Iron(III)-adriamycin and Iron(III)-daunorubicin complexes: physicochemical characteristics, interaction with DNA, and antitumor activity

Fe(III) complexes of two anthracyclines, adriamycin and daunorubicin, have been studied. Using potentiometric and spectroscopic measurements, we have shown that adriamycin and daunorubicin form two well-defined species with Fe(III), which can be formulated as respectively Fe(HAd)3 and Fe(HDr)3. In these formulas, HAd and HDr stand for adriamycin and daunorubicin in which the 1,4-dihydroxy-anthraquinone moiety is half-deprotonated. Both complexes are six-membered chelates. The stability constant is beta = (2.5 +/- 0.5) X 10(28) for both complexes. Interaction with DNA has been studied showing that, despite strong coordination to Fe(III), anthracyclines are able to intercalate between DNA bases pairs, releasing the metal. These complexes display antitumor activity against P 388 leukemia that compares with that of the free drug. Fe(HAd)3, unlike adriamycin, does not catalyze the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase. Moreover, it is shown that the triferric adriamycin compound so called "quelamycin" is in fact a mixture of Fe(HAd)3 and polymeric ferric hydroxide.     

Ferric Ion and Oxygen Reduction at the Surface of Protoplasts and Cells of Acer pseudoplatanus

This work was undertaken to verify whether surface NADH oxidases or peroxidases are involved in the apoplastic reduction of Fe(III). The reduction of Fe(III)-ADP, linked to NADH-dependent activity of horseradish peroxidase (HRP), protoplasts and cells of Acer pseudoplatanus, was measured as Fe(II)-bathophenanthrolinedisulfonate (BPDS) chelate formation. In the presence of BPDS in the incubation medium (method 1), NADH-dependent HRP activity was associated with a rapid Fe(III)-ADP reduction that was almost completely inhibited by superoxide dismutase (SOD), while catalase only slowed down the rate of reduction. A. pseudoplatanus protoplasts and cells reduced extracellular Fe(III)-ADP in the absence of exogenously supplied NADH. The addition of NADH stimulated the reduction. SOD and catalase only inhibited the NADH-dependent Fe(III)-ADP reduction. Mn(II), known for its ability to scavenge O^?₂, inhibited both the independent and NADH-dependent Fe(III)-ADP reduction. The reductase activity of protoplasts and cells was also monitored in the absence of BPDS (method 2). The latter was added only at the end of the reaction to evaluate Fe(II) formed. Also, in this case, both preparations reduced Fe(III)-ADP. However, the addition of NADH did not stimulate Fe(III)-ADP reduction but, instead, lowered it. This may be related to a re-oxidation of Fe(II) by H₂O₂ that could also be produced during NADH-dependent peroxidase activity. Catalase and SOD made the Fe(III)-ADP reduction more efficient because, by removing H₂O₂ (catalase) or preventing H₂O₂ formation (SOD), they hindered the re-oxidation of Fe(II) not chelated by BPDS. As with the result obtained by method 1, Mn(II) inhibited Fe(III)-ADP reduction carried out in the presence or absence of NADH. The different effects of SOD and Mn(II), both scavengers of O^?₂, may depend on the ability of Mn(II) to permeate the cells more easily than SOD. These results show that A. pseudoplatanus protoplasts and cells reduce extracellular Fe(III)-ADP. Exogenously supplied NADH induces an additional reduction of Fe(III) by the activity of NADH peroxidases of the plasmalemma or cell wall. However, the latter can also trigger the formation of H₂O₂ that, reacting with Fe(II) (not chelated by BPDS), generates hydroxyl radicals and converts Fe(II) to Fe(III) (Fenton's reaction).     

