Peptides mimicking the N-terminal Cu(II)-binding site of bovine serum albumin: synthesis, characterization and coordination with Cu(II) ions

The N-terminal region of bovine serum albumin (Asp-Thr-His-Lys) is known to provide a specific binding site for Cu(II) ions, with the histidine residue thought to be mainly responsible for the specificity. Thiomolybdates have been found to increase the binding affinity of Cu(II) to some serum albumins. As part of a series of studies to study the interactions between Cu(II), thiomolybdates and bovine serum albumin, we have performed the syntheses and characterization of small model peptides such as His-Lys, Thr(Ac)-His-Lys and Thr-His-Lys. Proton NMR spectra have been monitored in H(2)O solution as a function of pH and added Cu(II) concentration. Reliable K(a) values for His-Lys and Thr(Ac)-His-Lys have been established. Probable binding sites of Cu(II) and the relative strengths of binding to these peptides are also discussed.     

Selective binding behavior of zinc(II) and copper(II) ions to their native sites of apo-bovine superoxide dismutase

The four binding constants of zinc(II) ions to apo-bovine superoxide dismutase were measured by the method of equilibrium dialysis. The binding constants (10(11.1)-10(10.9) M-1) of zinc ions to the native zinc sites were much larger than those to the native copper sites (10(7.8)-10(6.5) M-1) at pH 6.25. The competitive reaction between copper(II) and zinc(II) ions for the native copper sites of copper free bovine superoxide dismutase was also investigated. The native copper sites of bovine superoxide dismutase selectively react with copper ions, because the binding constants of copper ions for the native copper sites were much larger (10(6) times) than those of zinc ions.     

Mg(II) binding by bovine prothrombin fragment 1 via equilibrium dialysis and the relative roles of Mg(II) and Ca(II) in blood coagulation

The first direct equilibrium dialysis titration of the blood coagulation protein bovine prothrombin fragment 1 with Mg(II) is presented. Fragment 1 has fewer thermodynamic binding sites for Mg(II) than Ca(II), less overall binding affinity, and significantly less cooperativity. Several nonlinear curve fitting models were tested for describing the binding of fragment 1 with Mg(II), Ca(II), and mixed metal binding data. The Mg(II) data is represented by essentially five equivalent, noninteracting sites; for Ca(II), a model with three tight, cooperative sites and four "loose", equal affinity, noninteracting sites provides the best model. Based on the reported equilibrium dialysis data and in conjunction with other experimental data, a model for the binding of Ca(II) and Mg(II) to bovine prothrombin fragment 1 is proposed. The key difference between the binding of these divalent ions is that Ca(II) apparently causes a specific conformational change reflected by the cooperativity observed in the Ca(II) titration. The binding of Ca(II) ions to the three tight, cooperative sites establishes a conformation that is essential for phospholipid X Ca(II) X protein binding. The filling of the loose sites with Ca(II) ions leads to charge reduction and subsequent phospholipid X Ca(II) X protein complex interaction. Binding of Mg(II) to bovine prothrombin fragment 1 does not yield a complex with the necessary phospholipid-binding conformation. However, Mg(II) is apparently capable of stabilizing the Ca(II) conformation as is observed in the mixed metal ion binding data and the synergism in thrombin formation.     

Synthesis and physicochemical characterization of Gd-DTPA-B(sLex)A, a new MRI contrast agent targeted to inflammation

A new magnetic resonance imaging (MRI) contrast agent designed to mimic sialyl Lewis X (sLeX) and to target inflammation has been synthesized and characterized. The evolution of its proton longitudinal relaxivity as a function of the magnetic field (NMRD) and temperature has been studied. The exchange rate of the water coordinated to the metal has been assessed by oxygen-17 relaxometry. The transmetalation by zinc(II) ions and the noncovalent binding to human serum albumin have been evaluated. The results show no limitation by the residence time of the coordinated water molecule above room temperature, a higher stability of the complex versus transmetalation by zinc(II) ions than a parent complex, the clinically used Gd-DTPA-BMA, and negligible interaction with human serum albumin.     

