Influence of Lanthanide(III) Ions on the Reaction System Tryptophan—H2O2–Fe(II)

Chemiluminescence (CL) studies were carried out with luminescent lanthanide ions as probes to a tryptophan-oxidation reaction at pH 6. The redox system consisted of tryptophan, hydrogen peroxide, and Fe(II) ions (catalysts of H2O2 decomposition). The luminescent lanthanide(III) ions used were Eu(III), Tb(III), Gd(III), and Dy(III). In the case of the reaction system with the Tb(III) ion a significant increase in the chemiluminescence intensity and its duration was observed over the other Ln(III) ions. The CL spectrum registered for this system shows emission bands typical of Tb(III) ions with maxima at λ 490 and 550 nm, corresponding to the electronic transitions of D4 F6 and D4 F5, respectively. The presence of emission bands characteristic of the Ln(III) ions was also observed in the systems containing Eu(III) and Dy(III) ions. These studies revealed a strong influence of the chemiluminescence intensity associated with the tryptophan oxidation, on the concentration of Ln(III) ions. On the basis of the results obtained, a possible mechanism is proposed for reaction of the systemml: Ln(III)—tryptophan—H2O2–Fe(II), taking into consideration an energy transfer process from the tryptophan oxidation products to the Tb(III), Dy(III), or Eu(III) ions.


INTRODUCTION
Due to their luminescent properties lanthanide(III) ions, particularly europium(III) and terbium(III), have been used in a large number of investigations involving systems of biological importance, including aminoacids and proteins [1][2][3][4].In these studies an important role is played by processes of energy transfer from organic chromophors to the Ln(III) ions.Tryptophan, used in these investigations, is an aromatic aminoacid-a naturally occurring chromophor.Energy transfer from tryptophan to Ln(III) ions has been widely studied through the use of spectrofluorimetric methods in which the observed emission was the result of tryptophan excitation [3].
In this paper, the Ln(III) ions were used as probes to study the reaction system of Ln(III)-tryptophan-H 2 O 2 Fe(II).Results of chemiluminescence, that is, CL intensities, obtained from the oxidation of tryptophan, strongly depend on the pH of the solutions.An intense luminescence intensity has been observed in strongly alkaline [5,6] and strongly acidic media [7].However, a smaller number of investigations refers to this reaction in solutions of pH near neutral because of the low luminescence intensities occurring in these systems.In this paper studies of the reaction system containing tryptophan-H 2 O 2 Fe(II) were carried out in solutions of pH ∼ 6, using Tb(III), Dy(III), and Eu(III) ions as luminescent probes for chemiluminescence.

EXPERIMENTAL
Chemiluminescence measurements were performed on the experimental set-up presented in Scheme 1 that is sensitive to ultra-low intensity irradiation using the stationary method.The absorption spectra were measured by means of a Shimadzu UV-2401 PC spectrophotometer, and fluorescence spectra were recorded using a Perkin-Elmer MP3 spectrofluorimeter.
Chemicals used in this study were tryptophan (analytical grade, Fluka), trypamine hydrochloride (analytical grade, Fluka) (Figure 1), chlorides of Eu(III), Tb(III), Gd(III), and Dy(III) obtained by dissolving an appropriate oxide (spectroscopically pure, prepared in the Laboratory of Rare Earths, Faculty of Chemistry, Adam Mickiewicz University, Pozna ń) in hydrochloric acid (spectroscopically pure, Fluka), hydrogen peroxide (30% solution, analytical grade, Merck), thenoyltrifluoroacetone TTA, (analytical grade, International Journal of Photoenergy Fluka); NaOH (analytical grade); FeCl 2 (analytical grade, Fluka).All solutions were prepared with the use of doubly distilled water, except for TTA, which was dissolved in 95% ethanol (pure for analysis).Chelate solutions of europium(III) with TTA were obtained by mixing solutions of EuCl 3 and TTA in the molar ratio 1 : 3. The ethanol content in > 1% v/v did not affect the CL intensities.All of the chemiluminescence studies were performed in the same manner.Hydrogen peroxide (2 ¡ 10 2 mol/l) was added to solutions containing tryptophan or trypamine (1 ¡ 10 4 mol/l), Fe(II) ions (6 ¡ 10 6 mol/l), and Ln(III) (4¡10 5 to 1¡10 2 mol/l), or europium(III) chelates (5¡10 6   to 1 ¡ 10 4 mol/l).The experiments were conducted in near neutral solutions (pH ∼ 6), because all initial solutions were adjusted to pH ∼ 6.This pH value was attained by the addition of NaOH or HCl solutions.For all solutions studied, curves of CL decay were obtained, and the CL light intensity sums were calculated as the area under these curves (S = t t0 IΔt; where I CL intensity, t-measurement duration).