An Unusual Nad(P)H-Dependent Oi-Generating Redox System in Hepatoma 22a Nuclei

Nuclear membranes from many tumors contain an unusual redox chain discovered originally in the Hepatoma 22a nuclear membranes⁷ which catalyzes superoxide dismutase-sensitive adrenaline oxidation to adrenochrome in the presence of either NADPH or NADH as electron donor, the reaction being inhibited by cyanide and azide. This redox chain can reduce anthracycline antitumor antibiotics adriamycin and carminomycin to their free radical states under anaerobic conditions. Evidence has been obtained for a higher stability of the carminomycin radical as compared to that of adriamycin. Operation of the nuclear membrane-bound redox chain can be a source of oxygen radical-mediated single strand breaks in DNA. The role of the nuclear membrane-associated electron transfer chain in augmenting the anticancer action of the anthracycline antibiotics is discussed.     

Interaction of 5-iminodaunorubicin with Fe(III) and with cardiolipin-containing vesicles

5-Iminodaunorubicin is an anthracycline derivative exhibiting promising antitumor activity. Using potentiometric and spectroscopic measurements we have shown that 5-iminodaunorubicin forms with Fe(III) a complex in which three molecules of drug are bound to one Fe(III) ion. Each molecule is chelated through the C-12-carbonyl and the C-11-phenolate oxygen atoms. The stability constant is 1.6 X 10(34). Using circular dichroism measurements we have studied the interactions of 5-iminodaunorubicin with cardiolipin-containing vesicles. We have shown that cardiolipin could bind one molecule of drug without penetration of the dihydroanthraquinone moiety into the bilayer.     

Metal anthracycline complexes as a new class of anthracycline derivatives. Pd(II)-adriamycin and Pd(II)-daunorubicin complexes: physicochemical characteristics and antitumor activity

Pd(II) complexes of two anthracyclines, adriamycin and daunorubicin, have been studied. Using potentiometric absorption, fluorescence, and circular dichroism measurements, we have shown that adriamycin can form two complexes with Pd(II). The first complex (I) involves two molecules of drug per Pd(II) ion; one of the molecules is chelated to Pd(II) through the carbonyl oxygen on C12 and the phenolate oxygen on C11, and the other one is bound to Pd(II) through the nitrogen of the amino sugar. This complexation induces a stacking of the two molecules of drug. In the second complex (II), two Pd(II) ions are bound to two molecules of drug (A1 and A2). One Pd(II) is bound to the oxygen on the carbons C11 and C12 of molecule A1 and the amino sugar of molecule A2 whereas the second Pd(II) ion is bound to the oxygen on C11 and C12 of molecule A2 and the amino sugar of molecule A1. The same complexes are formed between Pd(II) and daunorubicin. The stability constant for complex II is beta = (1.3 +/- 0.5) X 10(22). Interaction with DNA has been studied, showing that almost no modification of the complex occurred. This complex displays antitumor activity against P-388 leukemia that compares with that of the free drug. Complex II, unlike adriamycin, does not catalyze the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase.     

Partition coefficients of the iron(III) complexes of pyridoxal isonicotinoyl hydrazone and its analogs and the correlation to iron chelation efficacy

Pyridoxal isonicotinoyl hydrazone and its analogs are orally effective Fe(III) chelators which show potential as drugs to treat iron overload disease. The present investigation describes the measurement of the partition coefficient of the apochelator and Fe(III) complex of 20 of these ligands. These measurements have been done to investigate the relationship between lipophilicity and the efficacy of iron chelation in rabbit reticulocytes loaded with non-heme ⁵⁹Fe. The results demonstrate a linear relationship between the partition coefficient (P) of the apochelator and its Fe(III) complex, and a simple equation has been derived relating these two parameters. Experimental data in the literature are in agreement with the equation. The relationship of the partition coefficients of the iron chelators and of their Fe(III) complexes to the effectiveness of the ligands in mobilizing iron in vitro and in vivo is also discussed.     