The metallomics approach: use of Fe(II) and Cu(II) footprinting to examine metal binding sites on serum albumins

Metal binding to serum albumins is examined by oxidative protein-cleavage chemistry, and relative affinities of multiple metal ions to particular sites on these proteins were identified using a fast and reliable chemical footprinting approach. Fe(ii) and Cu(ii), for example, mediate protein cleavage at their respective binding sites on serum albumins, in the presence of hydrogen peroxide and ascorbate. This metal-mediated protein-cleavge reaction is used to evaluate the binding of metal ions, Na(+), Mg(2+), Ca(2+), Al(3+), Cr(3+), Mn(2+), Co(2+), Ni(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Ce(3+) to albumins, and the relative affinities (selectivities) of the metal ions are rapidly evaluated by examining the extent of inhibition of protein cleavage. Four distinct systems Fe(II)/BSA, Cu(II)/BSA, Fe(II)/HSA and Cu(II)/HSA are examined using the above strategy. This metallomics approach is novel, even though the cleavage of serum albumins by Fe(II)/Cu(II) has been reported previously by this laboratory and many others. The protein cleavage products were analyzed by SDS PAGE, and the intensities of the product bands quantified to evaluate the extent of inhibition of the cleavage and thereby evaluate the relative binding affinities of specific metal ions to particular sites on albumins. The data show that Co(II) and Cr(III) showed the highest degree of inhibition, across the table, followed by Mn(II) and Ce(III). Alakali metal ions and alkaline earth metal ions showed very poor affinity for these metal sites on albumins. Thus, metal binding profiles for particular sites on proteins can be obtained quickly and accurately, using the metallomics approach.     

Spectroscopic and thermodynamic determination of three distinct binding sites for Co(II) ions in human serum albumin

Human serum albumin (HSA) is the most abundant protein of blood serum, involved in the transport of metal ions, including Co(II). Using circular dichroism spectroscopic titrations we characterized three distinct Co(II) binding sites in HSA. Applying Cu(II), Ni(II) and Cd(II) ions as competitors we determined that these sites are identical with three binding sites known for other metal ions. We ordered these sites according to their binding affinities as cadmium site B (CdB) > multi-metal binding site (MBS) > N-terminal binding site (NTS). Using isothermal titration calorimetry (ITC) we confirmed the presence of these three binding sites and determined their conditional binding constants at pH 7.4 as 9 ± 5, 1.1 ± 0.5, and 0.9 ± 0.3 × 10⁴ M⁻¹, respectively. The impact of these results on the albumin cobalt binding (ACB) clinical assay for myocardial ischemia is discussed.     

Zn(II)-113Cd(II) and Zn(II)-Mg(II) hybrids of alkaline phosphatase. 31P and 113Cd NMR

Methods have been developed for the addition of different metal ion species to the three distinct pairs of metal sites (A, B, and C) found in the dimer of apoalkaline phosphatase. This allows the preparation of hybrid alkaline phosphatases in which A and B sites of each monomer contain two different species of metal ion or the A and B sites of one monomer contain the same species of metal ion, while the adjacent monomer contains a second species. The following hybrids have been characterized in detail: (Zn(II)ACd(II)B)2 alkaline phosphatase, (Zn(II)AMg(II)B)2 alkaline phosphatase, (Cd(II)AZn(II)B)2 alkaline phosphatase, and (Zn(II)AZn(II]B)(Cd(II)ACd(II)B) alkaline phosphatase. 31P and, where appropriate, 113Cd NMR have been used to monitor the behavior of the covalent (E-P) and noncovalent (E X P) phosphointermediates and of the A and B metal ions. From the pH dependencies of the E-P in equilibrium E X P in equilibrium E + Pi equilibria, it is clear that A site metal is the dominant influence in dephosphorylation of E-P and may have a coordinated water molecule, which ionizes to ZnOH- at a low pH providing the nucleophile for dephosphorylation. A site metal also serves to coordinate phosphate in the E X P complex. B site metal has a much smaller effect on dephosphorylation rates, although it does dramatically alter the Pi dissociation rate, which is the rate-limiting step for the native enzyme at alkaline pH, and is probably important in neutralizing the charge on the phosphoseryl residue, thus potentiating the nucleophilic attack of the OH- bound at A site. Phosphate dissociation is slowed markedly by replacement of B site zinc by cadmium. There is clear evidence for long range effects of subunit-subunit interactions, since metal ion and phosphate binding at one active center alters the environments of A and B site metal ions and phosphoserine at the other active site.     

