The use of ESI-MS to probe the binding of divalent cations to calmodulin

Proteins have evolved with distinct sites for binding particular metal ions. This allows metalloproteins to perform a myriad of specialized tasks with conformations tailor-made by the combination of its primary sequence and the effect on this of the ligated metal ion. Here we investigate the selectivity of the calcium trigger protein calmodulin for divalent metal ions. This ubiquitous and highly abundant protein exists in equilibrium between its apo and its holo form wherein four calcium ions are bound. Amongst its many functions, calmodulin modulates the calcium concentration present in cells, but this functional property renders it a target for competition from other metal ions. We study the competition posed by four other divalent cations for the calcium binding sites in calmodulin using electrospray ionization mass spectrometry (ESI-MS). We have chosen two other group II cations Mg²⁺, Sr²⁺, and two heavy metals Cd²⁺, Pb²⁺. The ease with which each of these metals binds to apo and to holo CaM[4Ca] is described. We find that each metal ion has different properties with respect to calmodulin binding and competition with calcium. The order of affinity for apo CaM is Ca²⁺ ≫ Sr²⁺ ∼ Mg²⁺ > Pb²⁺ ∼ Cd²⁺. In the presence of calcium the affinity alters to Pb²⁺ > Ca²⁺ > Cd²⁺ > Sr²⁺ > Mg²⁺. Once complexes have been formed between the metal ions and protein (CaM:[xM]) we investigate whether the structural change which must accompanies calcium ligation to allow target binding takes place for a given CaM:[xM] system. We use a 20 residue target peptide, which forms the CaM binding site within the enzyme neuronal nitric-oxide synthase. Our earlier work (Shirran et al. 2005) [1] has demonstrated the particular selectivity of this system for CaM:4Ca²⁺. We find that along with Ca²⁺ only Pb²⁺ forms complexes of the form CaM:4M²⁺:nNOS. This work demonstrates the affinity for calcium above all other metals, but also warns about the ability of lead to replace calcium with apparent ease.     

A New Approach for Titration Calorimetric Data Analysis on the Binding of Magnesium Ion with Myelin Basic Protein

The interaction of the myelin basic protein (MBP) from the bovine central nervous system with divalent magnesium ion was studied by isothermal titration calorimetry at 27 °C in aqueous solution. A simple rapid method for determination of the dissociation binding constants for Mg²⁺-MBP interaction was introduced using the isothermal titration calometric data. The binding isotherm for Mg²⁺-MBP interaction is easily obtained by carrying out a titration calorimetric experiment using only one set of concentrations of MBP. There are two identical independent intrinsic association constants equal to 0.021 μmol⋅L⁻¹ in the first- and second-binding sites, respectively.     

Molecular Vise Approach to Create Metal‐Binding Sites in MOFs and Detection of Biomarkers

We report a new approach to create metal‐binding site in a series of metal–organic frameworks (MOFs), where tetratopic carboxylate linker, 4′,4′′,4′′′,4′′′′‐methanetetrayltetrabiphenyl‐4‐carboxylic acid, is partially replaced by a tritopic carboxylate linker, tris(4‐carboxybiphenyl)amine, in combination with monotopic linkers, formic acid, trifluoroacetic acid, benzoic acid, isonicotinic acid, 4‐chlorobenzoic acid, and 4‐nitrobenzoic acid, respectively. The distance between these paired‐up linkers can be precisely controlled, ranging from 5.4 to 10.8 Å, where a variety of metals, Mg²⁺, Al³⁺, Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Ag⁺, Cd²⁺ and Pb²⁺, can be placed in. The distribution of these metal‐binding sites across a single crystal is visualized by 3D tomography of laser scanning confocal microscopy with a resolution of 10 nm. The binding affinity between the metal and its binding‐site in MOF can be varied in a large range (observed binding constants, K_obs from 1.56×10² to 1.70×10⁴ L mol^?1), in aqueous solution. The fluorescence of these crystals can be used to detect biomarkers, such as cysteine, homocysteine and glutathione, with ultrahigh sensitivity and without the interference of urine, through the dissociation of metal ions from their binding sites.     