Inhibition of phagocytic activity of ARPE-19 cells by free radical mediated oxidative stress

Oxidative stress is a main factor responsible for key changes leading to the onset of age-related macular degeneration (ARMD) that occur in the retinal pigment epithelium (RPE), which is involved in phagocytosis of photoreceptor outer segments (POS). In this study, hydrogen peroxide (H₂O₂), H₂O₂ and iron ions (Fe) or rose Bengal (RB) in the presence of NADH and Fe were used to model free radical mediated oxidative stress to test if free radicals and singlet oxygen have different efficiency to inhibit phagocytosis of ARPE-19 cells. Free radical mediated oxidative stress was confirmed by HPLC-EC(Hg) measurements of cholesterol hydroperoxides in treated cells. Electron paramagnetic resonance (EPR) spin trapping was employed to detect superoxide anion. Cell survival was analyzed by the MTT assay. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS and non-specific phagocytosis of fluorescent beads were measured by flow cytometry. HPLC analysis of cells photosensitized with RB in the presence of NADH and Fe indicated substantial increase in formation of free radical-dependent 7α/7β-hydroperoxides. EPR spin trapping confirmed the photogeneration of superoxide anion in samples enriched with RB, NADH and Fe. For all three protocols sub-lethal oxidative stress induced significant inhibition of the specific phagocytosis of POS. In contrast, non-specific phagocytosis was inhibited only by H₂O₂ or H₂O₂ and Fe treatment. Inhibition of phagocytosis was transient and recoverable by 24?h. These results suggest that free radicals may exert similar to singlet oxygen efficiency in inhibiting phagocytosis of RPE cells, and that the effect depends on the location where initial reactive species are formed.     

Iron Reduction and Trans Plasma Membrane Electron Transfer in the Yeast Saccharomyces cerevisiae

The ferri-reductase activity of whole cells of Saccharomyces cerevisiae (washed free from the growth medium) was markedly increased 3 to 6 h after transferring the cells from a complete growth medium (preculture) to an iron-deficient growth medium (culture). This increase was prevented by the presence of iron, copper, excess oxygen, or other oxidative agents in the culture medium. The cells with increased ferri-reductase activity had a higher reduced glutathione content and a higher capacity to expose exofacial sulfhydryl groups. Plasma membranes purified from those cells exhibited a higher reduced nicotinamide adenine phosphate (NADPH)-dependent ferri-reductase specific activity. However, the intracellular levels of NADPH, NADH, and certain organic acids of the tricarboxylic acids cycle were unchanged, and the activity of NADPH-generating enzymes was not increased. Addition of Fe(III)-EDTA to iron-deprived and iron-rich cells in resting suspension resulted in a decrease in intracellular reduced glutathione in the case of iron-deprived cells and in an increase in organic acids and a sudden oxidation of NADH in both types of cells. The depolarizing effect of Fe³⁺ was more pronounced in iron-rich cells. The metabolic pathways that may be involved in regulating the trans-plasma membrane electron transfer in yeast are discussed.     

Role of iron in adriamycin biochemistry

Adriamycin forms a chelate with Fe(III) that exhibits complex redox chemistry. The drug ligand is able to directly reduce the bound Fe(III) with the concomitant production of a one-electron oxidized drug radical. This Fe(II) can reduce oxygen to hydrogen peroxide and cleave the peroxide to yield the hydroxyl radical. In addition, the drug X Fe complex can catalyze the transfer of electrons from reduced glutathione to molecular oxygen to yield superoxide, hydrogen peroxide, and hydroxyl radicals. The adriamycin X Fe complex binds to DNA to form a ternary drug X Fe X DNA complex, which is also able to catalyze the thiol-dependent reduction of oxygen and the formation of hydroxyl radical from hydrogen peroxide. As a consequence of this chemistry, the adriamycin X Fe complex can cleave DNA on the addition of glutathione or hydrogen peroxide. Although less well defined, the adriamycin X Fe complex can bind to cell membranes and cause oxidative destruction of these membranes in the presence of thiols or hydrogen peroxide.     

Methane monooxygenase component B and reductase alter the regioselectivity of the hydroxylase component-catalyzed reactions. A novel role for protein-protein interactions in an oxygenase mechanism.

