Determination of the Stoichiometry of Complex Formation Between Transition Metal Ions and Tyrosine Using UV Absorption Spectrophotometry

Samantha Harriss

Abstract

            L-tyrosine and various transition metals are vital to biological functions.  The coordination of L-tyrosine and Fe(II), Cu(II), Cd(II), and Zn(II) was investigated by ultraviolet absorption spectrophotometry.  Using the mole ratio method, a 2:1 complex of Cu(II):tyrosine was reconfirmed and a new 2:3 complex of Cu(II):tyrosine was proposed.  Fe(II) ions seemed to complex to tyrosine in the molar ratios of 2:1 and 2:3 Tyr:Fe(II).  Cd(II) and Zn(II) did not form complexes of spectroscopic sensitivity with tyrosine and were used as controls.  Proposed structures of the Fe(II) and Cu(II) complexes are given.

Introduction

            The amino acid L-tyrosine and various transition metals are important in the biological functions of humans, animals, and plants.  L-tyrosine is one of the twenty major amino acids and is considered an essential amino acid.  This amino acid is synthesized in the human body from phenylalanine and is a direct precursor of various hormones, biogenic amines, and neurotransmitters.  It is used by the thyroid and adrenal glands to synthesize thyroid hormones and adrenaline, respectively [3].  Tyrosine metabolizes to products such as melanin, the pigment found in hair and skin, estrogen, and encephalin.  Tyrosine, a precursor to dopamine, also plays a role in the neurotransmission signaling and regulation of depression.  Other non-human organisms utilize tyrosine in their biological processes.  Sea squirts contain a transport protein, transferrin that contains two tyrosine residues in the terminal lobe that aid in the coordination of vanadium [5].  Much of the tyrosine used in the laboratory is prepared from plants, namely sugar beets.  Other plants such as sweet potatoes contain tyrosine in the active sites of enzymes that catalyze the hydrolysis of phosphoric acid esters and phosphoric acid anhydrides [4].

Inorganic elements like transition metals are vital to the proper functioning of the body�s processes.  Metal ions have the ability to form strong bonds and be stable in more than one oxidation state.  Iron has a major role in biological reactions.  Iron in the blood chelates with protoporphyrin to form heme, a prosthetic group of proteins such as myoglobin, hemoglobin, catalase, peroxidase, and cytochrome c.  It also plays a role in enzyme activity.  Enzymes called oxygenases catalyze the cleavage or degradation of aromatic amino acid rings in biological systems.  For example the degradation of phenylalanine begins with its hydroxylation to tyrosine, a reaction catalyzed by phenylalanine hydroxylase.  The active site of this enzyme contains iron that is not part of heme or an iron-sulfur cluster [1].  Zinc is found only in the 2+ state in biological systems.  It is known to form complexes with amino acids such as L-serine, L-aspartic acid, L-lysine, and L-phenylalanine [8].  In a previous study, a zinc-tyrosine complex was synthesized in the core of encapsulated dendrimers [9].  Copper ions form a complex with tyrosine that inhibits demethlyation and stimulates oxidation of microsomal pigments in hepatic cells [12].  The complex acts as an electron acceptor which stimulates the oxidation process.  Copper ions have also been found to be required in the metabolic transformation of tyrosine [3].  Cadmium is a naturally occurring transition metal found in the earth�s crust.  Although it is naturally occurring in the earth�s crust, it produces toxic effects in humans.  Cadmium accumulates in the kidneys and at high concentrations can cause kidney failure.  The primary route for cadmium intake is ingestion.  Trace amounts are found in food products due to the use of phosphate fertilizers used on agricultural soil.  Cigarette smoking is also a means of cadmium exposure.  Complex formation of cadmium with tyrosine could be a means of elimination of this toxic metal.

