Anti- and Pro-Oxidant Effects on the Fenton

Introductionby Ryan Hutcheson, Mark D. Engelmann, and I. Francis Cheng.

ExperimentalAll cyclic voltammetric experiments were conducted on a Bioanalytical Systems CV-50W (West Lafayette, Indiana). The reference electrode was a Ag/AgCl, and the working electrode was a glassy carbon disk polished with an aqueous slurry of 1 m alumina powder between each voltammogram to ensure reproducibility.Stock solutions of 100mM ferric nitrate; 100mM ferrous sulfate; 10mM ferric nitrate and ethylenediaminetetraacetate, tetrasodium salt (EDTA); 10mM ferrous sulfate and EDTA; 100mM HEPES buffered to pH of 7.4 to 7.5; 228mM hydrogen peroxide; and 10mM zinc nitrate. The iron solutions were acidified with two drops of nitric acid. The EDTA and ferrous/ferric solutions were then mixed to give the desired iron to EDTA complex ratio. All stock solutions, with the exception of the ferrous/ferric solutions, were buffered to pH 7.4 to 7.5 with 100mM HEPES. The FeIII:EDTA and zinc nitrate solutions were diluted to 1mM for the electrocatalytic runs.Ferric samples: 1mL of 10mM FeIII:EDTA (1:10), various mixtures of 2x DI water and 100mM Ca(NO3)2 totaling 1mL, and 8mL of HEPES buffer.Electrocatalytic samples: 1mL of 1mM Fe(III):EDTA (1:10), various mixtures of 2x DI water and 10mM Ca(NO3)2 totaling 1mL, 1mL of hydrogen peroxide, and 7mL of HEPES buffer (less buffer to ensure same volume).

Literature concerning the two physiologically vital metal ions Ca2+ and Zn2+ generally indicates that zinc has antioxidant properties while calcium is often implicated as a pro-oxidant. However, both metals are not redox active. The effects of calcium and zinc on the Fenton reaction may explain these observations. The Fe[III]EDTA induced electrocatalytic reduction of hydrogen peroxide provides a tool for the investigation of the effects of Ca2+, Zn2+ in the presence of excess EDTA on this iron complex. A previous investigation demonstrated that Fe[II]EDTA is only Fenton active at low [L]:[M] ratios (~1:1)‡. At higher [L]:[M] ratios, no catalytic voltammetric reductive wave is observed. Calcium acts as a pro-oxidant by sequestering excess EDTA effectively reducing the [L]:[M] ratio for EDTA to iron to the pro-oxidant 1:1 case. Speciation calculations indicate that the CaEDTA complex, = 10.65, is not great enough to displace the Fe(III)EDTA complex, = 25.1, even at high calcium to EDTA ratios. In the case of Zn, speciation calculations and cyclic voltammetric studies reveal that the OH- competition for Fe(III) becomes important and Fe(III) is displaced by Zn.‡Robert T. Bobier, Mark D. Engelmann, Terrance Hiatt, and I. Francis Cheng* “Variability of the Fenton Reaction Characteristics of the EDTA, DTPA, and Citrate Complexes of Iron” Biometals, 2003 16: 519–527

Figure 3: Proposed Pro-oxidant Mechanism

Log β1 Ca+2 Zn+2 Fe+2 Fe+3

Ethylenediaminetetraacetic Acid (EDTA) (HOOCCH2)2NCH2CH2N(CH2COOH)2

10.65 16.5 14.32 25.1

Nitrilotriacetic Acid (NTA) (HOOCCH2)3N 6.3 10.45 8.9 15.9

Citric Acid

HOOCCH2CH(OH)(COOH)-CH2COOH

3.45 4.27 4.4 11.5

Table 1: Stability Constants for various M:L complexes

Stability constants () for Ca+2, Zn+2, Fe+2, and Fe+3 with EDTA, NTA, and Citrate. Values were obtained from the NIST Critically Selected Stability Constants of Metal Complexes Database V 6.0

Fe-Enzymes

Oxidative Burst

+ H2O2

Fe-Enzymes + HO.

Free Fe + apoenzymes

+ Excess Low-MW LigandsHigh [L]:[Fe] ratiosFe-ComplexesLow Fenton Reaction Activity

+ Ca2+

1:1 [L]:[Fe] ratiosFe-ComplexesHigh Fenton Reaction Activity

+Ca-L complexes

Schematic of the role of calcium in the oxidative burst mechanism

The Fenton Rxn Kinetics is Highly Dependent on [ligand]:[Fe]

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ent

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00

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Ratio Fe:EDTA

Curr

ent

uA

Figure 2: Effect of excess ligand on Fe:L Fenton reactivity

A

B

C

Maximum currents of various Fe:L ratio’s. A) 1mM Fe:DTPA, B) 1mM Fe:Citrate, C) 1mM Fe:EDTA1

-0.000015

-0.000005

0.000005

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0.000025

0.000035

0.000045

0.000055

0.000065

0.000075

-700-500-300-100100300

Potential (mV)

Cu

rren

t (A

)

Cyclic Voltammagram of A) 10mM FeIIIEDTA (1:1), B) 25mM H2O2, and C) 25mM H2O2 in the presence of 0.1mM FeIIIEDTA. =10 mV/S

Figure 1: Effect of FeIII/IIEDTA on H2O2 reduction.

