Speciation studies of ternary complexes of some essential divalent metal ions with L-histidine and L-glutamic acid in DMSO-water mixtures

Abstract

Chemical speciation of ternary complexes of Ca(II), Mg(II) and Zn(II) ions with L-histidine as the primary ligand (L) and L-glutamic acid as the secondary ligand (X) has been studied pH metrically in the concentration range of 0.0-60.0% v/v DMSO-water mixtures maintaining an ionic strength of 0.16 mol L^-1 using sodium chloride at 303.0 K. Titrations were carried out in different relative concentrations (M:L:X = 1.0:2.5:2.5, 1.0:2.5:5.0, 1.0:5.0:2.5) of metal (M) to L-histidine to L-glutamic acid with sodium hydroxide. Stability constants of ternary complexes were refined with MINIQUAD75. The best-fit chemical models were selected based on statistical parameters and residual analysis. The predominant species detected for Ca(II), Mg(II) and Zn(II) are ML₂XH₂, MLXH₂ and MLX₂. Extra stability of ternary complexes compared to their binary complexes was explained to be due to electrostatic interactions of the side chains of ligands, charge neutralisation, chelate effect, stacking interactions and hydrogen bonding. The species distribution with pH at different compositions of DMSO and the plausible equilibria for the formation of species are discussed.     

NMR studies on metal complexes of DNA oligomers

A short overview of NMR spectroscopic applications for the study of metal ion complexes of DNA oligomers is presented. One typical example is given to illustrate the scope of the methods: the NMR structure of a trans-DDP interstrand cross-linked duplex, d(CTCCTG*TGTCTC) x d(GAGATA*AGGAG). The solution structure of this double-stranded DNA oligonucleotide, containing a trans-diammineplatinum(II) interstrand cross-link, was determined using two-dimensional nuclear magnetic resonance (2D NMR) and NOE-restrained molecular dynamics and energy minimization refinement. The duplex is a non-palindromic 12/11-mer with a missing central residue in the lower strand and in addition it contains a GT mismatched base pair. The analysis indicated that an interstrand cross-link is established between G6-N7 of the upper strand and A18-N1 of the lower strand.     

Interactions of DNA with divalent metal ions. II. Proton relaxation enhancement studies

The extent and modes of binding of the divalent metal ions Mn²⁺ and Co²⁺ to DNA and the effects of salt on the binding have been studied by measurements of the effects of these paramagnetic metal ions on the longitudinal and transverse relaxation rates of the protons of the solvent water molecules, a technique that is sensitive to overall binding. The number of water molecules coordinated to the DNA–bound Mn²⁺ and Co²⁺ is found to be between five and six, and the electron spin relaxation times and the electron-nuclear hyperfine constants associated with Mn²⁺ and Co²⁺ are little or not affected by the binding. These observations indicate little disturbance of the hydration sphere of Mn²⁺ and Co²⁺ upon binding to DNA. An average 2–3-fold reduction in the exchange rate of the water of hydration of the bound metal ions and an order-of-magnitude increase in their rotational correlation time are attributed to hydrogen-bond formation with the DNA. The binding constants of Mn²⁺ to DNA, at metal concentrations approaching zero, are found to be inversely proportional to the second power of the salt concentration, in agreement with the predictions of Manning's polyelectrolyte theory. A remarkable quantitative agreement with the polyelectrolyte theory is also obtained for the anticooperativity in the binding of Mn²⁺ to DNA, although the experimental results can be well accounted for by another simple electrostatic model. The various modes of binding of divalent metal ions to DNA are discussed.     

Proton and phosphorus-31 NMR study of the dependence of diadenosine tetraphosphate conformation on metal ions

Adenosine 5'-tetraphospho-5'-adenosine (Ap4A) plays a role in cellular metabolism in a wide variety of organisms. Because the divalent cations Mg2+ and Zn2+ are involved in the synthesis and function of Ap4A, the effect of divalent cations on the dinucleotide's conformation is of interest. 1H and 31P chemical shift experiments were carried out as a function of Mg2+ concentration and pH. We propose that Mg2+ stabilizes the unusual ring-stacked conformation of Ap4A at pH greater than 2 by interacting with the beta-phosphates. To further probe conformational effects, stable complexes of Ap4A with Co3+ were studied using 1H and 31P NMR. Co3+ forms two different bidentate complexes with Ap4A, independent of whether the other four octahedral coordination sites are occupied by ammonia or trimethylenediamine. NMR results suggest that in one complex the Co3+ is coordinated to two beta-phosphates and ring stacking is stabilized. In the other complex, Co3+ is coordinated to an alpha-phosphate and its neighboring beta-phosphate and ring stacking is destabilized. These results further support the hypothesis that Mg2+ stabilizes the ring-stacked conformation by interacting symmetrically with the two beta-phosphate groups.     