Coordination chemistry of vitamin C. Part II. Interaction of L-ascorbic acid with Zn(II), Cd(II), Hg(II), and Mn(II) ions in the solid state and in aqueous solution

The reaction of L-ascorbic acid with the zinc group and manganese ions has been investigated in aqueous solution at pH 6-7. The solid salts of the type M (L-ascorbate)2.2H2O, where M = Zn(II), Cd(II) and Mn(II) were isolated and characterized by 13C NMR and Fourier Transform infrared (FT-IR) spectroscopy. Spectroscopic evidence showed that in aqueous solution, the bindings of the Zn(II) and Mn(II) ions are through the ascorbate anion O-3 and O(2)-H groups (chelation), while the Cd(II) ion binding is via the O-3 atom only. In the solid state, the binding of these metal ions would be through two acid anions via O-3, O-2 of the first and O-1, O-3 of the second anion as well as to two H2O molecules, resulting in a six-coordinated metal ion. The Hg(II) ion interaction leads to the oxidation of the ascorbic acid in aqueous solution.     

Investigation of Ternary Complex Formations of Polyacrylic Acid with Bovine Serum Albumin in the Presence of Metal Ions by Fluorescence and Dynamic Light Scattering Measurements

In this paper, the complex formation of bovine serum albumin (BSA) and polyacrylic acid (PAA) in the presence metal ions at pH = 7 has been examined by using fluorescence and dynamic light scattering measurements. It has been observed that the most stable complexes of polyacrylic acid and bovine serum albumin have occurred in the presence of copper(II) ions. The other ions have the ability to form weak complexes between polyions and bovine serum albumin. To prior characterizing the interaction between bovine serum albumin and polyacrylic acid, the dynamic light scattering technique have been applied to determine the intensity-size distributions of the solutions of bovine serum albumin, polyacrylic acid, and ternary mixtures containing various molar ratios of bovine serum albumin to polyacrylic acid (the molar ratios of bovine serum albumin to polyacrylic acid has been taken equal to 0.5, 1.0, 1.5, 2.0 and 2.5) prepared at different molar ratios of copper(II) ions/acrylic acid unit. When the molar ratio of copper(II) ions to acrylic acid in the ternary mixtures has been lower than and equals to 0.3, two peaks have been observed in the curves of the intensity-size distributions due to contents of free bovine serum albumin and ternary complexes of polyacrylic acid-copper(II)-bovine serum albumin whereas when the molar ratio of copper(II) ions to acrylic acid equals to 0.4, the hydrodynamic diameter has pointed out only one peak. This result indicates that soluble and stable ternary complexes has occurred when the molar ratio of copper(II) ions to acrylic acid has been taken equal to 0.4.     

Characterization of the copper(II)- and nickel(II)-transport site of human serum albumin. Studies of copper(II) and nickel(II) binding to peptide 1-24 of human serum albumin by 13C and 1H NMR spectroscopy

As a basis for understanding the role of albumin in the transport of metal ions, detailed investigations have been carried out to elucidate the structure of Ni(II)- and Cu(II)-binding site of the peptide residue corresponding to the NH2-terminal peptide fragment 1-24 of human serum albumin by 1H and 13C NMR spectroscopy. These studies have been conducted in aqueous medium at different pH values and at different ligand/metal ratios. The results show the following: (i) Diamagnetic Ni(II) complex and paramagnetic Cu(II) complex are in slow exchange NMR time scale. (ii) Titration results of Ni(II)-bound form of peptide 1-24 show the presence of a 1:1 complex in the wide pH range (6.0-11.0), and the same stoichiometry is proposed for Cu(II) as well. (iii) Analysis of the spectra suggests that both Ni(II) and Cu(II) have one specific binding site at the NH2-terminal tripeptide segment (Asp-Ala-His...) involving the Asp alpha-NH2, His N(1) imidazole, two deprotonated peptide nitrogens (Ala NH and His NH), and the Asp COO- group. (iv) Complexation of Ni(II) and Cu(II) causes conformational change near the metal-binding site of the polypeptide chain, but there is no other binding group involved besides those in the first three residues.     

