, formation rate constants, k_f (M^?1 s^?1) of Ni(II) complexes with citrate and oxalate were evaluated that compare with the values from Eigen mechanism.     

青海群加地区种植大黄根茎组织结构中微量元素特征

微量元素对于种植青海大黄的生长发育有着重要作用。采集青海群加地区种植大黄根茎,分为皮层、韧皮部、木质部和髓部等四部分,采用原子吸收光谱法分别测试了其不同组织中铜、锌、铁、锰、钴、镍等元素含量。结果表明,种植大黄根茎中铜、锌、铁、锰等元素主要分布在皮层,其次是木质部或髓部,元素含量较低的是韧皮部。随着生长年龄增加种植大黄对铁、锰、钴元素营养需求量有所增加,对锌、铜、镍元素营养的需求量略减。     

Analysis of phytochelatin-cadmium complexes from plant tissue culture using nano-electrospray ionization tandem mass spectrometry and capillary liquid chromatography/electrospray ionization tandem mass spectrometry.

Phytochelatins (PCs, also known as class III metallothioneins), a family of sulfhydryl-rich peptides with the formula (gamma-GluCys)(n)Gly(Pc(n), n = 2-11), are induced in plants, yeast and fungi exposed to heavy metals, and are thought to detoxify metals by forming PC- metal complexes. Although PCs have been detected, PC- metal complexes have not been well characterized. In this work, nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) and capillary liquid chromatography/electrospray ionization tandem mass spectrometry (capillary LC/ESI-MS/MS) methods were used to analyze PC - Cd complexes isolated from Datura innoxia, also known as Jimsonweed, cell culture exposed to Cd. With nano-ESI-MS/MS and capillary LC/ESI-MS/MS we could simultaneously detect the presence of PCs and PC - Cd complexes from plant cell extracts, unambiguously identify these species and elucidate the nature of individual PC - Cd complexes. Phytochelatins with n = 3-6 were detected, as were PC - Cd complexes with PC(3), PC(4) and PC(5). This is the first study to report the size and nature of native PC - Cd complexes from plant tissue samples. These results demonstrate that the direct analysis of plant extracts using nano-ESI-MS/MS and capillary LC/ESI-MS/MS methods is simple and sensitive to the range of PCs and PC - Cd complexes in plants. Hence these methods open up new opportunities for further quantitative analysis of PCs and PC - metal complexes in cell culture and plant systems to understand the relationship between the biosynthesis of these compounds and metal tolerance.     

Photocatalytic oxidation in aqueous titanium dioxide suspensions: the influence of dissolved transition metals

The influence of type, species distribution, standard reduction potential, and concentration of several transition metals on the rate of photocatalytic oxidation of toluene was investigated. A significant increase in reaction rate was observed in the presence of 10⁻⁵ M Cu(II), Fe(III), and Mn(II) at pH 3, with decreased rates at higher concentrations and pH values. There was no clear correlation between reaction rate and aqueous metal species distribution, nor did the oxidation states of Cu or Fe alter their effects on the reaction rate. Neither Ni(II) nor Zn(II) had a significant influence on the rate of organic oxidation. Negligible adsorption of metals onto TiO₂ was measured at the metal concentrations and pH values for which the highest reaction rates were observed, indicating that dissolved metals increase the reaction rate via a homogeneous pathway rather than a TiO₂ surface reaction. A mechanism involving formation of a reactive complex between the metal, the organic or its oxidation intermediate, and an oxygen-containing species is proposed to explain the experimental data. The rate of the photocatalytic reaction is described by a Langmuir—Hinshelwood rate form, modified to account for homogeneous catalytic pathways and decreased UV transmittance in the presence of dissolved metals.     

Analysis of the metal-binding selectivity of the metallochaperone CopZ from Enterococcus hirae by electrospray ionization mass spectrometry

Metallochaperones are soluble proteins involved in metal transport and regulation in vivo. Copper metallochaperones belong to a structural family of metal binding domains displaying a ferredoxin-like fold (betaalphabetabetaalphabeta) and a consensus metal-binding motif MXCXXC. The metal-binding selectivities for this class of proteins are poorly documented so far. The present study focuses on the measurement of the selectivity of the copper metallochaperone CopZ from Enterococcus hirae for different metal ions using an experimental approach based on electrospray ionization mass spectrometry (ESI-MS). All the metal cations tested, i.e. Cu(I), Cu(II), Hg(II), Cd(II) and Co(II), form specific metal complexes with CopZ. The study of a chemically modified CopZ as well as variants of CopZ in the active site demonstrated that the complexes observed by ESI-MS, i.e. in the gas phase, corresponded to the complexes previously observed by other analytical methods in solution. Competition experiments allowed the classification of the metal ions by increasing affinities for CopZ as follows: Co < Cd < Hg < Cu. A dissociation constant in the range of 20 microM was determined for cobalt. The affinity of CopZ for the other metals tested was found to be higher, with dissociation constants smaller than micromolar.     

