A high-performance liquid chromatography method for determining transition metal content in proteins

Transition metals are common components of cellular proteins and the detailed study of metalloproteins necessitates the identification and quantification of bound metal ions. Screening for metals is also an informative step in the initial characterization of the numerous unknown and unclassified proteins now coming through the proteomic pipeline. We have developed a high-performance liquid chromatography method for the quantitative determination of the most prevalent biological transition metals: manganese, iron, cobalt, nickel, copper, and zinc. The method is accurate and simple and can be adapted for automated high-throughput studies. The metal analysis involves acid hydrolysis to release the metal ions into solution, followed by ion separation on a mixed-bead ion-exchange column and absorbance detection after postcolumn derivatization with the metallochromic indicator 4-(2-pyridylazo)resorcinol. The potential interferences by common components of protein solutions were investigated. The metal content of a variety of metalloproteins was analyzed and the data were compared to data obtained from inductively coupled plasma-atomic emission spectroscopy. The sensitivity of the assay allows for the detection of 0.1-0.8 nmol, depending on the metal. The amount of protein required is governed by the size of the protein and the fraction of protein with metal bound. For routine analysis 50 microg was used but for many proteins 10 microg would be sufficient. The advantages, disadvantages, and possible applications of this method are discussed.     

Is salivary histatin 5 a metallopeptide?

Histatins are small histidine-rich salivary polypeptides which exhibit antimicrobial activity against Candida albicans. This antimicrobial activity has been ascribed in part to a high content of basic amino acids. However, unlike most other antimicrobial proteins histatins have a high content of histidine, tyrosine and acidic amino acids known to participate in metal ion coordination. This study was conducted to test whether histatin 5 could bind zinc and copper which are metals present in salivary secretions and whole saliva. Physical binding parameters and spectral properties of zinc- and copper-histatin complexes were investigated in order to obtain direct evidence of these interactions. A spectrophotometric competition assay using the metallochromic indicator murexide showed that histatin 5 dissociates metal indicator complexes containing zinc or copper ions. Absorption spectra of histatin 5 at increasing copper chloride concentrations resulted in higher absorbance in the 230-280 nm wavelength range and this spectral change was saturated at a peptide:metal molar ratio of approx. 1:1. A corresponding band was observed in the visible range of the spectrum with a maximum and molar extinction coefficient corresponding to that of copper binding to an ATCUN motif. Quantitative assessment of zinc and copper binding to histatin 5 using isothermal titration calorimetry revealed at least one high affinity site for each metal, with binding constants of 1.2x10(5) and 2.6x10(7) M(-1), respectively. These results indicate that histatin 5 exhibits metallopeptide-like properties. The precise biological significance of this has not yet been established but histatins may contribute significantly to salivary metal binding capacity.     

Vacuoles: main compartments of potassium, magnesium, and phosphate ions in Saccharomyces carlsbergenis cells.

The uneven distribution of Mg2+, K+, and phosphate in Saccharomyces carlsbergensis was demonstrated by the differential extraction of ions. Their concentrations were 5, 60, and 1 mM in the cytoplasm and 73, 470, and 110 mM in vacuoles, respectively. The intracellular gradients of these ions were 1:15, 1:8, and 1:110, respectively, across the tonoplast. The determination of free Mg2+ (1.35 mM in the cytosol and 20 mM in vacuoles) showed that the ion accumulation in vacuoles could not be explained by the higher degree of ion complexing in these organelles.     

Liquid scintillation counting of calcium-45 in biological material.

Several scintillator solutions were tested for direct mixing with biological material for determination of calcium-45 radioactivity. For each system the capacity for aqueous solutions and stable calcium, the interference of various ions present in biological material, the stability of counting samples, and the possibility of quenching correction were determined. The results showed that the best capacity for aqueous acid solutions had Insta-Gel and Unisolve-1 scintillants. Tritontoluene-based scintillant showed marked unstability when mixed with biological fluids. The strongest quenching effect was produced by the yellow color of the ferric ion, which could be eliminated by complexing with the fluoride ion. For biological material of sufficiently high radioactivity the most convenient method seems to be counting the samples on filter paper in a toluene-based scintillator, especially since self-absorption in such a system can be corrected for by the channel and external standard ratio.     

A spin-label assay for metal ion chelation and complex formation.

