Lanthanide Ion Binding Research Articles

Characterization of the Lanthanide Ion-Binding Properties of Calcineurin-B Using Laser-Induced Luminescence Spectroscopy

Calcineurin (CaN) is a Ca2+/calmodulin-dependent protein phosphatase found in brain and other tissues. It is a heterodimer consisting of a catalytic subunit (CaN-A) and a Ca(2+)-binding regulatory subunit (CaN-B). The primary structure of CaN-B indicates that it, like calmodulin, is an EF-hand protein and binds four Ca2+ ions. Eu3+, due to its favorable spectroscopic and chemical properties, has been substituted for Ca2+ in CaN-B to determine the metal ion-binding properties of this "calmodulin-like" protein. Excitation of the 7F0-->5D0 transition of Eu3+ results in a spectrum similar to that of calmodulin, consisting of three peaks. Analysis of the spectral titration curves reveals four Eu(3+)-binding sites in CaN-B. The affinities vary: sites I and II have dissociation constants of 1.0 +/- 0.2 and 1.6 +/- 0.4 microM, respectively; the values for sites III and IV are Kd = 140 +/- 20 and Kd = 20 +/- 10 nM, respectively. Binding of Tb3+ is slightly weaker. Tb3+ luminescence, sensitized by tyrosine, reveals that for lanthanides the highest affinity sites lie in the C-terminal domain. Energy transfer distance measurements between Eu3+ and Nd3+ in sites III and IV reveal a separation of 10.5 +/- 0.5 A, which suggests that these sites are arranged in a typical EF-hand pair. This information indicates that the overall structure of CaN-B is similar to the dumbbell-shaped proteins troponin-C and calmodulin, but is more like TnC in its metal-binding properties.

Self-Association of Ca2+-Binding Peptides Induced by Lanthanide Ions: A Fluorescence Study

In this study a Ca2+-binding 14mer peptide was synthesized with the sequence GDKNADGCIEFEEL, allowing covalent attachment of sulfhydryl-reactive fluorescent molecules at position 7 of the 12-residue, metal-binding loop (underlined). This provided the opportunity to select donor molecules with suitable spectral characteristics for sensitized excitation of chelated terbium (Tb3+) or europium (Eu3+) ions. N-(1-Pyrene)-iodoacetamide and 7-diethylamino-3-((4′-iodoacetyl-amino)phenyl)-4-methylcoumarin were attached to the peptide and titrations carried out with terbium or europium stock solutions. It was possible to observe lanthanide ion binding to the loop in stoichiometric quantities, but maximal lanthanide luminescence was achieved with a large excess of lanthanide present, due to metal-induced peptide association. Obtaining maximal lanthanide luminescence is important in the development of systems for use in sensitive clinical diagnostic and time-resolved luminescence-based immunoassay applications.

The metal sites on sarcoplasmic reticulum membranes that bind lanthanide ions with the highest affinity are not the ATPase Ca2+ transport sites.

We attempted to establish whether lanthanide ions, when added to sarcoplasmic reticulum (SR) membranes in the absence of nucleotide, compete with Ca2+ for binding to the transport sites of the Ca(2+)-ATPase in these membranes, or whether they bind to different sites. Equilibrium measurements of the effect of lanthanide ions on the intrinsic fluorescence of SR ATPase and on 45Ca2+ binding to it were performed either at neutral pH (pH 6.8), i.e. when endogenous or contaminating Ca2+ was sufficient to nearly saturate the ATPase transport sites, or at acid pH (pH 5.5), which greatly reduced the affinity of calcium for its sites on the ATPase. These measurements did reveal apparent competition between Ca2+ and the lanthanide ions La3+, Gd3+, Pr3+, and Tb3+, which all behaved similarly, but this competition displayed unexpected features: lanthanide ions displaced Ca2+ with a moderate affinity and in a noncooperative way, and the pH dependence of this displacement was smaller than that of the Ca2+ binding to its own sites. Simultaneously, we directly measured the amount of Tb3+ bound to the ATPase relative to the amount of Ca2+ and found that Tb3+ ions only reduced significantly the amount of Ca2+ bound after a considerable number of Tb3+ ions had bound. Furthermore, when we tested the effect of Ca2+ on the amount of Tb3+ bound to the SR membranes, we found that the Tb3+ ions which bound at low Tb3+ concentrations were not displaced when Ca2+ was added at concentrations which saturated the Ca2+ transport sites. We conclude that the sites on SR ATPase to which lanthanide ions bind with the highest affinity are not the high affinity Ca2+ binding and transport sites. At higher concentrations, lanthanide ions did not appear to be able to replace Ca2+ ions and preserve the native structure of their binding pocket, as evaluated in rapid filtration measurements from the effect of moderate concentrations of lanthanide ions on the kinetics of Ca2+ dissociation. Thus, the presence of lanthanide ions slowed down the dissociation from its binding site of the first, superficially bound 45Ca2+ ion, instead of specifically preventing the dissociation of the deeply bound 45Ca2+ ion. These results highlight the need for caution when interpreting, in terms of calcium sites, experimental data collected using lanthanide ions as spectroscopic probes on SR membrane ATPase.