复合物Ca+-RNA碱基的结构与稳定性

Gas-phase metal ion affinities and optimized structures of RNA nucleic acid bases for the Ca+ were determined at a density functional level employing the hybrid B3LYP exchange correlation potential in connection with the 6-311+G(2df,2p) basis set. All the molecular complexes, obtained by the interaction between several low-lying tautomers of RNA nucleic acid and Ca+ on the different binding sites, were considered. For Cytosine, the most stable complex was obtained starting from the most stable tautomer of the free nucleic acid base tautomers. As to thymine, the bond energy of the ion with the most stable tautomer of the free nucleic acid base is the weakest among the three tautomer’s complexes, and that of the ion with least stable tautomer of the free nucleic acid base is the strongest . Uracil is similar to thymine. The two kinds of relation, bond energy and total energy for the complex, are in disagreement, as the metal affinities of RNA bases for the Ca+ depend on binding sites, and total energy of complex (Ca+-RNA base) relies on all atoms and their relative positions in the complex.     

Mg<Superscript>2+</Superscript>/Ca<Superscript>2+</Superscript> binding to DNA bases: a quantum chemical method and ABEEMσπ/MM fluctuating charge model study

The interactions of Mg²⁺ and Ca²⁺ binding to adenine, cytosine, guanine, and thymine at various binding sites were studied by a high-level quantum chemical method and ABEEMσπ/MM fluctuating charge model. The geometries and binding energies of M²⁺-bases complexes were determined at CCSD(T)/6-311 ++G(2d,2p)//MP2/6-311 ++G(2d,2p) level of theory, with the basis set superposition error corrections for the binding energy calculations. In comparison with the ab initio results, an accurate classical metal cation–base interaction potential was constructed and parameterized in terms of ABEEMσπ/MM model. It is revealed that Mg²⁺/Ca²⁺ prefers to bind with bases at the bidentate position (between two nitrogen atoms or oxygen and nitrogen atoms in purine and pyrimidine), where the binding energy is the largest. Moreover, the distance between M²⁺ and the base increases from Mg²⁺ to Ca²⁺, while the binding energy of Mg²⁺–base is greater than that of Ca²⁺-base. The ABEEMσπ/MM potential gives reasonable geometries and binding energies compared with the present quantum chemical calculations, and the overall percentage RMSDs are 1.4 and 1.6% for geometries and binding energies, respectively. Furthermore, the transferability of the parameters of the new potential is validated by investigation of Mg²⁺/Ca²⁺ binding to tautomer of bases, and results from our potential also show quite good consistency with those of MP2/6-311 ++G(2d,2p)//B3LYP/6-311 ++G(d,p) method, with the overall percentage RMSDs of 2.2 and 4.7% for geometries and binding energies, respectively. This work will serve as a basis for further investigations of the mechanisms of cation effects on the structure and property of nucleic acids.     

Determination of the intrinsic affinities of multiple site-specific Mg(2+) ions coordinated to domain 6 of a group II intron ribozyme

Group II introns are large metallo-ribozymes that use divalent metal ions in folding and catalysis. The 3'-terminal domain 6 (D6) contains a conserved adenosine whose 2'-OH group acts as the nucleophile in the first splicing step. In the hierarchy of folding, D6 binds last into the active site. In order to investigate and understand the folding process to the catalytically active intron structure, it is important to know the individual binding affinities of Mg2+ ions to D6. We recently studied the solution structure of a 27 nucleotide long D6 (D6-27) from the mitochondrial yeast group II intron Sc.ai5gamma, also identifying five Mg2+ binding sites including the one at the 5'-terminal phosphate residues. Mg2+ coordination to the 5'-terminal di- and triphosphate groups is strongest (e.g., log KA,TP = 4.55 +/- 0.10) and is evaluated here in detail for the first time. The other four binding sites within D6-27 are filled simultaneously (e.g., log KA,BR = 2.38 +/- 0.06) and thus compete for the free Mg2+ ions in solution, having a distinct influence on the individual affinities of the various sites. For the first time, we take this competition into account to obtain the intrinsic binding constants, describing a method that is generally applicable. Our data illustrates that any RNA molecule undergoing tertiary contacts to a second RNA molecule first needs to be loaded evenly and specifically with metal ions to compensate for the repulsion between the negatively charged RNA molecules.     