Transformations of metal species in ageing humic hydrocolloids studied by competitive ligand and metal exchange

Transformations of metal species (particularly Al, Ca, Fe, Mg, Mn, Zn) in ageing humic hydrocolloids were studied, applying a competitive ligand and metal exchange approach. For this purpose, metal-containing hydrocolloids, freshly collected from humic-rich German bog lake waters (Hohlohsee (HO), Black Forest; Venner Moor (VM), Muensterland; Arnsberger Wald (AW), Northrhine-Westfalia) and conventionally pre-filtered through 0.45 m membranes, were subjected on-site to an exchange with EDTA and Cu(II) ions, respectively, as a function of time. EDTA complexes gradually formed, metal fractions exchanged by Cu(II) (as well as free Cu(II) concentrations) were operationally discriminated by means of a small time-controlled tangential-flow ultrafiltration unit (nominal cut-off: 1 kDa). Metal and DOM (dissolved organic matter) fractions obtained this way were determined off-site using instrumental methods (AAS, ICP-OES, carbon analyzer). After weeks of storage, the collected hydrocolloids were studied again by this approach. The EDTA availability of colloid-bound metals (particularly Al and Fe) exhibited different ageing trends, dependent on the sample (VM: decrease of Fe availability (98–76%), HO: increase of Fe availability (76–82%)). In contrast, the Cu(II) exchange equilibria of colloid-bound metals revealed merely low availability of Al (16–38%) and Fe (5–11%) towards Cu(II) ions, also dependent on ageing effects. In particular, the conditional copper exchange constants K_ex obtained from the exchange between Cu(II) ions and available metal species (such as Ca, Mg, Mn, Zn) exhibited a strong decrease (by a factor of 2–100) during sample storage, indicating considerable non-equilibria complexation of these metal ions in the original bogwaters studied on-site.     

On-site characterization of humic-rich hydrocolloids and their metal loading by means of mobile size-fractionation and exchange techniques

Humic-rich hydrocolloids and their metal loading in selected German bog-waters have been characterized by a novel on-site approach. By use of an on-line multistage ultrafiltration (MST-UF) unit equipped with conventional polyethersulfone (PES)-based flat membranes (nominal cut-off 0.45, 0.22, and 0.1 microm, or 100, 50, 10, 5, 3 kDa) the hydrocolloids could be fractionated on-site in both sub-particulate and macromolecular size ranges. Characterization (dissolved organic carbon (DOC), metals) of the colloid fractions obtained this way was performed off-site by use of conventional instrumental methods (carbon analyzer, AAS, ICP-OES, and TXRF (total reflection X-ray fluorescence)). Major DOC fractions of the hydrocolloids studied were found to be in the size range <5 kDa. The assessed metals (Al, Cu, Fe, Mn, Pb, and Zn) were, however, predominantly enriched in the macromolecular and sub-particulate range, depending on the metal and the sample, respectively. In addition, metal species bound to these hydrocolloids were kinetically characterized on-site by use of competitive ligand (EDTA (ethylenediaminetetraacetate)) and metal (Cu(II)) exchange; the EDTA complexes formed and the metal ions exchanged were separated by means of a small time-controlled tangential-flow UF unit (cut-off 1 kDa). Bound metal fractions, in particular Al and Fe, reacted only slowly (500 to 1000 min) with EDTA; the conditional availability was 60-99%, depending on the hydrocolloid. In contrast, the Cu(II) exchange of colloid-bound metal species approached equilibrium within 5-10 min, with characteristic exchange constants, Kex, of the order of 0.01 to 90 for the metals (Fe相似文献   

Application of capillary electrophoretic methods for the analysis of plant phloem and xylem saps composition: A review

Plant vascular tissue is essential for the exchange of water, nutrients, metabolic products, and signals among distant organs in cormophytes. The compositions of phloem and xylem saps are highly dependent on many internal and external factors, and thus their analysis provides a valuable insight into plant physiology, growth, and development as well as nutrition status or presence of biotic or abiotic stresses. Capillary electrophoresis characterized by highly efficient separations and minuscule sample requirements represents a suitable analytical technique for this purpose because the sap constitutes a complex mixture with generally minimal availability. This review aims at providing a comprehensive overview of published capillary electrophoretic methods for the analysis of primary components present in the phloem and xylem saps of higher plants.     