B

A

C

The sequence of reactions that give rise to the amplified electro-reduction wave (curve c) is due to:

Step 1(electrochemical reduction):E: Fe[III]EDTA + e- Fe[II]EDTA

Step 2 (Fenton Rxn):C’: Fe[II]EDTA + H2O2

Fe[III]EDTA + HO- + HO.

The Effect of Excess Ligand on Fenton Reaction Kinetics.

The following three curves taken from a previous study indicates that the presence of excess ligand (A:EDTA, B:citrate, C:DTPA) slows down the Fenton Reaction kinetics.

We believe that this mimics physiological conditions where10-12 < [Fe]chelatible <10-6. Relative to available binding sites [L]>>[Fe2+/3+]

Ca2+ release is part of the inflammatory response, which is observed to be pro-oxidant. We hypothesize that Ca2+ uptakes the excess binding capacity. The proposed scheme is presented in Figure 3.

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A B C D E

Cu

rren

t (

A

)

Electrocatalytic H2O2 reduction currents measured at –700 mV v. Ag/AgCl.Sweep Rate = 10 mV/s. All solutions contain 100mM HEPES buffer and 22.8mM

H2O2.

Figure 4: Catalytic reduction of H2O2 in different environments

0.1mM FeIII:EDTA (1:1)

0.1mM FeIII:EDTA (1:10)

0.1mM FeIII:EDTA:Zn+2 (1:1:1)

0.1mM FeIII:EDTA:Ca+2 (1:1:10)

Blank no metal-ligandcomplex.

The ligands used in this study are shown in table 1. They were selected for their well-known binding constants and electrochemical characteristics. They were also selected as suitable models for physiological binding sites for Ca2+, Fe3+/2+, & Zn2+. All three metal ions prefer hard binding sites, i.e RCOO-, amines, & phosphates. Indeed the LMW pool of ligands in physiological systems consists primarily of proteins, glutamate, nucleotides.

Ligands Used in This Study

Results and DiscussionFigure 4 demonstrates that the Fenton Reaction kinetics of the 1:1 [EDTA]:[Fe] complex (col. C) is reduced to nearly the background (col. E). On the other hand, the presence of Ca2+ (col. D) increases the Fenton Reaction kinetics of the slow 10:1 [EDTA]:[Fe] complex (col. B) to near that of the 1:1 complex (col. A), see also Figure 9.

0.00E+00

5.00E-09

1.00E-08

1.50E-08

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pH

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cent

ratio

n (M

)

behavior of Zinc and Calcium Reactivity of Iron Complexes

Department of Chemistry, University of Idaho, Moscow, ID 83843, ifcheng@uidaho.edu, 208-885-6387

ReferencesBobier, R., Engelmann, M., Cheng, I.F., “Variability of the Fenton Reaction Characteristics of the EDTA, DTPA, and Citrate Complexes of Iron”, Biometals, 2003 16: 519–527

Gans, P., Sabatini, A., Vacca A., “Investigation of equilibria in solution: Determination of equilibrium constants with the HYPERQUAD suite of programs”, Talanta, 43:1739 (1996)

Karck M, Tanaka S, Berenshtein E, Sturm C, Haverich A, Chevion M, “The push-and-pull mechanism to scavenge redox-active transition metals: a novel concept in myocardial protection.” J Thorac Cardiovasc Surg. 2001 Jun;121(6):1169-78.

ConclusionsResults show Ca to behave as a pro-oxidant by sequestering excess ligand when L:Fe ratios are high; restoring the Fenton active complex ratio.The pro-oxidant role of Ca+2 in the oxidative burst mechanism appears to be supported by the data.Speciation calculations are supportive of mechanism but are not conclusive. Speciations are based on macro stability constants, knowledge of micro-stability constants could give further insight to kinetic barrier of Fenton reaction.

Acknowledgements

University of Idaho Chemistry Department, Malcolm and Carol RenfrewNational Institutes of HealthEveryone in the Cheng research group.

Voltammetric measurements of Fenton active Cu complexes in the presence of Zn. This would help give insight into the role of Zn as a control for biological oxidations in Cu-Zn SOD.4 Preliminary speciation calculations show Zn to behave the same toward Cu as it does toward Fe. Voltammetric measurements of Fenton active Cu and complexes in the presence of Ca. This may give insight to oxidative DNA damage. Speciation calculations show Ca to behave in much the same way towards Cu as it does for Fe.Voltammetric and spectrophotometric measurements of Heme based molecules and polypeptides. Correlation between current studies and biological chemicals is an important step in understanding metal based oxidations in biological systems.