Comparison of Ca2+ and Mg2+ binding to calmodulin. An enthalpy,entropy, and gibbs free energy analysis

A consistent set of G _B , H _B , and S _B parameters have been determined from ion specific electrode, calorimetric, and spectrophotometric studies for the binding of Ca²⁺ and Mg²⁺ to bovine calmodulin at pH=7.0 and an ionic strength I of 0.113M. A non-linear least squares analysis of calcium specific ion electrode data yields, on a molar basis, four calcium dissociation constants: 10^–7 for the first site, 10^–5 for the fourth site, and two constants between these values. Both calorimetric experiments and an indicator method provide evidence that Mg²⁺ binds to calmodulin, probably at the same sites as Ca²⁺, but with affinities about 100 times smaller: 4×10^–5 for the first site and 2×10^–3 for the fourth. Calorimetric titrations on Ca²⁺ binding to calmodulin in three buffers are consistent with 0.46 protons released upon binding at all four sites and yield an average H _B per site of 5.6 and 7.9 kJ-mol^–1 for Ca²⁺ and Mg²⁺, respectively. The entropy of the system increases by 524 and 361 J-K^–1-mol^–1 when Ca²⁺ and Mg²⁺, respectively, bind to four sites on calmodulin, i.e., the selectivity of calmodulin for Ca²⁺ is primarily derived from entropy effects. Further analysis based on elimination of the entropy term for the metal ions demonstrates that calmodulin bound to Ca²⁺ has a larger entropy than the unbound calmodulin; the opposite is true for calmodulin bound to Mg²⁺. These analyses are consistent with the hypothesis that Ca²⁺ forms tight complexes at all sites on calmodulin and that release of waters of hydration upon binding is the source of the increase of entropy in the system.     

Gas-Phase studies of valinomycin-alkali metal cation complexes: attachment rates and cation affinities

Reactions of laser-desorbed Na⁺, K⁺, Rb⁺, and Cs⁺ with thermally vaporized valinomycin generate metal-ligand complexes in a Fourier transform ion cyclotron resonance trapping cell, proving that complexes can form via gas-phase ion-molecule reactions. Although desorption of intact pre-formed complexes cannot be ruled out, this route appears minor. Relative rate constants for the complexation reactions show strong dependence on the charge densities of the cations. Competition experiments between valinomycin and the synthetic ionophores 18-crown-6 (18C6) and [2.2.2]-cryptand ([2.2.2]) show that valinomycin has a higher intrinsic alkali metal cation affinity than either 18C6 or [2.2.2], in contrast to the complex formation constants observed in methanol, where K+ affinities are in the order [2.2.2] > 18C6 > valinomycin.     

Site-specific control of N7-metal coordination in DNA by a fluorescent purine derivative

A synthetic strategy that utilizes O6‐protected 8‐bromoguanosine gives broad access to C8‐guanine derivatives with phenyl, pyridine, thiophene, and furan substituents. The resulting 8‐substituted 2′‐deoxyguanosines are push–pull fluorophores that can exhibit environmentally sensitive quantum yields (Φ=0.001–0.72) due to excited‐state proton‐transfer reactions with bulk solvent. Changes in nucleoside fluorescence were used to characterize metal‐binding affinity and specificity of 8‐substituted 2′‐deoxyguanosines. One derivative, 8‐(2‐pyridyl)‐2′‐deoxyguanosine (2PyG), exhibits selective binding of Cu^II, Ni^II, Cd^II, and Zn^II through a bidentate effect provided by the N7 position of guanine and the 2‐pyridyl nitrogen atom. Upon incorporation into DNA, 2‐pyridine‐modified guanine residues selectively bind to Cu^II and Ni^II with equilibrium dissociation constants (K_d) that range from 25 to 850 nM ; the affinities depend on the folded state of the oligonucleotide (duplex>G‐quadruplex) as well as the identity of the metal ion (Cu>Ni?Cd). These binding affinities are approximately 10 to 1 000 times higher than for unmodified metal binding sites in DNA, thereby providing site‐specific control of metal localization in alternatively folded nucleic acids. Temperature‐dependent circular‐dichroism studies reveal metal‐dependent stabilization of duplexes, but destabilization of G‐quadruplex structures upon adding Cu^II to 2PyG‐modified oligonucleotides. These results demonstrate how the addition of a single pyridine group to the C8 position of guanine provides a powerful new tool for studying the effects of N7 metalation on the structure, stability, and electronic properties of nucleic acids.     

Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism

Background

Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1^st and 2^nd EF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants.

Results

The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca²⁺ binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 10⁵ and 5.0 × 10² M^-1 with ??Hs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1^st non-canonical EF-hand is largely enhanced by the binding of Ca²⁺ to the 2^nd canonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca²⁺ binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its ??-helix secondary structure content. All measurements exclude Mg²⁺-binding in NCaBD.

Conclusions

We demonstrated that the 1^st non-canonical EF-hand of NOX5 has very weak Ca²⁺ binding affinity compared with the 2^nd canonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca²⁺ binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca²⁺ specific.

Graphical abstract

     

Sorption of metal ions to untreated,alkali-treated and peroxide-bleached TMP

Knowledge about how different metal ions are bound to pulp fibers is very important for optimal metal management in pulping processes. A column chromatographic method was used to assess the differences in affinity of 14 metal ions to untreated, alkali-treated and peroxide-bleached thermomechanical pulp (TMP). A method of competition between cations in the column chromatographic experiments was used in the sorption experiments, with an excess of each metal ion compared to the total capacity of the pulp studied. The method is very sensitive and even small differences in affinities can be detected. By combining the results from sorption experiments with four different metal ion mixtures the following order of affinity was obtained: Pb²⁺ ≫ Cu²⁺ ≫ Cd²⁺ > Zn²⁺ > Ni²⁺ > Ba²⁺ > Ca²⁺ > Mn²⁺ > Sr²⁺ > Mg²⁺ ≫ Rb⁺ ≈ K⁺ > Na⁺ > Li⁺. All three types of pulps showed the same affinity order. Lead and copper ions were clearly most strongly bound to the pulp fibers. Within the alkali and alkaline earth metal groups the differences in affinity were quite small. The sorption of metal ions to pulp fibers takes place mainly by complexation, where the divalent metal ions are coordinated to functional groups (acid groups) in the fiber phase. Protonation constants and concentrations of acid groups were determined by potentiometric titration. A model with two carboxyl groups and two phenolic hydroxyl groups satisfied best the experimental data. By treatment with alkali and peroxide new acid groups were created and the total binding capacity of hydrogen ions increased from 137 μeq/g for untreated pulp to 187 and 228 μeq/g for alkali-treated and peroxide-treated pulp, respectively.     

The interaction model between metal cation and tropylium: a quantum chemistry predication

The aromatic cation tropylium, C₇H₇⁺, predicted at the MP2/6-31G** level, is capable of binding with metal cations Be²⁺ or Mg²⁺, forming M²⁺–C₇H₇⁺ complexes. The obstacle for their binding is almost electrostatic repulsion, and the binding is from polarization and charge transfer. The orbital interaction between the M²⁺ and C₇H₇⁺ is mainly the s–π and p–π interactions. Interestingly, Be²⁺ is possible to pass through the ring of C₇H₇⁺, while Mg²⁺ is not. The intrinsic IR band of the M²⁺–C₇H₇⁺ complex is below 600 cm⁻¹, which results from the vibration of the M²⁺ along the normal axis of C₇H₇⁺.     