The soluble methane monooxygenase (MMO) system, consisting of reductase, component B, and hydroxylase (MMOH), catalyzes NADH and O2-dependent monooxygenation of many hydrocarbons. MMOH contains 2 mu-(H or R)oxo-bridged dinuclear iron clusters thought to be the sites of catalysis. Although rapid NADH-coupled turnover requires all three protein components, three less complex systems are also functional: System I, NADH, O2, reductase, and MMOH; System II, H2O2 and oxidized MMOH; System III, MMOH reduced nonenzymatically by 2e- and then exposed to O2 (single turnover). All three systems give the same products, suggesting a common reactive oxygen species. However, the distribution of products observed for most substrates that are hydroxylated in more than one position is different for each system. For several of these substrates, addition of component B to Systems I, II, or III causes the product distributions to shift dramatically. These shifts result in identical product distributions for Systems I and III in which MMOH passes through the 2e- reduced state ([Fe(II).Fe(II)]) during catalysis. In contrast, System II (in which MMOH probably does not become reduced) generally gives a unique product distribution. It is proposed that changes in MMOH structure occurring upon diiron cluster reduction and/or component complex formation cause substrates to be presented differently to the activated oxygen species. Kinetic studies show that component B strongly activates System I and, in most cases, strongly deactivates System II. The effect of component B on product distribution of System I (and III) occurs at less than 5% of the MMOH concentration, while nearly stoichiometric concentrations are required to maximize the rate of System I. This shows that component B has at least two roles in catalysis. EPR monitored titration of reduced MMOH ([Fe(II).Fe(II)]) with component B suggests that the effect of substoichiometric component B on product distribution is due to hysteresis in the MMOH conformational changes.     

An NADH: Fe(III) EDTA oxidoreductase from Cryptococcus albidus: an enzyme involved in ethylene production in vivo?

An ethylene-forming enzyme which forms ethylene from 2-oxo-4-methylthiobutyric acid (KMBA) was purified to an electrophoretically homogeneous state from a cell-free extract of Cryptococcus albidus IFP 0939. The presence of KMBA, NADH, Fe(III) chelated to EDTA and oxygen were essential for the formation of ethylene. When ferric ions, as Fe(III)EDTA, in the reaction mixture were replaced by Fe(II)EDTA under aerobic conditions, the non-enzymatic formation of ethylene was observed. Under anaerobic conditions in the presence of Fe(III)EDTA and NADH, the enzyme reduced 2 mol of Fe(III) with 1 mol of NADH to give 2 mol of Fe(II) and 1 mol NAD+, indicating that the ethylene-forming enzyme is an NADH-Fe(III)EDTA oxidoreductase. The role of NADH:Fe(III)EDTA oxidoreductase activity in the formation in vivo ethylene from KMBA is discussed.     

Oxidation-Reduction Reactions of Iron Bleomycin in the Absence and Presence of DNA

Using the pulse radiolysis technique, we have demonstrated that bleomycin-Fe(III) is stoichiometrically reduced by CO₂^? to bleomycin-Fe(II) with a rate of (1.9 ± 0.2) × 10⁸M^?1s^?1. In the presence of calf thymus DNA, the reduction proceeds through free bleomycin-Fe(III) and the binding constant of bleomycin-Fe(III) to DNA has been determined to be (3.8 ± 0.5) x 10⁴ M^?1. It has also been demonstrated that in the absence of DNA O₂^?1 reacts with bleomycin-Fe(III) to yield bleomycin-Fe(II)O₂, which is in rapid equilibrium with molecular oxygen, and decomposes at room temperature with a rate of (700 ± 200) s^?1. The resulting product of the decomposition reaction is Fe(III) which is bound to a modified bleomycin molecule. We have demonstrated that during the reaction of bleomycin-Fe(II) with O₂, modification or self-destruction of the drug occurs, while in the presence of DNA no destruction occurs, possibly because the reaction causes degradation of DNA.     