Tyrosine has three important components: a carboxylic group, an amino group, and a phenolic group.  It is one of three amino acids with a bulky, uncharged, aromatic side group which gives tyrosine an absorption spectrum that can be observed by ultraviolet and visual spectrophotometry.  The remaining three groups give tyrosine the pK_a values of 2.2 (-COOH), 9.1 (-NH₃⁺), and 10.5 (-OH).  Due to these pK_a values, tyrosine has the ability to exist in the monoprotonated form, HL, over a pH range of 2.7 to 8.5 where neither the phenolic group nor the amino group has completely released their protons.  The carboxylic group is completely deprotonated over this pH range.  In a study by Renzo Carta, the solubility of tyrosine was examined at various pH values from 0.0 to 13.0 in aqueous solutions [2].  The experimental conditions of Carta�s study were similar to other reports because a broad pH range [3, 10, 11] and an aqueous solution [2, 3, 5, 10, 14-16] were used.  Tyrosine concentrations in solution ranged from as low as 10^-6M [11, 15] to a high of 10^-2M [14].

Many studies have been done showing that tyrosine has the ability to covalently bond to transition metals as well as bond noncovalently to alkali metals [3, 4, 10, 11, 13-16].  The majority of the coordination occurs at the amino nitrogen or carboxylic oxygen of tyrosine [10, 13, 14, 16] but is also believed to occur at the phenolic oxygen [13, 16].  The bulk of the tyrosine-metal studies have involved complexes with transition metals (binding ratio M:L): Cu²⁺ (1:2) [10, 15], Y³⁺ (1:1 and 1:2) [14], Hg²⁺ [11], Zn²⁺ [9] and Fe³⁺ [4].  Tyrosine does not directly bond but is involved in the coordination of the transition metal, vanadium [5].  Other studies have included metals from the lanthanide series (binding ratio M:L): La³⁺ (1:1) [14], Ce³⁺ (1:1) [14], and Eu³⁺ (1:2) [16].  Victor Ryzhov et al. performed a unique study of the ability of tyrosine to noncovalently bond with alkali metals Na⁺ and K⁺ [13].  However no studies have reported findings concerning the direct bonding of tyrosine with Fe²⁺, Cd²⁺, which are two of the four ions used in this study.

            Tyrosine-metal complexes have been studied using a wide variety of techniques.  Some analytical techniques used include voltammetry [15], amperometry [11], and potentiometry [3].  Thermodynamics and kinetics [13] were also utilized to study the complexes.  Spectrophotometric techniques applied included FT-IR [16], H-NMR [3, 16], C-NMR [16], and UV-Vis [3, 16].  From the results of these techniques, information such as the binding molar ratios [14-16], protonation constants [10, 13, 14], stability constants [3, 14], and formation constants [5, 10, 11, 13] were determined.

            In this study, ultraviolet-visible spectrophotometry is used to determine the stoichiometry of complex formation between tyrosine and the transition metal ions Fe(II) and Cu(II).  There are four types of transitions between quantized energy levels that are responsible for the UV-Vis spectra: σ→σ*, n→σ*, π→π*, and n→π*.  The two most important transitions are π→π* and n→π* because they involve functional groups that are characteristic of the analyte.  Tyrosine�s aromatic group contains mobile π electrons which correspond to the π→π* transition.  This transition has a characteristic wavelength range of 200-500nm, where the wavelength 274nm is specific to tyrosine.  Several previous studies have utilized UV-Vis spectrophotometry to measure tyrosine complexation with Al(III), Cu(II), and Eu(III) ions [3, 10,16].  Since no new peaks were formed by the complex, the reaction was observed by the enhancement of tyrosine�s absorption at 274nm.  This indicates that the complex absorbs at the same wavelength as tyrosine.

Analysis of the enhancement of the absorption was carried out via the mole ratio method.  One reactant is kept constant, while the moles of the other reactant are varied.  The absorbance is measured at a wavelength at which the metal-ligand complex absorbs and is then plotted against the ligand-to metal mole ratio.  The mole ratio corresponding to the intersection of the linear segments indicates the formula of the complex.  This method is also useful for reactions that occur in a stepwise fashion [6].  The mole ratio method was used by Xu and Chen [16] and was also used in this study in which tyrosine was held constant while varying the moles of metal ion.  The absorbance of the complex was measured at 274nm.