The electron spin resonance (ESR) lines of nitroxide spin labels are broadened by electron spin exchange reactions that take place during collisions with paramagnetic ions. The degree of line broadening is greatly reduced when the paramagnetic ion forms a coordination bond with certain functional groups on organic molecules. These observations form the basis for a spin-label assay for metal ion chelation and complex formation. This paper describes the characteristics of such an assay for divalent nickel ions and the spin label TEMPONE (2,2,6,6-tetramethylpiperidone-N-oxyl). The chelation of Ni²⁺ by cysteine and the interaction of Ni²⁺ with sodium dodecyl sulfate micelles and phospholipid vesicles are demonstrated. In addition to monitoring interactions of paramagnetic ions, the assay also allows the detection of interactions of nonparamagnetic ions that compete with the paramagnetic ions for binding sites. A kinetic analysis of competition between Ni²⁺ and Zn²⁺ ions for binding sites on phospholipid vesicles is presented. There are several advantages of the spin-label line-broadening assay compared to other conventional assays for metal chelation and complex formation. The line-broadening assay does not require that the sample be optically clear or chemically defined, it requires only very small quantities of material, it can detect as little as 0.4 to 1 μmol of complexing agent, and it may be utilized in complex biological systems including subcellular organelles and macromolecules.     

Interaction between trehalose and alkaline-earth metal ions

We investigated the interaction between trehalose and alkaline-earth metal ions. The nuclear relaxation times of carbon atoms of trehalose were shortened by addition of the alkaline-earth chloride salts, MgCl2, CaCl2, and SrCl2, indicating that trehalose formed metal-complexes with the alkaline-earth metal chlorides. From the data of the 1H-1H coupling constants of trehalose in the presence of the alkaline-earth chlorides, it appeared that trehalose formed complexes with MgCl2, and CaCl2 at the various complexing sites: Mg2+ was coordinated to O-4 and O-4' of trehalose, and Ca2+ to O-2 and O-3. We succeeded in the preparation of two types of crystals of the trehalose/CaCl2. One was a crystal consisting of trehalose, CaCl2, and water in a ratio of 1:1:1. The other was an anhydrous crystal containing trehalose and CaCl2 in a ratio of 1:2. Several applications of the complexing between trehalose and the metal ions for food processing are proposed.     

A survey of the available colorimetric indicators for Ca2+ and Mg2+ ions in biological experiments

The binding of Ca²⁺ and Mg²⁺ ions to commercially available and easily synthesizable metallochromic indicators has been systematically examined at pH 7.35, temperature 37°C, ionic strength 0.16, the conditions of blood plasma. The pCa and pMg midpoints of the colour changes of all the useful indicators are reported. In addition to the well-known indicators arsenazo III, chlorophosphonazo III, antipyrylazo III, and murexide for Ca²⁺, and Eriochrome Black T and Eriochrome Blue SE for Mg²⁺, we draw attention to the values of oxyacetazo I, carboxyazo III, tropolone, methylthymol blue, Mordant Black 32, and the tetracyclines.     

Spectrophotometric determination of photoreceptor cGMP-gated channel Mg2(+)-fluxes using dichlorophosphonazo III.

We have characterised the spectroscopic properties of the metallochromic dye dichlorophosphonazo III and describe its use for the determination of changes of Mg2+ concentration in the micromolar range. Using a previously described reconstitution procedure, we incorporated the cGMP-gated channel from bovine rod photoreceptors into magnesium-containing liposomes and used the dye to monitor cGMP-activated Mg2(+)-efflux. The Km and cooperativity of the cGMP-dependence were identical regardless of whether Mg2+ or Ca2+ was the transported ion, however, the vmax for Ca2+ was more than 2-fold higher than that for Mg2+. We thereby determined a channel selectivity (Ca2+:Mg2+) of 1.0:0.44 in the presence of symmetrical (30 mM) K+. We also describe conditions where Mg2+ or Ca2+ effluxes can be selectively monitored in the presence of each other. This allowed the demonstration that magnesium ions can flow through the cGMP-gated channel even in the presence of an identically directed calcium gradient. Together these results indicate that magnesium ions may enter the photoreceptor rod outer segment cytosol through the cGMP-gated channel under dark conditions, thereby alluding to the existence of an as yet unknown Mg2(+)-extrusion mechanism, distinct from the Na+/Ca2(+)-exchanger, in these cells.     

Interaction of metallochromic indicators with calcium sequestering organelles.

Examples are presented of the interaction between cell organelles and metallochromic indicators used in the measurement of ionized Ca2+. Sarcoplasmic reticulum was found to sequester murexide type indicators along with Ca2+ in the presence of ATP, but not to sequester arsenazo III and antipyrylazo III. The presence of a permeable anion suppresses the sequestration of murexide type indicators by the sarcoplasmic reticulum. In the presence of ruthenium red, both rat liver and beef heart mitochondria release sequestered Ca2+ with arsenazo III, but not with murexide.     