Different binding thermodynamics of Ni2+, Cu2+, and Zn2+ to bacitracin A1 determined by capillary electrophoresis

Thermodynamics of the binding of Ni(2+), Cu(2+) and Zn(2+) to bacitracin A(1) was studied by capillary electrophoresis measuring the peptide effective mobility at different pH in the presence of increasing concentration of the three ligands. The affinity follows the order Ni(2+) > Cu(2+) > Zn(2+), with association constant values of (2.3 +/- 0.1)x10(4), (4.9 +/- 0.2)x10(3), and (1.5 +/- 0.1)x10(3) M(-1), respectively. The only model able to rationalize mobility data implies that metal ion binds to the P(0) peptide form. Moreover, mobility values indicated a change of bacitracin A(1) acidic properties on Ni(2+) and Cu(2+) binding, with a shift of the pK(a) of N-terminal Ile-1 from 7.6 to about 5 and of the pK(a) of the delta-amino group of D-Orn-7 from 9.7 to about 7. Even though on Zn(2+) binding a shift of the N-terminal Ile-1 pK(a) was observed, restrictions in the pH range suitable for investigation, due to precipitation phenomena, did not allow establish if the shift of D-Orn-7 lateral chain pK(a) also occurred. Nonetheless, if present, the shift should be limited to the 7.8-9.7 range. Mobility data indicated that the Stokes radius of the complexes is ca. 3 A lower than that of the free peptide. The present results indicate that metal-ion binding to bacitracin A(1) is more complex than previously assumed.     

CE of phytosiderophores and related metal species in plants

Phytosiderophores (PS) and the closely related substance nicotianamine (NA) are key substances in metal uptake into graminaceous plants. Here, the CE separation of these substances and related metal species is demonstrated. In particular, the three PS 2'-deoxymugineic acid (DMA), mugineic acid (MA), and 3-epi-hydroxymugineic acid (epi-HMA), and NA, are separated using MES/Tris buffer at pH 7.3. Moreover, three Fe(III) species of the different PS are separated without any stability problems, which are often present in chromatographic analyses. Also divalent metal species of Cu, Ni, and Zn with the ligands DMA and NA are separated with the same method. By using a special, zwitterionic CE capillary, even the separation of two isomeric Fe(III) chelates with the ligand ethylenediamine-N,N'-bis(o-hydroxyphenyl)acetic acid (EDDHA) is possible (i.e., meso-Fe(III)-EDDHA and rac-Fe(III)-EDDHA), and for fast separations of NA and respective divalent and trivalent metal species, a polymer CE microchip with suppressed EOF is described. The proposed CE method is applicable to real plant samples, and enables to detect changes of metal species (Cu-DMA, Ni-NA), which are directly correlated to biological processes.     

Iron (Fe) speciation in xylem sap by XANES at a high brilliant synchrotron X-ray source: opportunities and limitations

The development of highly brilliant synchrotron facilities all around the world is opening the way to new research in biological sciences including speciation studies of trace elements in plants. In this paper, for the first time, iron (Fe) speciation in xylem sap has been assessed by X-ray absorption near-edge structure (XANES) spectroscopy at the highly brilliant synchrotron PETRA III, beamline P06. Both standard organic Fe-complexes and xylem sap samples of Fe-deficient tomato plants were analyzed. The high photon flux provided by this X-ray synchrotron source allows on one side to obtain good XANES spectra in a reasonable amount of time (approx. 15 min for 200 eV scan) at low Fe concentrations (sub parts-per-million), while on the other hand may cause radiation damage to the sample, despite the sample being cooled by a stream of liquid nitrogen vapor. Standard Fe-complexes such as Fe(III)-succinate, Fe(III)-α-ketoglutarate, and Fe(III)-nicotianamine are somehow degraded when irradiated with synchrotron X-rays and Fe(III) can undergo photoreduction. Degradation of the organic molecules was assessed by HPLC-UV/Vis analyses on the same samples investigated by X-ray absorption spectroscopy (XAS). Fe speciation in xylem sap samples revealed Fe(III) to be complexed by citrate and acetate. Nevertheless, artifacts created by radiation damage cannot be excluded. The use of highly brilliant synchrotrons as X-ray sources for XAS analyses can dramatically increase the sensitivity of the technique for trace elements thus allowing their speciation in xylem sap. However, great attention must be paid to radiation damage, which can lead to biased results.

Some Colloidal and Chemical Aspects of Biotransformation of Heavy Metal Citrate Complexes

It was established that chemically stable water-soluble citrate complexes of heavy metals, which are widespread forms of their occurrence under natural conditions, can be mineralized in the course of metabolism of citrate ligand by Basillus cereus AUMC 4368 metallophilic bacterial culture. Biodegradation of metal citrate complexes is not directly related to their stability constants and corresponds to the Ca (Mg, Sr) > Fe(III) > Zn (Cu, Ni, Co, Cd) > U(VI) series. As a result of biomineralization of water-soluble metal citrate complexes, pH of dispersion medium increases and water-insoluble salts, mainly carbonates and hydroxides, can be formed; correspondingly, the disperse state of metals can be changed. Exception is uranium(VI) that forms (at pH 8) water-soluble stable uranyl carbonate complex. It was shown that the bound forms of metals (water-soluble complexes, insoluble precipitants) are nontoxic for microorganisms.     