Spectroscopic studies on metal distribution in Co(II)/Zn(II) mixed-metal clusters in rabbit liver metallothionein 2.

Metal selectivity of metal-thiolate clusters in rabbit liver metallothionein (MT) 2 has been studied by examining the metal distribution of two similarly sized divalent metal ions, cobalt and zinc, which have different thiolate affinity. The forms of mixed-metal cluster species in (Co/Zn)7-MT generated with different ratios of both metal ions offered to the metal-free protein were investigated using EPR, ultraviolet/visible absorption and MCD spectroscopy. The results demonstrated that the distribution of these metals between the two metal-thiolate clusters is not random. Thus, the EPR absorption intensities of the bound Co(II) ions in the Zn-cluster matrix increased linearly up to a ratio of Co(II)/Zn(II) equivalents of 3:4, with the final EPR intensity of three non-interacting Co(II)-binding sites. This EPR behaviour is consistent with a binding scheme in which one Co(II) ion occupies a metal-binding site within the three-metal cluster and the remaining two Co(II) ions occupy two distinctly separate sites in the four-metal cluster. With four or more Co(II) ions in the cluster matrix, magnetic coupling between adjacent, sulphur-bridged Co(II) ions was observed. In previous studies on mixed-metal clusters in MT formed with Co(II)/Cd(II), Zn(II)/Cd(II) and Cd(II)/Fe(II), changes in the respective cluster volumes were shown to be a significant factor dictating the widely differing metal distributions in these systems. Based on the results of the current study, it is suggested that both the sizes of the two metal ions and their relative affinities towards the cysteine-thiolate ligands are important in the formation of mixed-metal clusters in MT.     

Binding of Cu(II) to non-prosthetic sites in ceruloplasmin and bovine serum albumin

The binding of Cu(II) to native human, porcine, bovine and ovine ceruloplasmin (Cp) and to bovine serum albumin (bSA) has been studied at pH 7.4, 30 mM barbital buffer. The results were analyzed for the strength and the number of binding sites using Scatchard plots. Evidence for additional copper binding sites in Cp and bSA was obtained suggesting a role for copper ion in the homeostatic regulation of Cu(II) and other metal ions in the serum. In the binding studies the Cp was freed of exogenous Cu(II) by passing it over a Chelex-100 column. Two flow rates were used, 4 ml/hr and 40 ml/hr, which removed Cu(II) of different affinities. Cp passed at the slower flow rate (Cp4) only contained the prosthetic copper atoms. Cp passed at the faster flow rate (Cp40) contained one additional copper atom with a Ka approximately 10(7) M-1. Another 2-6 Cu(II) ion could be added to the Cp40 with an average affinity of about Ka approximately 10(5) M-1. The Cu(II) ions found in Cp provide two distinguishable classes: (1) the prosthetic copper atoms and (2) the exogenous copper atoms that can be removed by Chelex-100. For bSA one copper atom was bound strongly with a Ka value approaching 10(12) - 10(13) M-1 and was not removed by Chelex-100 at any flow rate. A second copper atom was found with a Ka = 5.2 x 10(6) M-1 and was removed by Chelex-100 at 4 ml/hr. Three additional copper atoms were bound with a Ka = 1.6 x 10(5) M-1; they were readily removed by Chelex-100 at 40 ml/hr but were nondialysable.     

Macromolecularization of a tripeptide analogue of the Cu(II) binding site of human serum albumin: 2. Conformational and binding properties towards Cu(II) and Ni(II)ions of Gly-Gly-His derivatives of poly(l-lysine)