1H NMR studies on lanthanides substituted transferrins

The binding of lanthanide(III) ions to human serum apotransferrin has been investigated through 1H NMR spectroscopy. Several well resolved isotropically shifted signals have been observed between 100/-100 ppm for the Tm, Tb, Yb, and Dy derivatives. Significant spectroscopic inequivalence of the two metal binding sites has been revealed. Differences in the behavior of signals assigned to the C- and to the N-terminal site have been observed upon titration with sodium perchlorate.

Fluorescent probes for measuring the binding constants and distances between the metal ions bound to Escherichia coli glutamine synthetase.

TNS, 2-p-toluidinylnaphthalene-6-sulfonate, has been used as a fluorescent probe to determine the binding constants of metal ions to the two binding sites of Escherichia coli glutamine synthetase (GS). TNS fluorescence is enhanced dramatically when bound to proteins due to its high quantum yield resulting from its interactions with hydrophobic regions in proteins. The fluorescence energy transfer from a hydrophobic tryptophan residue of GS to TNS has been detected as an excitation band centered at 280 nm. Therefore, TNS is believed to be bound to a hydrophobic site on the GS surface other than the active site and is located near a hydrophobic Trp residue of GS. GS binds lanthanide ions [Ln(III)] more tightly than either Mn(II) or Mg(II), and the binding constants of several lanthanide ions were determined to be in the range (2.1-4.6) x 10(10) and (1.4-3.0) x 10(8) M-1 to the two metal binding sites of GS, respectively. The intermetal distances between the two metal binding sites of GS were also determined by measuring the efficiencies of energy transfer from Tb(III) to other Ln(III) ions. The intermetal distances of Tb(III)-Ho(III) and Tb(III)-Nd(III) were 7.9 and 6.8 A, respectively.

Location of high-affinity metal binding sites in the profile structure of the Ca+2-ATPase in the sarcoplasmic reticulum by resonance x-ray diffraction

Resonance x-ray diffraction measurements on the lamellar diffraction from oriented multilayers of isolated sarcoplasmic reticulum (SR) membranes containing a small concentration of lanthanide (III) ions (lanthanide/protein molar ratio approximately 4) have allowed us to calculate both the electron density profile of the SR membrane and the separate electron density profile of the resonant lanthanide atoms bound to the membrane to a relatively low spatial resolution of approximately 40 A. Analysis of the membrane electron density profile and modeling of the separate low resolution lanthanide atom profile, using step-function electron density models based on the assumption that metal binding sites in the membrane profile are discrete and localized, resulted in the identification of a minimum of three such binding sites in the membrane profile. Two of these sites are low-affinity, low-occupancy sites identified with the two phospholipid polar headgroup regions of the lipid bilayer within the membrane profile. Up to 20% of the total lanthanide (III) ions bind to these low-affinity sites. The third site has relatively high affinity for lanthanide ion binding; its Ka is roughly an order of magnitude larger than that for the lower affinity polar headgroup sites. Approximately 80% of the total lanthanide ions present in the sample are bound to this high-affinity site, which is located in the "stalk" portion of the "headpiece" within the profile structure of the Ca+2 ATPase protein, approximately 12 A outside of the phospholipid polar headgroups on the extravesicular side of the membrane profile. Based on the nature of our results and on previous reports in the literature concerning the ability of lanthanide (III) ions to function as Ca+2 analogues for the Ca+2 ATPase we suggest that we have located a high-affinity metal binding site in the membrane profile which is involved in the active transport of Ca+2 ions across the SR membrane by the Ca+2 ATPase.