Synthesis and characterization of metal ion imprinted nano-porous polymer for the selective recognition of copper

Selective recognition of metal ions utilizing metal ion-imprinted polymers (MIIPs) received much importance in diverse fields owing to their high selectivity for the target metal ions. In the present study, a copper ion imprinted polymer was synthesized without an additional complexing ligand or complex with a broad aim to avoid the conventional extra metal ion complexing ligand during the synthesis of MIIP. The complete removal of the copper metal ion from the MIIP was confirmed by AAS and SEM–EDX. SEM image of the MIIP exhibited nano-patterns and it was also found to be entirely different from that of non-imprinted polymer and polymer with copper metal ions. BET surface area analysis revealed more surface area (47.96 m²/g) for the Cu(II)-MIIP than non-imprinted control polymer (41.43 m²/g). TGA result of polymer with copper metal ion indicated more char yield (18.41%) when compared to non-imprinted control polymer (8.3%) and Cu(II)-MIIP (less than 1%). FTIR study confirmed the complexation between Cu(II)-MIIP and Cu(II) metal ion through carbonyl oxygen of acryl amide. The Cu(II)-MIIP exhibited an imprinting efficiency of 2.0 and it was showing 8% interference from a mixture of Zn, Ni and Co ions. A potentiometric ion selective electrode devised with Cu(II)-MIIP showed more potential response for Cu(II) ion than that was fabricated from non-imprinted polymer.     

Nucleotide pyrophosphatase from yeast. The presence of bound zinc.

Nucleotide pyrophosphatase from yeast was inhibited by thiols, o-phenanthroline, 8-hydroxyquinoline, EDTA, and 8-hydroxyquinoline-5-sulfonic acid. The inhibition by chelating agents was time and concentration dependent. Inhibition by EDTA was decreased by complexing the EDTA with metal ions before addition to the enzyme. The effectiveness of the metal ions in preventing inhibition by EDTA paralleled the stability constants of the EDTA-metal complexes. Partial recovery of EDTA-inhibited enzyme activity was achieved with Zn²⁺, Co²⁺, Fe²⁺, and Mn²⁺. Analyses for zinc in the purified enzyme by atomic absorption spectroscopy and by titration with 8-hydroxyquinoline-5-sulfonic acid revealed the presence of approximately 1 g atom/mol of enzyme (M_r 65,000). The data indicate that yeast nucleotide pyrophosphatase is a metalloenzyme in which the zinc plays some role in activity.     

X-ray scattering from labeled membranes.

We present a new method for the determination of structural parameters in biological membranes. Recording the continuous scattering of heavy-atom labeled membranes and applying elementary Fourier methods we obtain the scattering of the heavy-atom distribution alone. The details of this distribution are explored by developing a simple model and testing for cases relevant to biological membranes. We find that the intensity distribution is highly sensitive to many key parameters. The increased signal from heavy-atom labeling and the use of an improved x-ray system make it possible to record patterns from dilute membrane suspensions. Thus determination of these parameters is possible in the same environment where many membrane biochemical studies are performed. Application of the method is made to a model lipid bilayer membrane, dipalmitoyl phosphatidylcholine by labeling with UO2++ ions. We determine the precise distance between UO2++ layers on either side of the membrane as well as the width of the label on each side. This determination permits estimation of phosphate separation across single labeled bilayers in an aqueous suspension.     

Stoichiometry of the reactions of calcium with the metallochromic indicator dyes antipyrylazo III and arsenazo III.

A method for determining the stoichiometry of one-product reactions involving a metal ion and an organic ligand is presented and applied to the reactions of calcium and magnesium with the metallochromic dyes Antipyrylazo III and Arsenazo III. The method consists of fitting titration data, obtained in solutions buffered for the metal, with theoretical functions that include: (a) the dependence of product concentration on the concentration of both reactants, (b) the relationship between metal ion concentration and total amount added in the presence of the buffer, and (c) a correction for the amount of metal ion that binds to the organic ligand. It is shown that the products of the reactions of Antipyrylazo III with calcium and magnesium are CaD2 and MgD, respectively. The product formed between calcium and Arsenazo III at [Ca2+] over 20 microM is CaD2, other products accumulating at lower [Ca2+]. The kinetics of the Antipyrylazo III:Ca reaction are rapid under conditions in which this dye has been applied to measure calcium transients in skeletal muscle fibers. The present results provide a calibration for previous studies with Antipyrylazo III in muscle fibers.     