The conformational and binding properties towards Cu(II) and Ni(II) ions of Gly-Gly-His derivatives of poly(l-lysine) have been investigated mainly using circular dichroism (c.d.) spectroscopy. These derivatized polymers can be considered macromolecular analogues of the Cu(II) and Ni(II) binding site of human serum albumin. It has been shown that modification up to 53% of the ε-amino groups of lysine side chains by covalent binding of the tripeptide unit Gly-Gly-His does not induce appreciable alteration of the α-helix forming tendency of the polylysine backbone. The derivatized polymers exhibit strong affinity towards Cu(II) and Ni(II) ions. At neutral pH, complexes are formed in which each tripeptide chelating unit is linked to one metal ion. The spectral characteristics in the visible absorption region are consistent with a square planar geometry of the complexes, with deprotonated peptide groups and one imidazole nitrogen in the coordination sphere of the ion. C.d. measurements in the far u.v. indicate that complex formation in the side chains causes an increase of ordered structure of the peptide backbone at neutral pH. This fact is interpreted in terms of a reduced electrostatic repulsion among side chains due to charge neutralization in the tripeptide units linked to metal ions.     

Binding of cadmium(II) and zinc(II) to human and dog serum albumins. An equilibrium dialysis and 113Cd-NMR study.

The binding of Cd(II) and Zn(II) to human serum albumin (HSA) and dog serum albumin (DSA) has been studied by equilibrium dialysis and 113Cd(II)-NMR techniques at physiological pH. Scatchard analysis of the equilibrium dialysis data indicate the presence of at least two classes of binding sites for Cd(II) and Zn(II). On analysis of the high-affinity class of sites, HSA is shown to bind 2.08 +/- 0.09 (log K = 5.3 +/- 0.6) and 1.07 +/- 0.12 (log K = 6.4 +/- 0.8) moles of Cd(II) and Zn(II) per mole of protein, respectively. DSA bound 2.02 +/- 0.19 (log K = 5.1 +/- 0.8), and 1.06 +/- 0.15 (log K = 6.0 +/- 0.2) moles of Cd(II) and Zn(II) per mole of protein, respectively. Competition studies indicate the presence of one high-affinity Cd(II) site on both HSA and DSA that is not affected by Zn(II) or Cu(II), and one high-affinity Zn(II) site on both HSA and DSA that is not affected by Cd(II) or Cu(II). 113Cadmium-HSA spectra display three resonances corresponding to three different sites of complexation. In site I, Cd(II) is most probably coordinated to two or three histidyl residues, site II to one histidyl residue and three oxygen ligands (carboxylate), while for the most upfield site III, four oxygens are likely to be involved in the binding of the metal ion. The 113Cd(II)-DSA spectra display only two resonances corresponding to two different sites of complexation. The environment around Cd(II) at sites I and II on DSA is similar to sites I and II, respectively, on HSA. No additional resonances are observed in any of these experiments and in particular in the low field region where sulfur coordination occurs. Overall, our results are consistent with the proposal that the physiologically important high-affinity Zn(II) and Cd(II) binding sites of albumins are located not at the Cu(II)-specific NH2-terminal site, but at internal sites, involving mostly nitrogen and oxygen ligands and no sulphur ligand.     

A circular dichroic study of Cu(II) binding to bovine alpha-lactalbumin.

The visible and ultraviolet circular dichroic spectra resulting from the interaction of bovine alpha-lactalbumin with successive Cu(II) ions have been recorded under a variety of conditions. Analysis of the observed change-transfer and d-d band transitions can be made in terms of two kinds of binding sites: at a histidyl group and at the N-terminal amino group, respectively. At basic pH the amide nitrogens of the peptide backbone progressively take part in the coordination. The occupation of the high affinity calcium binding site by Ca(II) and Mn(II) does not influence the Cu(II) binding process, suggesting that there is no direct interaction between this site and the Cu(II) binding sites.     

Equilibrium studies on the binding of cadmium(II) to human serum transferrin

The binding of cadmium(II) to human serum transferrin in 0.01 M N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid with 5 mM bicarbonate at 25 degrees C has been evaluated by difference ultraviolet spectroscopy. Equilibrium constants were determined by competition versus three different low molecular weight chelating agents: nitrilotriacetic acid, ethylenediamine-N,N'-diacetic acid, and triethylenetetramine. Conditional equilibrium constants for the sequential binding of two cadmium ions to transferrin under the stated experimental conditions are log K1 = 5.95 +/- 0.10 and log K2 = 4.86 +/- 0.13. A linear free energy relationship for the complexation of cadmium and zinc has been prepared by using equilibrium data on 243 complexes of these metal ions with low molecular weight ligands. The transferrin binding constants for cadmium and zinc are in good agreement with this linear free energy relationship. This indicates that the larger size of the cadmium(II) ion does not significantly hinder its binding to the protein.     