RESULTS AND DISCUSSION
The chemiluminescence in the reaction systems under study arises as the result of tryptophan oxidation by hydrogen peroxide in the presence of terpositive lanthanide ions (Eu(III), Tb(III), Dy(III), and Gd(III)) in aqueous solutions at pH ∼ 6.The kinetic curves of CL decay in these systems are presented in Figure 2 The concentration of Tb(III) and Eu(III) ions was 1 ¡ 10 3 mol/l, Dy(III) ions was 6 ¡ 10 3 mol/l, and the initial concentration of being introducing into the solution under investigation.This CL emission intensity decayed to the baseline in 5 min.The presence of Gd(III) ions in this system caused no changes, while the presence of Tb(III), Dy(III), or Eu(III) resulted in an increase of CL intensity.The latter ions also lead to considerable lengthening of the duration of the CL (up to 2 hours).The kinetic curves of CL decay were independent of the presence of the Ln(III) ion introduced.They all have a similar time dependence, that is, the same maximal intensity at the moment of the reaction initiation.Moreover, the measurements indicated that changes of pH in the range from 5.5 to 6.6 did not influence the CL intensity.Spectral analysis of the chemiluminescence in the systems studied was performed using cut-off filters as described earlier [8].Because of the very low CL intensity of the reaction mixture tryptophan-H 2 O 2 Fe(II), its spectral distribution could only be measured using a 10-fold higher concentration of tryptophan and H 2 O 2 than in the systems containing Ln(III) ions.The spectral distribution of the system tryptophan-H 2 O 2 Fe(II) shows a broad emission band in the range of 430-520 nm with a maximum at ∼460 nm (spectrum in, Figure 3(a)).This spectrum is consistent with the fluorescence spectrum of N-Formylkynurenine [9], which is presented in a series of publications as a product of tryptophan oxidation by H 2 O 2 [5,6,10].An identical spectrum was also recorded for this reaction mixture containing additionally Gd(III) ions.However, in the systems tryptophan-H 2 O 2 Fe(II) Eu(III), tryptophan-H 2 O 2 Fe(II) Tb(III), and tryptophan-H 2 O 2 Fe(II) Dy(III) recorded 3 min after introduction of H 2 O 2 , the CL spectra show, in addition to this band (λ max ∼ 460 nm), the luminescence bands characteristic of the Ln(III) ions with maxima at λ ∼ 600 nm for Eu(III), 550 nm for Tb(III), and 580 nm for Dy(III), respectively [11] (Figure 3).The CL spectra recorded at longer times following the introduction of H 2 O 2 show only the bands characteristic for lanthanide(III) ions, which indicates that the long-lived CL occurring in the systems of tryptophan-H 2 O 2 Fe(II) Ln(III) is due to the Ln(III) emission.
