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DiMarco End of semester Communication

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DiMarco End of semester Communication

  1. 1. ElectrochemicalStudies of the Coordination of Antioxidant Molecules with Iron and Copper Kayla DiMarco, Matthew Zimmerman Clemson University May 1, 2013 ABSTRACT: Reactive oxidative species (ROS) often created through the Fenton Reaction is a well-known cause of oxidative damage to DNA. Cell death and mutation contribute to many diseases as well as aging. Antioxidants containing sulfur and selenium can prevent this through various mechanisms. Glutathione (GSH), L-Cysteine, and Methionine were all effective antioxidants toward DNA damage at low concentrations.. Cyclic voltammetry (CV) studies became the next step in determining the mechanisms used by these antioxidants. The results showed that the antioxidants were not ROS scavengers and used metal coordination as its mechanism of preventing DNA damage. These studies further the understanding of antioxidant mechanisms. Introduction Figure 1 The Fenton Reaction [2-4] The Hydroxyl radical produced by the Fenton reaction is a powerful oxidant, which can destroy DNA and lead to disease and aging. In fact, the metal mediated *OH formation is the leading cause of cell death. [5-6] The Fenton reaction generates a radical incredibly close to DNA through the oxidation of iron(II) or copper (I) then the reduction of hydrogen peroxide, as depicted in figure 1. The Fenton reaction as it occurs in living organisms can only run when in the range of -160 mV to 460 mV (at a pH of 7). [1] One possible mechanism of sulfur and selenium antioxidant activity may be caused by an ability to shift the oxidation (or reduction) of iron and copper out of this range. Results and Discussion Combined copper or iron in metal:ligand samples. For example, copper (which has 4 binding sites) was combined with methionine (Met) in 600 micromolar increasing concentrations. 600 micromolar Copper was paired with 600 micromolar Met, then 1200 micromolar met, then 1800 micromolar Met and so on until reaching a 1:5 ratio. Iron was combined with all three ligands in this same way. To keep the samples in as close to biological conditions as possible, the samples were treated with buffers (MOPS for copper and MES for iron) which kept the solution at pH 7. Each sample was prepared as quickly as possible to ensure that they did not oxidize or reduce during the process of prepping the samples (in other words, each sample was made completely within 45 minutes). Parameters for CV Initial 1 V High 1 V Low -1 V Polarity negative Scan rate .1s Sleep 4s Interval .001 V Quiet time 5s Sensitivity 1e -005
  2. 2. Electrochemical Results Figure 2 L-Cysteine and copper Epa = 0.387 V CuI/II Figure 2 Figure 3 L-Cysteine and iron Epc = -0.257 V FeII/III The Gel electrophoresis studies conducted previously by other members of the team determined that Cysteine and methionine prevent DNA damage through copper coordination. They used [Cu(bipy)2]+ to fully coordinate with copper and measured the effectiveness of the antioxidants if they were unable to coordinate. The results showed that they could not even in high concentrations. When [Fe(EDTA)]2- was studied, the results showed that despite the inability of the ligands to coordinate with the iron, there was still damage prevention, although significantly less. They suspected that at higher concentrations, the DNA inhibition was most likely be due to ROS scavenging. -1.0 -0.5 0.0 0.5 1.0 -0.00004 -0.00002 0.00000 0.00002 0.00004 0.00006 0.00008 Current,I(A) Potential, E(V) -1.0 -0.5 0.0 0.5 1.0 -0.000015 -0.000010 -0.000005 0.000000 0.000005 0.000010 0.000015 Current,I(A) Potential, E(V)
  3. 3. 3 For Glutathione, the group determined through DNA damage assays that its antioxidant activity is not attributed to GPx activity. Based on the results, metal-antioxidant binding was the primary mechanism and ROS scavenging was a secondary mechanism for iron-mediated damage. [7] The results of the L-Cysteine on copper and iron are depicted in figures 2 and 3. These results were similar for all 3 ligands. Even with the addition of the ligands and high concentrations, oxidation of each of these metals is within the range for the Fenton reaction to still occur. There was a slight shift of no more than .05 V from the oxidation of iron or copper without the ligands. This shows that there was still binding of the ligands, but not a significant amount. The previous studies suspected that the ligands may participate in ROS scavenging. When measuring the electrochemical activity of the ligands, they showed that they were not electrochemically active. This prevents them from being likely candidates for ROS scavenging. Conclusions The gel studies showed that the antioxidants studied were effective at preventing DNA damage. The CV studies show that ROS scavenging was not the reason for this. Metal coordination would be the likely antioxidant mechanism for methionine, glutathione, and L-cysteine. References [1] Pierre, J. L.; Fontecave, M. Biometals 1999, 12, 195-9. [2] Rai, P.; Wemmer, D.; Linn, S. Nucleic Acid Res. 2005, 33, 497-510. [3] Bar-Or, D.; Thomas, G. W.; Rael, L. T.; Lau, E. P.; Winkler, J. V. Biochem. Biophys. Res. Commun. 2001, 282, 356-60. [4] Rae, T. D.; Schmidt, P. J.; Pufahl, R. A.; Culotta, V. C.; O'Halloran, T. V. Science 1999, 284, 805-8. [5] D. Bar-Or, G. W. Thomas, L. T. Rael, E. P. Lau and J. V. Winkler, 
 Biochem. Biophys. Res. Commun., 2001, 282, 356–360. [6] S. Park and J. A. Imlay, J. Bacteriol., 2003, 185, 1942–1950. [7] Metallomics, 2011, 3, 503–512

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