Aluminum determination in biological fluids and dialysis concentrates via chelation with 8-hydroxyquinoline and solvent extraction/fluorimetry

We describe a simple, rapid, and sensitive fluorescence method for measurement of aluminum (Al) in human biological fluids, in dialysis solutions, and in tap water, which uses 8-hydroxyquinoline for ion chelation. The fluorescence intensity of the toluene-extracted metal chelate (excitation wavelength, 380 nm; emission wavelength, 504 nm) remains unchanged for over 48 h at room temperature. Fluorescence intensity is a linear function of the concentration of Al in the 2-1000 microg/L range with detection limits of 0.7-2 microg/L. A large excess of other ions normally found in biological fluids does not interfere in Al determination. The method developed was successfully used in assaying Al in serum and urine of reference subjects, in serum samples from patients undergoing long-term dialysis, and in dialysis solutions. Al concentrations, measured by this fluorimetric procedure, were compared with those obtained by Zeeman graphite-furnace atomic absorption spectrometry. A correlation coefficient of 0.98 was obtained. The proposed method could be used for routine analysis in clinical laboratories for accurate determination of aluminum in aqueous or biological fluids.     

Metal-ion-directed site-specificity of hydroxyl radical detection.

A wide variety of .OH detectors are in use for determination of biological .OH production. The chemical generation of .OH is site-specific with respect to the metal-binding site, and thus .OH detectors with metal-binding properties may affect the biological damage and bias .OH detection. The present study shows that both salicylate and phenylalanine, added as low molecular weight .OH indicators, decreased Cu(II) binding to erythrocyte ghosts. In a cell-free system, Cu(II) complexed to both salicylate and phenylalanine. Phenylalanine is a stronger Cu(II) chelator than salicylate, both when competing for Cu(II) bound to ghosts and when competing directly with each other. When OH radicals were generated by ascorbate and Cu(II), the amount of .OH detected as dihydroxybenzoates was proportional to the amount of .OH produced. However, when phenylalanine was added to this system, the efficiency of .OH detection by salicylate strongly decreased, concomitant with the transfer of Cu(II) binding from salicylate to the amino acid. This decrease was larger than that predicted by calculations for random competition of the two detectors for .OH. Deoxyribose and mannitol, which do not bind copper appreciably, competed poorly with salicylate for the .OH. Hydroxylation of phenylalanine, on the other hand, was only slightly affected by the presence of salicylate and unaffected by deoxyribose and mannitol. These results suggest that the detection of .OH by low molecular weight .OH indicators was related to the relative affinity of the detectors for the catalyzing metal, and thus partially site-specific. Furthermore, glutamate, which does not contain an aromatic ring but binds Cu(II) with considerable affinity, competed strongly with salicylate for the .OH, indicating that metal-binding properties rather than the presence of an aromatic ring were the cause of the deviation from random competition. The results indicate that .OH indicators with metal-binding properties affect the distribution of catalytic metal ions in a biological system, causing a shift of free radical damage and localizing a site-specific reaction of .OH on these detectors, with a resulting positive bias in the apparent .OH production.     

Spectrophotometric determination of reaction stoichiometry and equilibrium constants of metallochromic indicators. II. The Ca2+-arsenazo III complexes

The analytical method described in the preceding article was applied to spectrophotometric Ca2+-titrations of the metallochromic indicator arsenazo III (Ar). At various reactant concentrations it was determined that Ar forms 1:1,1:2 and 2 : 1 complexes with calcium. The equilibrium constants and extinction coefficients at 602 nm were determined. Corrected to zero ionic strength at 293 K and pH 7.0, the reactions Ca + Ar = CaAr, CaAr + Ar = CaAr2 and CaAr + Ca = Ca2Ar are associated with dissociation equilibrium constants k(11) = 1.6 x 10(-6)M, K12 = 3.2 x 10(-4)M and K21 = 5.8 x 10(-3)M. respectively. The extinction coefficient of unbound indicator is (602) = 9.6 (+/-0.3) x 10(3) cm(-1) M(-1). Arscnazo III complexes with monovalent ions like Na+ and K+ : at zero ionic strength, the dissociation constant of the Na+-Ar complex is about 0.1 M.     