Approach combining on-line metal exchange and tangential-flow ultrafiltration for in-situ characterization of metal species in humic hydrocolloids

This paper deals with the development and optimization of an analytical procedure using ultrafiltration and a flow-injection system, and its application in in-situ experiments to characterize the lability and availability of metal species in humic-rich hydrocolloids. The on-line system consists of a tangential flow ultrafiltration device equipped with a 3-kDa filtration membrane. The concentration of free ions in the filtrate was determined by atomic-absorption spectrometry, assuming that metals not complexed by aquatic humic substances (AHS) were separated from the complexed species (M–AHS) retained by the membrane. For optimization, exchange experiments using Cu(II) solutions and AHS solutions doped with the metal ions Ni(II), Mn(II), Fe(III), Cd(II), and Zn(II) were carried out to characterize the stability of the metal–AHS complexes. The new procedure was then applied in-situ at a tributary of the Ribeira do Iguape river (Iguape, São Paulo State, Brazil) and evaluated using the ions Fe(III) and Mn(II), which are considered to be essential constituents of aquatic systems. From the exchange between metal–natural organic matter (M–NOM) and the Cu(II) ions it was concluded that Cu(II) concentrations >485 μg L^?1 were necessary to obtain maximum exchange of the complexes Mn–NOM and Fe–NOM, corresponding to 100% Mn and 8% Fe. Moreover, the new analytical procedure is simple and opens up new perspectives for understanding the complexation, transport, stability, and lability of metal species in humic-rich aquatic environments.     

Retention of heavy metal ions on SBA-15 mesoporous silica functionalised with carboxylic groups

Three samples of SBA-15 functionalised with -(CH(2))(3)COOH groups have been prepared by co-condensation, starting from solutions of TEOS and 4-(triethoxysilyl)butyronitrile, acting as -(CH(2))(3)COOH precursor, of different molar compositions. Materials were characterised by X-ray diffraction, nitrogen adsorption, and FT-IR spectroscopy. The pK(a) and the acidic capacity were measured for all samples by potentiometric titration. The acidic capacity increases with increasing amount of -COOH precursor in the synthesis mixture only up to 10% molar of total alkoxysilane. The value for the pK(a)(4.75) is independent of the acidic capacity of the material. The sample prepared starting from an amount of -COOH precursor equal to 10% molar of total alkoxysilane was chosen to test selective interactions with heavy metals of environmental importance (Ag(+), Cd(2+), Fe(3+), Cu(2+), Zn(2+)) at different pH values and ionic strengths. The significant and selective adsorption exhibited by the material has been exploited in a preliminary cation-exchange chromatographic application showing the possibility of eluting the metal ions at different retention times.     

Study of metal ion labeling of the conformational and charge states of lysozyme by ion mobility mass spectrometry

The efficiency of Zn(2+), Cu(2+), Ni(2+), Co(2+), Fe(2+) or Mn(2+) labeling of the conformational and charge states of lysozyme was studied in H(2)O solvent at pH 2.5-6.8. Labeling of lysozyme was conducted with 50 M, 100 M and 500 M excess of the metal ion, resulting in the number of metal ions attached to lysozyme increasing two-fold over this range. At pH 6.2-6.8, Zn(2+), Cu(2+), Ni(2+), Co(2+) and Mn(2+) labeled the highly folded 7+ conformer and the 8+ and 9+ partially unfolded conformers of lysozyme with the same number of metal ion tags, with only Fe(2+) exhibiting no labeling. Lysozyme conserved its charge after metal ion labeling which shows at each charge state the divalent metal ion is replacing two protons. As the pH is lowered to 4.7-5.0 and 2.5-2.9, the labeling of lysozyme by Zn(2+), Cu(2+), Ni(2+), Co(2+) or Mn(2+) decreased in efficiency due to increased competition from protons for the aspartate and glutamate binding sites. The metal ions preferentially labeled the highly folded 7+ and partially unfolded 8+ conformers, but labeling decreased as the charge of lysozyme increased. In contrast to the other metal ions, Fe(2+) exhibited labeling of lysozyme only at the lowest pH of 2.8. At higher pH, the oxidation of Fe(2+) and formation of hydroxy-bridged complexes probably make the Fe(2+) unreactive towards lysozyme.