First-principles electronic structure study of rhizoferrin and its Fe(III) complexes

We have determined the structure and coordination chemistry of rhizoferrin (Rf), which is a particular type of siderophore, and its Fe(III) complexes using density functional theory calculations. Our results show that the Fe(III) ion binds in an octahedral coordination, with a low-spin (S = 1/2) charge-neutral chiral complex having the largest binding energy of the investigated complexes. We have also calculated nuclear magnetic resonance parameters, such as chemical shifts for ¹H and ¹³C, and indirect nuclear spin–spin couplings for ¹H–¹H and ¹³C–¹H in free Rf and in a low-spin neutral Rf metal complex, as well as nuclear quadrupole interaction parameters, such as asymmetry parameter and nuclear quadrupole coupling constants for ¹⁴N. Our calculated values for the chemical shifts for free Rf are in excellent agreement with experimental data while the calculated NMR parameters for Fe(III) complexes are predictions for future experimental work.     

Theoretical investigation of the ring opening process of verdoheme to biliverdin in the presence of dioxygen

The conversion of ferrous verdoheme to ferric biliverdin in the presence of O₂ was investigated using the B3LYP method. Both 6-31G and 6-31G (d) basis sets were employed for geometry optimization calculation as well as energy stabilization estimation. Three possible pathways for the conversion of iron verdoheme to iron biliverdin were considered. In the first route oxygen and reducing electron were employed. In this path formation of ferrous verdoheme-O₂ complex was followed by the addition of one electron to the ferrous-oxycomplex to produce ferric peroxide intermediate. The ferric peroxide intermediate experienced an intramolecular nucleophilic attack to the most positive position at 5-oxo carbons on the ring to form a closed ring biliverdin. Subsequently the ring opening process took place and the iron (III) biliverdin complex was formed. Closed ring iron biliverdin intermediate and open ring iron biliverdin formed as a product of verdoheme cleavage were respectively 13.20 and 32.70 kcal mol⁻¹ more stable than ferric peroxide intermediate. Barrier energy for conversion of ferric peroxide to closed ring Fe (III) biliverdin and from the latter to Fe (III) biliverdin were respectively 8.67 and 3.35 kcal mol⁻¹. In this path spin ground states are doublet except for iron (III) biliverdin in which spin state is quartet. In the second path a ferrous-O₂ complex was formed and, without going to a one electron reduction process, nucleophilic attack of iron superoxide complex took place followed by the formation of iron (III) biliverdin. This path is thermodynamically and kinetically less favorable than the first one. In addition, iron hydro peroxy complex or direct attack of O₂ to macrocycle to form an isoporphyrin type intermediate have shown energy surfaces less favorable than aforementioned routes.     

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction

Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP⁺ reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k _cat/K _m value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k _cat/K _m value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.     

Synthesis and in vitro antioxidant activity study of some new piperazinyl flavone compounds

Flavones exhibit a variety of beneficial effects and are well known for their medicinal importance in several diseases, including cardiovascular, neurodegenerative and cancer. The inclusion of the piperazine ring to the flavone backbone is an important strategy in drug discovery but only a few studies have synthesized piperazinyl flavone compounds to test their biological activity. While there is a major focus on the antioxidant properties of drugs in therapy of several diseases of inflammatory origin, we synthesized a series of the novel piperazinyl flavone analogues bearing the phenyl ring with different substituents. The analogues were evaluated for in vitro antioxidant activity against superoxide anion radical, hydroxyl radical, 2,2‐diphenyl‐1‐picrylhydrazyl radical, and hydrogen peroxide scavenging properties. The total antioxidant status based on the absorbance of the 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) radical cation (ABTS^+?) and total antioxidant capacity using the Fe(III)‐ferrozine complex were also monitored. The results of the above studies showed that the compounds synthesized were found possessed moderate radical scavenging potential, and that their interaction with reactive oxygen species is complex and depends on their structural conformation and the type of substituent R in the piperazine ring being attached. Best antiradical activity were found for the compounds with methoxy groups on the phenyl ring of substituent R, whereas the presence of methoxy or trifluoromethyl groups in substituent R resulted in higher ABTS^+? and ion Fe(III) reduction. These compounds are promising molecules to be used for their antioxidant properties and may be regarded, after improvement of the antioxidant potential, to control diseases of free radical etiology.     