Experimental

            Reagents and solutions:

            Stock solutions of 1mM FeCl₂ (Fisher), CdCl₂ (Aldrich), ZnCl₂ (Natural Science), and CuCl₂ (Alfa Aesar), and 0.5mM tyrosine (Aldrich) were prepared by directly dissolving the required amount of substance in distilled water.  All solutions were stored in a refrigerator.

            Apparatus:

            All spectra were obtained on a SP-2000UV UV-Vis spectrophotometer.  Only the ultraviolet wavelength range (200-400nm) was utilized.

            Procedure:

            The absorbance spectrum of tyrosine was scanned from 200nm to 300nm to ensure that the peak absorbance was found at 274nm, which is specific for tyrosine.  The absorbance spectra of the stock solutions of transition metal salts were scanned from 260nm to 280nm to check that they did not absorb at 274nm.  Twenty-one sample solutions were made by adding from 0mL to 2.0mL of 1mM metal salt solution in 0.1mL increments to 2.0mL of 0.5mM tyrosine and diluting to a total of 4.0mL.  All solutions were diluted with distilled water.  The solution�s absorbance was then measured at 274nm with the UV spectrophotometer.  All experiments were carried out at room temperature.

Data and Results

Figure 1 shows the absorption spectrum of 1mM tyrosine; the peak specific to tyrosine is located at 274nm which is consistent with its literature value.  The absorption spectra of FeCl₂, CdCl₂, ZnCl₂, and CuCl₂ stock solutions are given in figure 2.  The stock solutions showed no significant absorbance.

Upon plotting the spectroscopic data, the following absorbance vs. mole ratio curves were obtained for the Tyr-Fe(II), Tyr-Cd(II), Tyr-Zn(II), and Tyr-Cu(II) solutions. 

            Figures 3 and 4 show the absorbance vs. mole ratio curves for Tyr-Cd(II) and Tyr-Zn(II), respectively.  These two graphs basically show a straight line of constant tyrosine molecule absorption; the absorption range is only a slight 0.01 difference, as expected.  Therefore, Cd(II) and Zn(II) act as appropriate controls for this experiment.

Figure 5 illustrates the complex formation curve for tyrosine and Fe(II) ions.  The absorbance gradually rises from 0 to 0.5 mole ratio of Fe(II):Tyr, followed by an interval of relatively constant absorbance.  The gradual increase in absorbance up to 0.5 mole ratio corresponds to a complex formation between two tyrosine molecules and one Fe(II) ion.  Another gradual increase in absorbance between the mole ratios of 1.0 and1.5 followed by a plateau is also observed.  This similar behavior indicates another complex formation with two tyrosine molecules and three Fe(II) ions at higher Fe(II) concentrations.  Comparing the absorbance increases of the two mole ratios shows that the 2:1 Tyr:Fe(II) and 2:3 Tyr:Fe(II) complexes, correspond to a 0.04  and 0.02 increase in absorbance, respectively.  The higher increase in absorption for 2:1 complex implies a greater amount of the 2:1 complex formation.

The complex formation curve for tyrosine and Cu(II) ions is given in figure 6 and is similar to the tyrosine and Fe(II) formation complex curve in figure 5.  The absorbance in figure 6 rapidly increases from 0 to 0.5 mole ratio and then plateaus.  This increase and leveling off corresponds to two tyrosine molecules coordinating with one Cu(II) ion.  Another increase and leveling occurs at a mole ratio of 1.5 Cu(II):Tyr.  At this mole ratio, a complex of two tyrosine molecules coordinates with three Cu(II) ions at higher Cu(II) concentrations.  The absorbance increases by 0.04 for the 2:1 Tyr:Cu(II) complex, but only increases by 0.02 upon the formation of the 2:3 Tyr:Cu(II) complex which implies a smaller amount of the 2:3 complex formation again.