Future Work

Speciation diagram of 5nM FeII:EDTA(aq) (1:10)in the presence of 50M Ca+2(aq).

Insoluble species are omitted due to slow kinetics. The red line indicates physiological pH(7.4). Diagram produced with Hyperquad Simulation and Speciation5.

Figure 6: Speciation diagram of 1:10 FeII:EDTA in the presence of excess Ca+2

0.00%

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Ca:L ratio

%E

C'm

ax

EDTA

Citrate

NTA

Figure 9: Effect of Ca on Fenton reactivity in the presence high (10:1) [EDTA]:[FeIII] ratios.

[EDTA] = 1mM; [Fe3+] = 0.1 mM. At this ratio Fe[III]L complexes are Fenton Reaction inactive. Currents are reported as percentages of maximum EC’ current at low L:M ratios. All currents are measured at –700 mV (Ag/AgCl). 100% represents the measured electrocatalytic current measured for 1:1 [EDTA]:[FeIII], 1:1 [citrate]:[FeIII], & 2:1 [NTA]:[FeIII]. These are ratios that are optimal for Fenton Reaction kinetics.

Speciation diagram of 5nM FeIIIEDTA(aq) (1:10) in the presence of 50M Ca+2(aq).

Insoluble species are omitted due to slow kinetics. The red line indicates physiological pH(7.4).Diagram produced with Hyperquad Simulation and Speciation5.

Figure 5: Speciation diagram of 1:10 FeIII:EDTA in the presence of excess Ca+2

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cent

ratio

n (M

)

CaEDTA

H2EDTA

FeIIIEDTA [Fe(OH)4]-1Fe(OH)3

FeIII(OH)EDTA

HEDTA

CaHEDTA

H3EDTA

H4EDTA

H5EDTA

FeIIIHEDTA

CaEDTA

FeIIEDTA FeII(OH)2EDTA

FeII(OH)EDTA

[Fe(OH)]+

HEDTA

CaHEDTA

H2EDTA

H3EDTA

H4EDTA

H5EDTA

-6.00E-06

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Potential (mV)

Cu

rren

t (A

)

Figure 8: Effect of excess ligand on Fe(III)/(II) reduction current and potential

Cyclic Voltammagram of A) 1mM FeIIIEDTA (1:1) and B) 1mM FeIIIEDTA (1:10). Solutions were buffered at a pH of 7.4 to 7.5 with 100mM HEPES and a scan rate of 10mV/s was used.

A

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FeII(OH)2EDTA

[Zn(OH)4]-2

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cent

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Fe+2

ZnEDTA

ZnHEDTA

FeIIEDTA

FeII(OH)EDTA

Zn(OH)EDTA

[Zn(OH)3]-

[Fe(OH)3]-

Zn(OH)2

Fe(OH)+

Fe(OH)2Zn(OH)+

Speciation diagram of 5nM FeIIEDTA(aq) in the presence of 5nM Zn+2(aq). Insoluble species are omitted due to slow kinetics. The red line indicates physiological pH(7.4). Diagram produced with Hyperquad Simulation and Speciation5.

Zn+2

H4EDTA

H2EDTA

H3EDTA

H5EDTA HEDTA

Figure 7:Speciation diagram of 1:1 FeIII:EDTA in the presence of Zn+2

Figure 8 – The presence of excess ligand does not have an effect on the Fe+3/Fe+2 redox potential but has a siginificant effect on the catalytic reduction of H2O2 by the iron center. Suggesting that there is a kinetic barrier to H2O2 reduction as opposed to a thermodynamic barrier.

This kinetic barrier is most probably a cooperative coordination of the iron sphere by the excess ligand, preventing the interaction between the iron and hydrogen peroxide. This barrier is alleviated by the addtion of Ca+2.

Figures 5 & 6 – Speciation calculations generated from thermodynamic binding constants. The additon of Ca2+ beyond an equivalence of the ligand does not interfere with the formation of the Fenton reactive FeEDTA complex. This is due to weak binding of the ligand by Ca2+. On the other hand, Ca2+ takes up excess EDTA and allows for increases in Fenton Reaction kinetics (see also figure 4).

Figure 7 – In the case of Zn2+, there is a displacement of the iron center by zinc and a subsequent loss of the pro-oxidant FeEDTA complex. This transition is aided by the hydrolysis properties of iron. Thus displacement occurs despite the higher apparent EDTA binding constant for Fe3+ over Zn2+.

Figure 9 – Increases in [Ca2+]:[Fe3+] ratios demonstrates that higher values are able to recover the Fenton Reaction kinetics of the 10:1 [EDTA]:[Fe3+] complex (see B in figure 4)

Metal-Ligand pH Speciation Considerations.