Macromolecularization of a tripeptide analogue of the Cu(II) binding site of human serum albumin: 2. Conformational and binding properties towards Cu(II) and Ni(II)ions of Gly-Gly-His derivatives of poly(l-lysine)

The conformational and binding properties towards Cu(II) and Ni(II) ions of Gly-Gly-His derivatives of poly(l-lysine) have been investigated mainly using circular dichroism (c.d.) spectroscopy. These derivatized polymers can be considered macromolecular analogues of the Cu(II) and Ni(II) binding site of human serum albumin. It has been shown that modification up to 53% of the ε-amino groups of lysine side chains by covalent binding of the tripeptide unit Gly-Gly-His does not induce appreciable alteration of the α-helix forming tendency of the polylysine backbone. The derivatized polymers exhibit strong affinity towards Cu(II) and Ni(II) ions. At neutral pH, complexes are formed in which each tripeptide chelating unit is linked to one metal ion. The spectral characteristics in the visible absorption region are consistent with a square planar geometry of the complexes, with deprotonated peptide groups and one imidazole nitrogen in the coordination sphere of the ion. C.d. measurements in the far u.v. indicate that complex formation in the side chains causes an increase of ordered structure of the peptide backbone at neutral pH. This fact is interpreted in terms of a reduced electrostatic repulsion among side chains due to charge neutralization in the tripeptide units linked to metal ions.     

Site-specific substituted cobalt(II) horse liver alcohol dehydrogenases. Preparation and characterization in solution, crystalline and immobilized state.

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1.1.1.1) has been studied with dissolved, crystalline and agarose-immobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives. In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier. Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 microM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure-stabilizing role of the catalytic metal ions. The enzyme immobilised on agarose, with unchanged content of active sites [Schneider-Bernl?hr et al. (1978) Eur. J. Biochem. 41, 475--484], was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogenous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.     

Zinc(II) binding to apo-(bovine erythrocyte superoxide dismutase).

The binding of zinc(II) ions to apo-(bovine erythrocytes superoxide dismutase) was studied by 1H n.m.r. spectroscopy. Two zinc(II) ions bind to each subunit of the apoenzyme, and the first has a binding constant at least an order of magnitude larger than the second. The nature of the spectral changes that occur on binding the first zinc(II) ion are interpreted in terms of a change in the structure of the protein around the active site to one very similar to that of the holoenzyme, thus pre-forming the second zinc(II) binding site. The binding of the second zinc(II) ion effects changes in the environment of only those residues involved in its co-ordination.     

Independent and mutual interactions of copper(II) and zinc(II) ions with phytic acid

The interactions of phytic acid with Cu(II) and Zn(II) ions were examined as functions of metal ion concentrations and pH. Cu(II) ion-selective potentiometric and electron spin resonance (ESR) experiments provide strong evidence for the binding of Cu(II) ions to the phytic acid molecule at low pH (2.4–3.4) values. The relative stabilities of the copper and zinc phytates at low pH values were found to be very similar. For systems with metal ion:phytic acid molar ratios of 1:1–4:1 and 5:1–6:1 and pH values in the 3.4–5.9 and 3.4–5.0 ranges, respectively, Zn(II) ions were found to form complexes with phytic acid that were more stable than those of Cu(II) ions with phytic acid. The phytic acid molecule, however, was found to accommodate Cu(II) ions more readily than Zn(II) ions. For example, in systems containing equal amounts of Cu(II) and Zn(II) ions, 2 Zn(II) ions and 2, 3, 4, or 4.5 Cu(II) ions were found per phytic acid molecule depending upon metal ion:phytic acid molar ratios in the systems and pH. Total metal ion:phytic acid molar ratios and pH affected resultant metal ion solubilities and were factors influencing the effects of Zn(II) and Cu(II) ions on the binding of each other by phytic acid. Zn(II) and Cu(II) ions were observed to potentiate the binding of each other by phytic acid in some systems and compete with each other for phytate binding sites in others.