The interaction of metal ions with nucleic acids. Ternary complexes of copper(II) with peptides and nucleosides

Difference electronic absorption and electron paramagnetic resonance spectroscopy were used to monitor the formation of the ternary complexes of Cu(II) ions with nucleosides and dipeptides containing Gly, Leu and Trp residues. Stability constants of these mixed-ligand complexes of Cu(II)-peptides with nucleosides were found to decrease in the following order: 6-ketopurines greater than 6-aminopurine greater than pyrimidines. Interpretation of the EPR data indicated that the covalent nature of the copper-ligand bond also decreases in the same order. The EPR findings suggest that nucleosides are bonded in the equatorial position of the Cu(II)-peptide complexes, however, in the case of pyrimidine nucleosides weak axial bonding also seems to occur.     

A multicomponent self-diffusion NMR study of aggregation of nucleotides, nucleosides, nucleic acid bases and some derivatives in aqueous solution with divalent metal ions added

The self-aggregation of the mononucleotides AMP, CMP, and UMP with Mg2+ added (nucleotide concentration = Mg2+ concentration) up to 0.4 molal or to their solubility limit in 2H2O has been monitored through self-diffusion measurements, using the Fourier transform NMR pulsed-gradient spin-echo multicomponent-self-diffusion technique. Also, purine, cytidine, uridine, purine with Mg2+ added and both cytidine and uridine with Mg2+, Zn2+ or Cd2+ added, were studied in the same way. The experimental data were fitted to two different aggregation models. For the mononucleotides with Mg2+ added a cooperative indefinite aggregation model, where the first (dimerization) aggregation constant is a magnitude lower than those for the higher aggregation step gives the best agreement between simulations and experiment. Typical values are 0.3 and 12 kg mol(-1), respectively. The latter value is about twice that found for the uncomplexed nucleotides. Also, purine and the nucleosides, cytidine and uridine, with divalent metal ions added fit best with this model. The degree of aggregation is increased upon metal ion addition, as previously shown for the mononucleotides. For purine, cytidine and uridine without metal ions added an 'isodesmic', indefinite aggregation model, with the aggregation constant for each step equal, fits the data as well. Here the application of the 'semi-isodesmic' model results in a higher first (dimerization) aggregation constant than is found for the nucleotides. The typical value is 2 kg mol(-1). In this case, the evaluated aggregation constants for the higher step become only about twice as large as those of the first step. The same measurements on isopropylcytidine, isopropyluridine and theophylline-7-acetic acid in water show that these three compounds aggregate to the same extent as the nucleosides, cytidine and uridine. Pyrimidine diffusion data reveal no aggregation at all; the application of either model results in essentially zero aggregation constants.     

Structure of mutagen nucleic acid complexes in solution. Proton chemical shifts in 9-aminoacridine complexes with dG-dC, dC-dG, and dA-dT-dG-dC-dA-dT

The influence of self-complementary oligodeoxynucleotides on the chemical shifts of protons of the mutagenic acridine dye 9-aminoacridine has been measured. Upfield shifts indicative of intercalative binding are found in the cases of dG-dC, dC-dG, and dA-dT-dG-dC-dA-dT but not in dA-dT. Geometries for the complexes that are compatibile with the chemical-shift data and the X-ray structure of the complex between ri5C-rG and 9-aminoacridine determined by Sakore et al. [Sakore, T.D., Jain, S.C., Tsai, C., and Sobell, H.M. (1977), Proc. Natl. Acad. Sci. U.S.A. 74, 188--192] can be identified using recent theoretical estimates of shifts induced by nucleotide bases.     

Proton NMR studies of vesicles incorporating glycophorin

The effects of incorporation of glycophorin. the major sialoglycoprotein or the human crythrocyte membrane, on the lipid of small vesicles have been studied using proton NMR and electron microscopy. In contrast to the incorporation of other peptides, the major effect is apparently the clustering of vesicles without fusion. The relative mobility of lipids of the vesicle. monitored by changes in proton spin-lattice time, is only moderately effected by the presence of protein. The methylene protons of the lipid chains are subject to a somewhat greater restriction of motion following the incorporation of glycophorin than are the protons of the head group.     

Quantification of single-stranded nucleic acid and oligonucleotide interactions with metal ions by affinity capillary electrophoresis: part I

The interactions between oligonucleotides and inorganic cations have been measured by capillary zone electrophoresis. With increasing concentrations of divalent cations (Ca²⁺, Mg²⁺, Mn²⁺ and Ni²⁺) in the running buffer, the migration behavior was evaluated by calculation of the binding constants. Besides these fundamental studies of binding equilibria, different buffer components, tris(hydroxymethyl)aminomethane and 3-(N-morpholino)propanesulfonic acid, have been investigated and their effects on metal ion binding quantified.     