An Unusual Nad(P)H-Dependent Oi-Generating Redox System in Hepatoma 22a Nuclei

Nuclear membranes from many tumors contain an unusual redox chain discovered originally in the Hepatoma 22a nuclear membranes⁷ which catalyzes superoxide dismutase-sensitive adrenaline oxidation to adrenochrome in the presence of either NADPH or NADH as electron donor, the reaction being inhibited by cyanide and azide. This redox chain can reduce anthracycline antitumor antibiotics adriamycin and carminomycin to their free radical states under anaerobic conditions. Evidence has been obtained for a higher stability of the carminomycin radical as compared to that of adriamycin. Operation of the nuclear membrane-bound redox chain can be a source of oxygen radical-mediated single strand breaks in DNA. The role of the nuclear membrane-associated electron transfer chain in augmenting the anticancer action of the anthracycline antibiotics is discussed.     

Trace metal complexation by the triscatecholate siderophore protochelin: structure and stability

Although siderophores are generally viewed as biological iron uptake agents, recent evidence has shown that they may play significant roles in the biogeochemical cycling and biological uptake of other metals. One such siderophore that is produced by A. vinelandii is the triscatecholate protochelin. In this study, we probe the solution chemistry of protochelin and its complexes with environmentally relevant trace metals to better understand its effect on metal uptake and cycling. Protochelin exhibits low solubility below pH 7.5 and degrades gradually in solution. Electrochemical measurements of protochelin and metal–protochelin complexes reveal a ligand half-wave potential of 200 mV. The Fe(III)Proto³⁻ complex exhibits a salicylate shift in coordination mode at circumneutral to acidic pH. Coordination of Mn(II) by protochelin above pH 8.0 promotes gradual air oxidation of the metal center to Mn(III), which accelerates at higher pH values. The Mn(III)Proto³⁻ complex was found to have a stability constant of log β₁₁₀ = 41.6. Structural parameters derived from spectroscopic measurements and quantum mechanical calculations provide insights into the stability of the Fe(III)Proto³⁻, Fe(III)H₃Proto, and Mn(III)Proto³⁻ complexes. Complexation of Co(II) by protochelin results in redox cycling of Co, accompanied by accelerated degradation of the ligand at all solution pH values. These results are discussed in terms of the role of catecholate siderophores in environmental trace metal cycling and intracellular metal release.     

Preparation, crystal structure and luminescent properties of lanthanide picrate complexes with N-benzyl-2-{2′-[(benzyl-methyl-carbamoyl)-methoxy]-biphenyl-2-yloxy}-N-methyl-acetamide (L)

A new amide-based ligand derived from biphenyl, N-benzyl-2-{2′-[(benzyl-methyl-carbamoyl)-methoxy]-biphenyl-2-yloxy}-N-methyl-aceamide (L) was synthesized. Solid complexes of lanthanide picrates with this new ligand were prepared and characterized by elemental analysis, conductivity measurements, IR and electronic spectroscopies. The molecular structure of [Eu(pic)₃L] shows that the Eu(III) ion is nine-coordinated by four oxygen atoms from the L and five from two bidentate and one unidentate picrates. All the coordinate picrates and their adjacent equivalent picrates form intermolecular π-π stacking. Furthermore, the [Eu(pic)₃L] complex units are linked by the π-π stacking to form a two-dimensional (2-D) netlike supramolecule. Under excitation, the europium complex exhibited characteristic emissions. The lifetime of the ⁵D₀ level of the Eu(III) ion in the complex is 0.22 ms. The quantum yield Φ of the europium complex was found to be 1.01 × 10⁻³ with quinine sulfate as reference. The lowest triplet state energy level of the ligand indicates that the triplet state energy level of the ligand matches better to the resonance level of Eu(III) than Tb(III) ion.