Metal-binding characteristics of the amino-terminal domain of ZntA: binding of lead is different compared to cadmium and zinc

ZntA from Escherichia coli, a P1-type ATPase, specifically transports Pb(II), Zn(II), and Cd(II). Most P1-type ATPases have an N-terminal domain that contains one or more copies of the conserved metal-binding motif, GXXCXXC. In ZntA, the N-terminal domain has approximately 120 residues with a single GXXCXXC motif, as well as four additional cysteine residues as part of the CCCDGAC motif. The metal-binding specificity and affinity of this domain in ZntA was investigated. Isolated proteins, N1-ZntA and N2-ZntA, containing residues 1-111 and 47-111 of ZntA, respectively, were characterized. N1-ZntA has both the CCCDGAC and GXXCXXC motifs, while N2-ZntA has only the GXXCXXC motif. ICP-MS measurements showed that N1-ZntA can bind both divalent metal ions such as Cd(II), Pb(II), and Zn(II) and monovalent metal ions such as Ag(I), with a stoichiometry of 1. N2-ZntA can bind Zn(II) and Cd(II) with a stoichiometry of 1 but not Pb(II). The affinity of N1-ZntA for Zn(II), Pb(II), and Cd(II) was measured by competition titration with metallochromic indicators. Association constants of approximately 10(8) M(-)(1) were obtained for Zn(II), Pb(II), and Cd(II) binding to N1-ZntA. To investigate whether the CCCDGAC sequence has an important role in binding specifically Pb(II), a mutant of ZntA, which lacked the first 46 residues, was constructed. This mutant, Delta46-ZntA, had the same activity as wtZntA with respect to Cd(II) and Zn(II). However, its activity with Pb(II) was similar to the mutant DeltaN-ZntA, which lacks the entire N-terminal domain (Mitra, B., and Sharma, R. (2001) Biochemistry 40, 7694-7699). Thus, binding of Pb(II) appears to involve different ligands, and possibly geometry, compared to Cd(II) and Zn(II).     

Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC

Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ (Kd approximately 20 microM, 6 microM and 12 microM respectively) and selectivity over Mg2+ (Kd approximately 2 mM for mag-Fura-2, > 10 mM for Fura-2FF and BTC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca2+ binding, susceptibility to photodamage, and sensitivity to alterations in pH. All three indicators bound other metal ions including Zn2+, Cd2+ and Gd3+. Interestingly, only in the case of BTC were spectral differences apparent between Ca2+ and other metal ions. For example, the presence of Zn2+ increased BTC fluorescence 6-fold at the Ca2+ isosbestic point, suggesting that this dye may be used as a fluorescent Zn2+ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca2+ indicator for most imaging applications. BTC may be useful if experimental conditions require visible wavelength excitation or sensitivity to other metal ions including Zn2+.     

Coordination chemical studies on metalloenzymes. Measurement of binding constant between apo-tyrosinase and copper ion

The behavior of complexing agents for the copper removal reaction was studied by the equilibrium dialysis method. In the copper removal reaction, complexing agents are divided into two types: those that are reducing agents and those that are not. Sodium cyanide and sodium thiosulfate are of the first type, and 8-hydroxyquinoline-5-sulfonic acid, 2,2′-bipyridyl, and picolinic acid are of the second type. From equilibrium dialysis with the first type of complexing agent, the apparent binding constant (pH 6.0) between cuprous ions and apotyrosinase was calculated to be 10¹⁵m^?1. Similarly, the apparent binding constant (pH 6.0) between cupric ions and apo-tyrosinase was about 10¹³m^?1, which was calculated from equilibrium dialysis with the second type of complexing agent. The apparent binding constant between cuprous ions and apo-tyrosinase was larger than that between cupric ions and apo-tyrosinase.     

Influence of complexing agents on stability and activity.

Metal ion-complexing agents, like KCN, EDTA etc., inactivate alkaline phosphatase of pig kidney. This inactivation is reversible at low concentrations of the complexing agents and irreversible at high concentrations. The reversible inhibition is probably due to removal of Zn2+ ions from the active site, where they are necessary for catalytic action, whereas the irreversible inhibition results from the removal of Zn2+ ions necessary for preservation of the structure. The inactivation is pseudo-first order. It depends on the concentration, size and charge of the complexing agents. Beta-Glycerophosphate and Mg2+ ions protect the enzyme from inactivation by complexing agents. Quantitative examination of the effect of substrate leads to a model that is similar to the "sequential model" proposed by D.E. Koshland, G. Nemethy & D. Filmer (1966) (Biochemistry 5, 365-385) to explain allosteric behavior of enzymes. It describes the sequential addition of two substrate molecules at two active centres of the dimer enzyme. The binding of the substrate molecules is accompanied by changes in the conformation, which lead to stabilization of the enzyme against attack by complexing agents.