Antioxidant properties of complexes of flavonoids with metal ions

The formation of complexes of metal ions with the flavonoids quercetin (L1), rutin (L2), galangin (L3) and catechin (L4) has been investigated by UV-visible spectroscopy. The antioxidant activities of the compounds were evaluated by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicalscavenging method. In this work, we have shown that the complexed flavonoids are much more effective free radical scavengers than the free flavonoids. We suggest that the higher antioxidant activity of the complexes is due to the acquisition of additional superoxide dismutating centers. Radical scavenging activities of the compounds were also investigated from an electrochemical point of view. There is a relationship between the logarithm of the antioxidant activity (represented by EC50) and the oxidation potential. The synergic effect of the complexes and ascorbic acid were studied by [13C]-NMR analyses. The results show that ascorbic acid can protect flavonoids from oxidative degradation, and reveal antioxidant synergies between ascorbic acid and the compounds.     

Further structural analysis of GnRH complexes with metal ions

MALDI-TOF mass spectrometry, 1H NMR spectrometry, the continuous variation method and molecular modeling by MM3 calculation confirmed our earlier studies showing that gonadotropin-releasing hormone (GnRH) forms complex with copper(II) ion with the binding ratio 1:1. The copper(II) complex formed at physiological pH has a square planar configuration and GnRH complexes with nickel(II) and cobalt(II) ions are less stable than that of copper(II).     

Proton NMR studies of myohemerythrin from Themiste zostericola

 One- and two-dimensional NMR experiments have been carried out on different forms of myohemerythrin (MHr), a monomeric 13.9-kDa oxygen carrier, focusing on paramagnetically shifted proton resonances. Compared to the corresponding forms of octameric hemerythrin (Hr), all of the MHr forms exhibit spectra with better resolution and signal-to-noise ratios. The metMHr spectra allow the differentiation of the signals from the N_δ-H protons of the five N_ε-coordinated His ligands and those from the bridging Asp and Glu ligands. The 1D spectra of deoxyMHr exhibit a number of relatively sharp features including three solvent-exchangeable peaks that account for five protons. One of these His N-H protons exchanges more slowly with solvent than the other four and is assigned to His 54, which, by analogy to the crystal structure of deoxyHr, is the only His ligand that is hydrogen-bonded to an amino acid residue, Glu24 in this case. One-dimensional NOE results on the non-exchangeable signals clearly show the connectivities among the α and β protons of the bridging Asp111, and the α, β, and γ protons of the bridging Glu58 ligands. One-dimensional NOE experiments performed on the N-H proton signals of the coordinated His ligands, together with the COSY results, help to identify the geminal β protons of the His ligands. Upon the binding of N₃ ^– to one of the Fe(II) sites in deoxyMHr, the overlapping His N_δ-H proton signals observed in the deoxyMHr spectrum are resolved into individual signals; these have been correlated to the corresponding signals in deoxyMHr by saturation transfer experiments. Similarly, all five His N-H protons are resolved in the ¹H NMR spectrum of the deoxy form of the single point mutant L103N MHr. However, all five N-H protons readily exchange with solvent, indicating that the mutation affects the hydrogen-bonding interaction between His54 and Glu24. Received: 20 May 1996 / Accepted: 24 October 1996     

NMR studies of the interaction of bleomycin with (dC-dG)3.

The interaction of bleomycin A2 and Zn(II)-bleomycin A2 with the oligonucleotide (dC-dG)3 has been monitored by nuclear magnetic resonance spectroscopy. Binding of the drug to the oligonucleotide is indicated by an upfield shift of the bithiazole proton resonances consistent with partial intercalation of this group between base pairs. The effect of temperature and ionic strength on the binding of both free bleomycin and the Zn(II) complex has been studied. Consistent with earlier studies on polynucleotides, the rate of exchange between the free drug and the drug-oligonucleotide complex is rapid on the 1H NMR chemical shift time scale. Binding of the oligonucleotide induced changes in resonances assigned to protons in the metal-binding region of Zn(II)-bleomycin. Intermolecular nuclear Overhauser effect enhancements between bleomycin and the oligonucleotide have not been detected.     

Metal coordination core of bleomycin : comparison of metal complexes between bleomycin and its biosynthetic intermediate.

On the basis of electron spin resonance results, the 1:1 Cu(II), Co(II), Co(II)-O₂, and Ni(III) complexes of bleomycin(BLM) have been compared with the corresponding metal complexes of its biosynthetic intermediate(P-3A). The present study suggests that (1) P-3A is an useful ligand for the clarification of metal-binding sites of BLM; (2) the secondary amine, pyrimidine ring nitrogen, deprotonated peptide nitrogen of histidine residue, and histidine imidazole groups as planar ligand donors, and the α-amino group as axial donor, are substantially important for metal-coordination of BLM; and (3) the sugar and bithiazole portions of BLM probably contribute to stabilization of Co(II)-O₂ adduct complex and axial sixth coordination of Cu(II) and Ni(III) complexes.