Effect of Hydrogenation on Ring C of Flavonols on Their Affinity for Bovine Serum Albumin

In this paper, the effect of hydrogenation on ring C of flavonols on the affinity for bovine serum albumin was investigated. Two differently substituted B-ring hydroxylation flavonols (myricetin and quercetin) and their dihydrides (dihydromyricetin and dihydroquercetin) were used to study their affinities for BSA by quenching the intrinsic BSA fluorescence in solution. From the spectra, the bimolecular quenching constants, the binding constants, the number of binding sites and the binding distances were calculated. The hydroxylation on ring B and hydrogenation on ring C of flavonols significantly affected the binding/quenching process; in general, the hydroxylation increased the affinity and the hydrogenation decreased the affinity. For myricetin and quercetin, the binding constants (K _a) for BSA were 1.84×10⁸ L⋅mol⁻¹ and 3.83×10⁷ L⋅mol⁻¹. For dihydromyricetin, the binding constant was 1.36×10⁴ L⋅mol⁻¹, while dihydroquercetin hardly quenched the BSA intrinsic fluorescence. These results showed that hydrogen bonding and conjugative effects may play an important role in binding of flavonols to BSA. These results also showed that the properties of flavonols are related to their chemical structure.     

Ditopic Aza-Scorpiand Ligands Interact Selectively with ds-RNA and Modulate the Interaction upon Formation of Zn2+ Complexes

Nucleic acids are essential biomolecules in living systems and represent one of the main targets of chemists, biophysics, biologists, and nanotechnologists. New small molecules are continuously developed to target the duplex (ds) structure of DNA and, most recently, RNA to be used as therapeutics and/or biological tools. Stimuli-triggered systems can promote and hamper the interaction to biomolecules through external stimuli such as light and metal coordination. In this work, we report on the interaction with ds-DNA and ds-RNA of two aza-macrocycles able to coordinate Zn²⁺ metal ions and form binuclear complexes. The interaction of the aza-macrocycles and the Zn²⁺ metal complexes with duplex DNA and RNA was studied using UV thermal and fluorescence indicator displacement assays in combination with theoretical studies. Both ligands show a high affinity for ds-DNA/RNA and selectivity for ds-RNA. The ability to interact with these duplexes is blocked upon Zn²⁺ coordination, which was confirmed by the low variation in the melting temperature and poor displacement of the fluorescent dye from the ds-DNA/RNA. Cell viability assays show a decrease in the cytotoxicity of the metal complexes in comparison with the free ligands, which can be associated with the observed binding to the nucleic acids.     

Molecular Dynamics Simulations of Metal Ion Binding to the His‐tag Motif

In our previous study, we have observed that the chelation of various metal ions to the His‐tag motifs mostly involves the i and i+2 His residues for Ni²⁺, Cu²⁺, Zn²⁺ and Co²+. In the present study, various 200 ps molecular dynamics simulations were further conducted to investigate the chelating pathway of various metal ions to the His‐tag motif with 6 His residues (His‐tag6) and the binding affinities of these metal binding pockets towards these metal ions. The results indicate that His‐tag6 with the chelated metal ion located in positions His(2,4) or His(3,5) exhibits the strongest affinity for Ni²+ and Cu²+.K⁺ was found to be preferred to chelate in His(1,3) and His(3,5) coordinations. However, Fe³+ was found to have higher affinity towards His(1,3) and His(2,4) binding pockets. Our results also suggest that Ni²⁺ exhibits the highest binding affinity towards His‐tag6 over the other metal ions. Most of the structural variations of the His‐tag6 motif were from the Histidyl side chains during metal ion binding. In addition, there is an inverse linear correlation between the final chelated distance and the charge/volume ratio of metal ion. There is a negative correlation between the metal binding affinity and the averaged potential energy generated from the MD simulations.     

High Ammonia Uptake of a Metal–Organic Framework Adsorbent in a Wide Pressure Range

Although numerous porous adsorbents have been investigated for NH₃ capture applications, these materials often exhibit insufficient NH₃ uptake, low NH₃ affinity at the ppm level, and poor chemical stability against wet NH₃ conditions. The NH₃ capture properties of M₂(dobpdc) complexes (M=Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺, and Zn²⁺; dobpdc⁴⁻=4,4-dioxidobiphenyl-3,3-dicarboxylate) that contain open metal sites is presented. The NH₃ uptake of Mg₂(dobpdc) at 298 K was 23.9 mmol g⁻¹ at 1 bar and 8.25 mmol g⁻¹ at 570 ppm, which are record high capacities at both pressures among existing porous adsorbents. The structural stability of Mg₂(dobpdc) upon exposure to wet NH₃ was superior to that of the other M₂(dobpdc) and the frameworks tested. Overall, these results demonstrate that Mg₂(dobpdc) is a recyclable compound that exhibits significant NH₃ affinity and capacity, making it a promising candidate for real-world NH₃-capture applications.     