In order to determine the role of the Ln(III) ions in the reaction mixtures, measurements were made of the fluorescence decays of tryptophan in solutions with and without Ln(III) ions.In addition, studies were made of chemiluminescence in systems containing the same initial concentrations of tryptophan and H 2 O 2 and various concentrations of the Ln(III) ions.In aqueous solutions, tryptophan emits fluorescence with a maximum at λ ∼ 360 nm [6].For all of the reaction systems, tryptophan-H 2 O 2 Fe(II) and tryptophan-H 2 O 2 Fe(II) Ln(III), the same fluorescence decay of the tryptophan band was observed after addition of H 2 O 2 (Figure 4).A very strong tryptophan emission decay was observed in the time period ∼100 after the reaction initiation.Following this initial time period, the tryptophan emission decay was much slower as seen in Figure 4.The fluorescence decays obtained for tryptophan in the reaction mixtures are in agreement with the kinetics of the CL decays in these systems.
In the reaction mixtures of tryptophan-H 2 O 2 Fe(II) Ln(III), an increase in the emission intensity with an increase in the Ln(III) concentration was observed; a particularly striking increase occurred in the case of Tb(III).The maximal value of the light-intensity sum in the system was observed for the molar ratio tryptophan: Tb(III) = 1 : 40 (Figure 5).A similar tendency was found for Dy(III) ions.However, in the case of the tryptophan-H 2 O 2 Eu(III), an increase of the CL intensity was observed up to the molar ratio Eu(III): tryptophan = 1 : 1.However, increases in the Eu(III) concentration did not influence the CL intensity.The spectral distributions of the CL from the systems contained the bands characteristic of the trivalent lanthanide ions.This observation suggests that the CL intensity increases were related to the emission of the Ln(III) ions.However, independent of the concentrations of the Ln(III) ions, the CL decay profiles were the same as in the fundamental International Journal of Photoenergy  system.The lack of an influence of the Ln(III) ions on the decay of the tryptophan fluorescence band following addition of H 2 O 2 and the observation that the CL kinetic curves of the systems with and without Ln(III) ions are of the same shape independent of the Ln(III) concentration prove that the emission from these systems is related to the H 2 O 2 oxidation of tryptophan and that the kinetics of this process are independent of the Ln(III) ions.Aminoacids form complexes with the Ln(III) ions in aqueous solutions, independent of the metal: ligand ratio, but mainly ML species are created [12][13][14][15].Furthermore, in systems generating excited carbonyl groups (decay of dioxoethans) [16] in the presence of either uncomplexed Eu(III) and Tb(III) ions, excitation of Ln(III) was achieved only in the case of Tb(III).These two observations from the literature [12][13][14][15][16] suggest that the differences observed in our luminescence intensities between Tb(III) and Eu(III) ions are likely the result of energy transfer only to complexed Eu(III)-tryptophan ions in the system containing Eu(III).
In order to confirm the role of Eu(III)-tryptophan.complexes in the observed chemiluminescence and studies using tryptamine were carried out.Tryptamine contains in its structure an imidazole ring (similar to tryptophan), which is sensitive to oxidation by H 2 O 2 .However, in solutions of pH < 9, tryptamine does not form complexes with the Ln(III) ions because the amino group of tryptamine is protonated.The system tryptamine-H 2 O 2 Fe(II) shows very low emission intensity which is also of short duration.Introduction of Eu(III) ions into this reaction mixture did not cause any changes, but the presence of Tb(III) resulted in an increased emission intensity characteristic of the Tb(III) ions.Lack of Eu(III) emission in solutions of tryptamine-H 2 O 2 Fe(II) Eu(III) supports the notion that energy transfer from the excited (C=O) £ groups to Eu(III) takes place in the complexes of Eu(III)-tryptophan.In contrast to Eu(III) systems, there was excitation of uncomplexed Tb(III) ions in the system tryptamine/H 2 O 2 .This observation along with the observations of maximal emission intensities in the tryptophan-H 2 O 2 Fe(II) Ln(III) (Ln = Tb, Dy) systems even when there was a considerable excess of Tb(III) and Dy(III) relative to tryptophan prove that energy was being transferred from the excited carbonyl groups to the uncomplexed Tb(III) and Dy(III) ions.The significantly lower emission intensities seen in the systems with Dy(III) compared to the ones with Tb(III) are related to the differences in the intrinsic emission quantum yields of these two ions.