Conclusion and Discussion

            This study has shown that the complex of tyrosine with Fe(II) and Cu(II) ions can be observed using UV spectrophotometry.  Tyrosine exhibits an absorption spectrum in the UV region because it contains mobile π electrons in its aromatic ring.  Excitation occurs when light energy is absorbed by the electrons in the π bonding orbital causing them to move up to the π antibonding orbital.  A similar effect is taking place in the Cu(II) and Fe(II)-tyrosine complexes.  According to the ligand field theory, coordination of a ligand like tyrosine disrupts the five-fold degeneracy of transition metal�s valence d-orbitals.  When a field of negative charges from a ligand surrounds a metal ion, the symmetry of the field is not spherical and therefore the d-orbital energies split [7].  If the metal ions in figures 7 and 9 are placed in cubes, the four ligands approaching the metal ion from alternate corners form a tetrahedral geometry about it.  The d-orbitals in a tetrahedral symmetry split into two closely spaced degenerate energy levels.  When the metal-tyrosine complex absorbs UV light, the electrons in the lower energy d-orbitals become excited and occupy the higher energy d-orbitals.  In addition to the absorption from the tyrosine molecules, the coordinated metal complex may absorb light at the same wavelength enhancing the detected absorption.  This is the case observed with Cu(II) and Fe(II) complexes in this study.  Cd(II) and Zn(II) do not display an absorption spectrum due to their completely filled d-orbitals.  The absence of an absorption spectrum makes them suitable control metal ions for this study.

Neutral solutions of pH around 7 are desirable for biological systems because they are neither too acidic nor too basic.  The solutions in this study were prepared at pH 7, mimicking a biological pH, where the carboxylic acid and amine groups were significantly deprotonated and the phenolic group was only slightly deprotonated.  This deprotonation allowed for the coordination of the metal ions at these sites.  In tyrosine, as well as other amino acids, the amine and carboxylic acid functionalities take part in the metal coordination forming a stable five-membered ring with the metal ion [9].  Cu(II) ions were previously found to bind with the tyrosine molecule in a 2:1 Tyr:Cu(II) ratio using voltammetry [10, 15].  Results using UV absorption are in agreement with this ratio.  The proposed structure of the 2:1 tyrosine-Cu(II) complex is given in figure 7.  The 2:1 Tyr:Cu(II) complex implies a tetrahedral geometry which is common for transition metal complexes.  However, in this study, evidence for another complex at mole ratio 2:3 Tyr:Cu(II) was also suggested.  The proposed structure of the 2:3 tyrosine-Cu(II) complex is given in figure 8.  As seen in figures 7 and 8, two stable five membered rings are formed between the metal ion, two amino, and two carboxylic groups.  A much smaller amount of complex at 1.5 mole ratio (2:3 Tyr:Cu(II)) is formed in solution compared to the amount of complex at 0.5 mole ratio (2:1 Tyr:Cu(II)).  The small amount of 2:3 Tyr:Cu(II) complex is due to the very small fraction of deprotonated phenolic group due to a high pK_a of 10.5.  This study suggests that at high metal concentrations, the possibility of 2:3 Tyr:Cu(II) complex formation.

Tyrosine molecules were previously found to be ligands for Fe(III) ions [4], however the binding of Fe(II) ions with tyrosine molecules have not been reported.  In this study, two complex formations at the same mole ratios of Cu(II) ions were found for Fe(II) ions.  Fe(II) forms a 2:1 Tyr:Fe(II) complex with tetrahedral geometry.  The structure for the 2:1 complex is shown in figure 9.  A complex formation of 2:3 Tyr:Fe(II) was also found.  The proposed structure is given in figure 10.  Again a stable five membered ring is formed in both complexes.  Similar to Cu(II), the complex formed at 0.5 mole ratio is much greater than the complex formed at 1.5 mole ratio due to the small percentage of deprotonated phenolic group.

The results of this basic study may imply some significance in the metabolism and transport mechanisms in biological systems.  The finding of the binding of tyrosine with Fe(II) ions in the 2:1 Tyr:Fe(II) ratio might help to elucidate the structures of Fe²⁺ containing active sites in enzymes.  Also, the results of this study showed that both Cu(II) and Fe(II) ions coordinated with tyrosine at a 2:3 Tyr:M(II) ratio, which has not been reported for any metal ions studied before.  Tyrosine might take part in the metabolism of excess iron and copper in the blood stream by coordinating with free metal ions, as well as providing a transport system for copper and iron ions.  The results of this study suggest the formation of interesting new complexes that may prove to be of biological importance.
 

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