Conformational study of L‐lysine in aqueous solution by lanthanide shift probes

AbstractConformation of L‐lysine in aqueous solution was investigated by lanthanide shift probes (Dy, Ho, Er, Tm and Yb). Reilley's method was employed to separate the contact and dipolar components of the 13C paramagnetic shifts. This study reveals that Cα shift has the largest contact contribution while the other carbon shifts are dominated by the dipolar contribution. The average overall conformation of L‐lysine in aqueous solution is extended with the molecular backbone in trans form. In the complex, lanthanide ion coordinates to the carboxyl group with Ln—O bond length 2.2 Å and the whole ligand is located outside the zero‐dipolar shift cone of the lanthanide ion. The electronic spin density distribution on the ligand nuclei shows that the spin polarization is the predominant mechanism of the contact interaction for nuclei in close proximity to the bound lanthanide ion.

Binding of divalent and trivalent cations with crotoxin and with its phospholipase and its non-catalytic subunits: effects on enzymatic activity and on the interaction of phospholipase component with phospholipids

We have studied the interaction of divalent and trivalent with a potent phospholipase A(2) neurotoxin, crotoxin, from Crotalus durissus terrificus venom. The pharmacological action of crotoxin requires dissociation of its catalytic subunit (component B) and of its non-enzymatic chaperone subunit (component A), then the binding of the phospholipase subunit to target sites on cellular membranes and finally phospholipid hydrolysis. In this report, we show that the phospholipase A(2) activity of crotoxin and of component B required Ca2+ and that other divalent cations (Sr2+, Cd2+ and Ba2+) and trivalent lanthanide ions are inhibitors. The lowest phospholipase A(2) activity was observed in the presence of Ba2+, which proved to be a competitive inhibitor of Ca2+. The binding of divalent cations and trivalent lanthanide ions to crotoxin and to its subunits has been examined by equilibrium dialysis and by spectrofluorimetric methods. We found that crotoxin binds two divalent cations per mole with different affinities; the site presenting the highest affinity (K(d) in the mM range) in involved in the activation (or inhibition) of the phospholipase A(2) activity and must therefore be located on component B, the other site (K(d) higher than 10 mM) is probably localized on component A and does not play any role in the catalytic activity of crotoxin. We also observed that crotoxin component B binds to vesicular and micellar phospholipids, even in the absence of divalent cations. The affinity of this interaction either does not change or else increases by an order of magnitude in the presence of divalent cations.

Interactions of spin-labeled calmodulin with trifluoperazine and phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III)

Bovine calmodulin analogues, spin-labeled at methionine and tyrosine residues, have been utilized in electron paramagnetic resonance (EPR) studies designed to investigate calmodulin interactions with the antipsychotic drug trifluoperazine and the calmodulin-binding protein 3',5'-cyclic nucleotide phospho-diesterase. Trifluoperazine titrations of spin-labeled calmodulin analogues were carried out in the presence of Ca(II), Cd(II), and Tb(III). Similar experiments were performed with the phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III). EPR signals from the methionine-directed probe proved to be more sensitive to the binding of target molecules than signals from the tyrosine-directed probe, perhaps indicating that the spin-labeled methionine is at a site close to the target molecule binding site. While the binding of TFP, as monitored by EPR spectral changes in the methionine spin-labeled calmodulin, was in evidence with Ca(II), Cd(II), and all the lanthanides examined, no binding of phosphodiesterase to calmodulin could be detected in the presence of the lanthanide ions, perhaps due to inactivation of the phosphodiesterase by lanthanide ion binding. The abilities of the spin-labeled calmodulins to activate phosphodiesterase were also investigated. The spin-labeled tyrosine calmodulin was able to activate phosphodiesterase as well as native calmodulin, while a lower degree of activation was found when the spin-labeled methionine analogue was used.

The thermodynamics of lanthanide ion binding to adriamycin

We have examined the thermodynamics of lanthanide ion binding to adriamycin by monitoring the effects of variations in temperature on the dissociation constants of various lanthanide ion complexes of the drug. These constants were obtained by analyzing the extent of quenching of the fluorecence of adriamycin in the presence of lanthanide ions in terms of an equilibrium binding process. Our binding model included the following features, all of which are supported by evidence derived from previous published reports, vide infra. The lanthanides form 1:1 complexes with adriamycin. The binding is dependent on the pH of the solution, indicating that only the nonprotonated amine form of the drug participates in lanthanide ion binding. And finally the drug self-associates in solution to for a dimeric species. Our present results indicate that the binding process is almost completely independent of temperature, indicating that the enthalpy of complex formation is extremely small. The entropy terms are consistent with the formation of a complex in which the adriamycin acts as a bidentate ligand. Our results suggest that the lanthanide complexes are isostructural, at least as far as the adriamycin is concerned, throughout the lanthanide series.