Application of an extended solvation theory to study on the binding of magnesium ion with myelin basic protein

Binding properties of myelin basic protein (MBP) from bovine central nervous system due to the interaction by divalent magnesium ion (Mg²⁺) was investigated at 27°C in aqueous solution using isothermal titration calorimetry (ITC) technique. An extended solvation model was used to reproduce the enthalpies of Mg²⁺-MBP interaction over the whole Mg²⁺ concentrations. It was found that there is a set of two identical and noninteracting binding sites for Mg²⁺ ions. The dissociation equilibrium constant is K _d=45.5 μM. The molar enthalpy of binding site is identical for both sites; ΔH=−15.24 kJ mol⁻¹. The solvation parameters recovered from the solvation model were attributed to the structural change of MBP due to the metal ion interaction.     

Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit

In bacteria, the binding between the riboswitch aptamer domain and ligand is regulated by environmental cues, such as low Mg²⁺ in macrophages during pathogenesis to ensure spatiotemporal expression of virulence genes. Binding was investigated between the flavin mononucleotide (FMN) riboswitch aptamer and its anionic ligand in the presence of molecular crowding agent without Mg²⁺ ion, which mimics pathogenic conditions. Structural, kinetic, and thermodynamic analyses under the crowding revealed more dynamic conformational rearrangements of the FMN riboswitch aptamer compared to dilute Mg²⁺‐containing solution. It is hypothesized that under crowding conditions FMN binds through an induced fit mechanism in contrast to the conformational selection mechanism previously demonstrated in dilute Mg²⁺solution. Since these two mechanisms involve different conformational intermediates and rate constants, these findings have practical significance in areas such as drug design and RNA engineering.     

Template‐Directed Approach Towards the Realization of Ordered Heterogeneity in Bimetallic Metal–Organic Frameworks

Controlling the arrangement of different metal ions to achieve ordered heterogeneity in metal–organic frameworks (MOFs) has been a great challenge. Herein, we introduce a template‐directed approach, in which a 1D metal–organic polymer incorporating well‐defined binding pockets for the secondary metal ions used as a structural template and starting material for the preparation of well‐ordered bimetallic MOF‐74s under heterogeneous‐phase hydrothermal reaction conditions in the presence of secondary metal ions such as Ni²⁺ and Mg²⁺ in 3 h. The resulting bimetallic MOF‐74s were found to possess a nearly 1:1 metal ratio regardless of their initial stoichiometry in the reaction mixture, thus demonstrating the possibility of controlling the arrangement of metal ions within the secondary building blocks in MOFs to tune their intrinsic properties such as gas affinity.     

Influence of metal ions on the interaction between gatifloxacin and calf thymus DNA

To study the interaction between gatifloxacin (GT), metal ions (Cu²⁺, Cd²⁺, Co²⁺, Mg²⁺) and calf thymus DNA under condition of physiology pH, UV absorption and fluorescence methods were adopted. Result shows that metal ions and DNA are able to react with GT in ground state. In further research, by studying the influence of metal ions on binding of GT with DNA in metal ions–GT–DNA ternary system, we found that influential mechanism of Mg²⁺ on the binding of GT with DNA may be different from the other three. Mg²⁺ can act as a bridge in the binding of GT's carboxyl/carbonyl with DNA phosphate in certain concentration range; while Cu²⁺, Cd²⁺, Co²⁺ can combine directly with GT by reaction between GT carboxyl/carbonyl and DNA base, and enhance the binding ability of GT with DNA. The influence extent and type depend not only on the binding site of DNA with metal ions (phosphate or base), but also the binding ability of which. The stronger the binding ability of metal ions with DNA base is, the larger their promotion to binding of GT with DNA is. The order of metal ions’ influential ability on the binding of GT–DNA is identical to the binding ability order of metal ions with DNA base, that is: Cu²⁺ > Cd²⁺ > Co²⁺ > Mg²⁺.