In order to confirm that excited organic molecules (C=O) £ participate in the energy transfer process, studies using the Eu(III)/TTA chelate were carried out.β-diketonate complexes of Ln(III) ions, among them TTA, are widely utilized in studies involving interactions between excited organic ligands and metal complexes [17][18][19][20].A good energy matching between the triplet states of TTA (∼20300 cm 1 [21]) and excited products of the dioxane decomposition enables an efficient triplet-triplet energy transfer.The excitation of europium is the result of the intermolecular energy transfer (triplet-triplet) from the excited (C=O) £ groups to the ligand TTA (complexed with Eu), and consequently the intramolecular energy transfer to Eu(III) [4].Introduction of this chelate into the fundamental system, as presented in Figure 6, stimulated an increase in the CL intensity.The emission intensity in a solution containing the Eu/TTA complex was 70 times higher than in a system containing the same concentration of Eu(III) concentration, and it was 30 times higher than in a solution with Tb(III) ions.Taking into consideration the results of our previous studies in which Eu/TTA was used as an effective sensitizer of the H 2 O 2 decay in a strongly alkaline solution [22], studies were carried out in the Eu(III)/TTA-H 2 O 2 system at pH of ∼6.In these experiments the complex concentrations ranged from 5 ¡ 10 5 to 3 ¡ 10 4 mol/l.The CL intensity could not be detected for lower concentrations of Eu(III)/TTA, and, even for the concentration of 3 ¡ 10 4 mol/l, the CL intensity was very weak.After introducing tryptophan (2 ¡10 4 mol/l) into these systems, the CL intensity increased by two orders of magnitude.A linear dependence was observed for the CL intensity of the system tryptophan-H 2 O 2 Eu(III)/TTA as a function of the tryptophan concentration.In these experiments the initial concentration of hydrogen peroxide and chelate was held constant while the tryptophan concentra-tion varied between 5 ¡ 10 6 and 1 ¡ 10 4 mol/l.The presence of Eu(III)/TTA complex in these reaction mixtures was confirmed on the basis of its absorption band at λ max =∼350 nm [21][22][23].The absorption spectra of the reaction mixture before and after introducing H 2 O 2 were similar.Because the 350 nm absorbance of tryptophan and H 2 O 2 is very small in the concentration ranges used, obtaining comparable absorption values for this wavelength in the mixture after the reaction proves that the Eu(III)/TTA concentration is constant during this process.
The mechanism for the oxidation of tryptophan using hydrogen peroxide or peroxynitrous acid ONOOH has been discussed in several papers, and it can be presented as in Scheme 2 [6,24].
The mechanism describing the processes under study is proposed and presented in Scheme 3.This scheme takes into consideration the tryptophan-oxidation mechanism in Scheme 2, and our chemiluminescence results where emissions were observed with Eu(III), Tb(III), and Dy(III) but none was seen with Gd(III), which has a high lying emitting level (∼32000 cm 1 ) that is located above the singlet and triplet states of most organic ligands.

CONCLUSIONS
In this work ions of Eu(III), Tb(III), Dy(III), and Gd(III) were used for the studies of chemiluminescence accompanying the reaction of tryptophan oxidation by hydrogen peroxide in solution at pH 6.Strong chemiluminescence intensities characteristic of Eu(III) and Tb(III) ions in the systems of tryptophan-H 2 O 2 Fe(II) Ln(III) compared to the emission from the system tryptophan-H 2 O 2 Fe(II) prove that the Ln(III) ions play a role as energy mediators of the CL accompanying tryptophan oxidation by hydrogen peroxide.
In the case of Eu(III), the energy transfer process from the excited carboxylic groups to the Eu(III) ion occurs within the Eu(III)-tryptophan complex (pathway B in Scheme 3) whereas, in the system with Tb(III) ions, energy transfer also occurs with the involvement of uncomplexed Tb(III) ions (pathways B and C, Scheme 3).
Scheme 3: Processes occurring in the reaction system of tryptophan-H 2 O 2 Fe(II) Ln(III).