Metal-ion binding to parvalbumin. A 113Cd-n.m.r. study of the binding of different lanthanide ions.

113Cd-n.m.r. studies were used to investigate the binding of the lanthanide ions La3+, Gd3+, Tb3+, Yb3+ and Lu3+ to parvalbumins. It was shown that lanthanide ions with a smaller ionic radius bind sequentially to Cd2+-saturated parvalbumin, whereas those with a larger ionic radius bind with similar affinity to both the CD site and the EF site. The smallest ion, Lu3+, does in fact not compete significantly with Cd2+ for the CD site in carp parvalbumin, but appears to bind only to the EF site. This preference of the smaller lanthanide ions for the EF site was used to assign the n.m.r. signals for protein-bound 113Cd. By using Cd n.m.r. and Tb3+ fluorescence it was also shown for alpha-lineage parvalbumin from pike that these proteins possess a third site that can bind lanthanide ions. This site is, however, much weaker than in the beta-lineage parvalbumins. It was used to assign the 113Cd resonances from protein-bound Cd2+ ions in the spectrum of pike pI5.0 parvalbumin.

Europium(III) luminescence excitation spectroscopy. A species-specific method for the quantitation of lanthanide ion binding to chelating agents. Complexes of (1,2-ethanediyldioxy)diacetate

Etude de la complexation de l'EDA (ethanediyldioxy-1,2) diacetate avec des ions Lanthanide (III) par spectroscopie d'excitation laser. Mesure de la constante de formation du bis complexe Eu (EDA) 2 −

A fluorimetric study of the lanthanides binding to Concanavalin A

1. 1. The bincling of Tb 3+ and other lanthanides to Con A has been studied by sensitized Tb 3+ luminescence, by quenching of intrinsic fluorescence and by activity measurements. 2. 2. In all the experimental conditions tested, it was found that holo and apo Con A bind lanthanide ions at a site different from the bincling sites of the constitutive metals, Mn 2+ and Ca 2+. 3. 3. The bound lanthanide did not affect the saccharide bincling ability and the hemoagglutinating ability of Con A. 4. 4. The intrinsic fluorescence of Con A is quenched by the bincling of Tb 3+ and Gd 3+. The same quenching is obtained by shifting the pH of Con A from pH 6.5 to 4.5. 5. 5.It is proposed that H + and Ln 3+ completely quench a tryptophan, perhaps the residue 88 or 182.

The solution structure of kringle 4: NMR studies on native and several chemically modified kringle 4 species of human plasminogen

Kringle 4 of human plasminogen has been studied by NMR spectroscopy to define the solution structure of the kringle-fold and to characterize the ω-aminocarboxylic acid binding site. Aromatic and aliphatic resonances of the NMR spectrum have been identified with the aid of spin-decoupling and NOE procedures as well as pH-titration and metal ion probe studies. Comparison of the NMR spectrum of kringle 4 with the spectra of various kringle 4 species chemically modified at defined positions permitted the assignment of several resonances to specific residues in the kringle 4 sequence. The NOE studies revealed that Leu 45 is in close proximity of the sequentially distant Trp 25/Trp 61 residue pair, thus delineating a definite structural feature of the kringle-fold. The binding of 6-aminohexanoic acid to kringle 4 was shown to cause shifts in the resonances of several aromatic residues, including those of Trp 71, suggesting that several aromatic residues may be lining the ω-aminocarboxylic acid binding site. The binding of the ligand is competitive with the binding of lanthanide ions which reveal much detail of this site.

Europium(III) binding to bovine prothrombin residues 1-39 and to bovine prothrombin fragment 1.

Changes in the Eu3+ luminescence decay constant observed for Eu3+ bound to prothrombin fragment 1 and the peptide containing residues 1-39 of prothrombin result solely from changes in the number of water molecules in the primary hydration sphere of the ion. Highly coordinated sites which bind Eu3+ in a different manner from simple Gla-containing peptides exist on both the fragment 1 and 1-39 molecules. Metal ions bound at some of these sites do not appear to undergo rapid exchange with metal ions in solution. The binding of other lanthanide ions and calcium ions in the presence of europium ions cause a change in the conformation of the macromolecules, resulting in even tighter coordination of fragment 1 or residues 1-39 to Eu3+. Hydrophilic groups other than the actual Eu3+ binding sites on fragment 1 help to maintain its water solubility when complexes to Eu3+ or Ca2+. Because the 1-39 peptide does not contain as many of these hydrophilic groups, the peptide precipitates upon binding to di- and trivalent cations. Self-association of the 1-39 molecules appears to occur at high peptide concentrations which result in a peptide concentration effect on Eu3+ binding not seen in fragment 1. pH titration yields results which suggest a role for Gla carboxyl groups in Eu3+ complexation to both fragments 1 and 1-39. Although the two molecules are not identical with regard to Eu3+ binding behavior, enough similarities exist that residues 1-39 appear to be a reasonable model of the fragment 1 molecule for the purposes of metal ion complexation studies.

Spectroscopic studies of lanthanide ion binding to multidentate ligands in aqueous solution. 3. Optically active Tb(EHPG)(L)n and Tb(DPA)m(L)n complexes

Spectroscopic studies of lanthanide ion binding to multidentate ligands in aqueous solution. 3. Optically active Tb(EHPG)(L)n and Tb(DPA)m(L)n complexes

Spectroscopic studies of lanthanide ion binding to multidentate ligands in aqueous solution. 2. N,N'-Bis(2-hydroxyphenyl)ethylenedinitrilo-N,N'-diacetate

Spectroscopic studies of lanthanide ion binding to multidentate ligands in aqueous solution. 2. N,N'-Bis(2-hydroxyphenyl)ethylenedinitrilo-N,N'-diacetate

Spectroscopic studies of lanthanide ion binding to multidentate ligands in aqueous solution. 1. Malic acid

Spectroscopic studies of lanthanide ion binding to multidentate ligands in aqueous solution. 1. Malic acid

Time-resolved europium(III) excitation spectroscopy: a luminescence probe of metal ion binding sites.

A laser-induced luminescence technique is introduced for probing the structure and equilibria of lanthanide complexes and lanthanide ion binding to macromolecules. The method involves the excitation of the 7F0 leads to 5D0 transition between nondegenerate levels in the europium(III) ion by means of an intense pulsed dye laser source. Excitation profits obtained by scanning the laser through the transition region reveal distinct peaks characteristic of individual europium(III) ion environments. The technique may be used to characterize the species present in complex equilibria in solution or to study europium(III) binding to macromolecules. Distinct europium(III) binding sites in thermolysin with long and short excited state lifetimes are observed.

The interaction of various lanthanide ions and some anions with phosphatidylcholine vesicle membranes A 31P NMR study of the surface potential effects

The interaction of various lanthanide ions with vesicles of phosphatidyl-choline from egg yolk has been followed by 31P NMR at 30°C. From known magnetic properties of these ions, separation of the paramagnetic shift into a pure contact and a pseudo-contact part was carried out. Binding curves for the contact contribution ( F curves) were obtained from vesicles in solutions of sodium salts with monovalent anions over a wide concentration range. These curves should be insensitive to any conformational effects due to ion binding. Indication of a conformational change in the lipid head group at low ion binding was obtained by studying the ratio between the contact and the pseudo-contact contributions. Besides the adsorption of lanthanide ions, specific anion binding to the surface was introduced to account for the enhanced chemical shifts ( Cl − < Br − < NO 3 − ). The results were analyzed in terms of the theory for the diffuse double layer (Gouy-Chapman-Grahame) with equilibrium conditions for the adsorbing cations and anions. Simulations of the titration curves furnished parameters for the ion-lipid interactions. The synergism between the cations and anions follows from the potential effects. Comparison of results with lanthanide ions and Ca 2+ indicates that the anion adsorption probably depends on the nature of the adsorbed cation. Lanthanide ion binding to l-glycerophosphorylcholine is not influenced by sodium salts. The binding constant for this complex is weaker than with phosphatidylcholine. The chemical shifts for the lanthanide ion complexes with these two phosphorus compounds seem to be about the same.