Short Abstract
The reaction of chromium(VI) with four thiols,
b-mercaptoethanol, dithiothreitol, glutathione, and cysteine at pH 7.0 and 37 °C was studied in order to determine differences in their reactions with chromium(VI). It was shown that the reduction of chromium(VI) by cysteine and glutathione was very fast, and very little chromium(V) was detectable. Reduction of chromium(VI) by b-mercaptoethanol or dithiothreitol, however, proceeded much more slowly, and relatively large levels of chromium(V) were detectable. All chromium(V) species observed displayed EPR spectra indicative of complexes possessing axial geometries. The presence of DNA or chelating agents in reactions involving b-mercaptoethanol resulted in a large increase in chromium(V) concentration seen over time. It was shown that the levels of chromium(V) formed in the presence of the four thiols studied is related to the amount of chromium bound to DNA in the presence of these thiols.The reduction of chromium(VI) by ethanethiol, propanethiol, dimercaptosuccinic acid, and glutathione, typically lead to the formation of thiyl radical, whereas reduction by cysteine and cysteamine led to formation of hydroxyl radical. Reduction of chromium(VI) by ethanethiol, propanethiol, dimercaptosuccinic acid, or glutathione, also led to the formation of chromium(V). Reaction of ethanethiol or propanethiol with chromium(VI) resulted in the formation of very similar chromium(V) species, and the presence of 5,5-dimethyl-1-pyrroline-N-oxide in these reactions altered the relative intensities of the chromium(V) species formed. It has been proposed that this may be due to removal of reactive radical species from the reaction sphere by 5,5-dimethyl-1-pyrroline-N-oxide.
The reaction of bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) or bis(2-hydroxy-2-methylbutyrato)oxochromate(V) with reducing agents such as
b-mercaptoethanol, dithiothreitol, glutathione, oxidized glutathione, thioglycolic acid, glycine, oxalic acid, and ascorbic acid was studied. In non-aqueous solvents where reduction was retarded, reactions between the initial chromium(V) complex and dithiothreitol or thioglycolic acid resulted in ligand substitution, and the formation of mono- or bis-(thiol)chromium(V) complexes.Abstract
The overall goal of this research has been to illuminate the possible fate of chromium(VI) and chromium(V) in biological systems, by studying in vitro reactions of chromium(VI) and chromium(V) with low molecular weight biological and model reductants, as a means of more fully understanding the processes involved in the genotoxicity of chromium. The main tool for this study has been Electron Paramagnetic Resonance Spectroscopy
The reaction of chromium(VI) with four thiols,
b-mercaptoethanol, dithiothreitol, glutathione, and cysteine at pH 7.0 and 37 °C was studied in order to determine differences in their reactions with chromium(VI). These experiments have revealed important structural and rate information on the chromium species formed during these reactions. Specifically, all chromium(V) species observed displayed EPR spectra indicative of complexes possessing axial geometries. It was shown that the reduction of chromium(VI) by cysteine and glutathione was very fast, with most of the chromium(VI) reduced to chromium(III) within 2 minutes, and very little chromium(V) was detectable. Reduction of chromium(VI) by b-mercaptoethanol or dithiothreitol, however, proceeded much more slowly, and relatively large levels of chromium(V) were detectable. The reaction of b-mercaptoethanol with chromium(VI) in the presence of DNA or chelating agents resulted in a large increase in chromium(V) concentration seen over time, vs. reactions of b-mercaptoethanol and chromium(VI) alone. It was shown that the levels of chromium(V) formed in the presence of the four thiols studied is related to the amount of chromium bound to DNA in the presence of these thiols. Dithiothreitol and b-mercaptoethanol produced very high levels of chromium-DNA adducts and also produced high levels of chromium(V), whereas much lower levels of both chromium-DNA adducts and chromium(V) were produced with glutathione and cysteine.The reduction of chromium(VI) by thiols, i.e., ethanethiol, propanethiol, dimercaptosuccinic acid, and glutathione, typically lead to the formation of thiyl (•SR) radical, and in the cases of cysteine and cysteamine to the formation of hydroxyl (•OH) radical. For the thiols ethanethiol, propanethiol, dimercaptosuccinic acid, or glutathione, the rate of the overall chromium(VI)
Æ chromium(III) reduction was slower than for cysteine or cysteamine, and the formation of chromium(V) was observed in the EPR spectra. Reaction of ethanethiol or propanethiol with chromium(VI) resulted in the formation of very similar chromium(V) species, and the presence of 5,5-dimethyl-1-pyrroline-N-oxide in these reactions altered the relative intensities of the chromium(V) species formed. It has been proposed that this may be due to removal of reactive radical species from the reaction sphere by 5,5-dimethyl-1-pyrroline-N-oxide.The reaction of bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) or bis(2-hydroxy-2-methylbutyrato)oxochromate(V) with reducing agents such as
b-mercaptoethanol, dithiothreitol, glutathione, oxidized glutathione, thioglycolic acid, glycine, oxalic acid, and ascorbic acid was studied. It was shown that in most cases reduction occurred before any appreciable ligand substitution on the chromium(V) center took place. However, especially in non-aqueous solvents where reduction was retarded, reactions between the initial chromium(V) complex and dithiothreitol or thioglycolic acid resulted in ligand substitution, and the formation of mono- or bis-(thiol)chromium(V) complexes.Acknowledgments
This thesis is due in large part to Professor Karen Wetterhahn, and I would like to thank her for her guidance through my graduate student years. I am very fortunate to have had her as a mentor and scientific role-model; her knowledge, expertise, and dedication to science is inspiring and is a goal I will have to strive for many, many years to approach.
The Wetterhahn research group, Jayshree Aiyar, Joy Alcedo, Kim Borges, Holly Berkovitz, Andrew Standeven, Josh Hamilton, and Sam Brauer have all been great to work with; Jayshree, Joy and Kim have been especially helpful with biochemistry-related questions, and have taught me not a few techniques.
Most of this work involved EPR spectroscopy, and I would like to give thanks to Wayne Casey for doing a great job keeping the EPR equipment up and running; without his help with the hardware I could not have completed this work. In a similar vein, Professor Dean Wilcox has been especially helpful in answering my questions about EPR spectroscopy in general.
The other members of my committee, Professors Russell Hughes, Gordon Gribble, and Thomas Curphey have given me very helpful and insightful comments through the years. Professor Robert Cantor (Dr. Bob) has been quite helpful with my many computer/programming questions, and also was kind enough to agree to be a substitute on my committee.
I can thank my high school science teacher, Sister Carol Anne Murray, for believing in me and fostering my interest in science. She is the most dedicated teacher I have ever had, and she had the sense to push me to do my best. Thanks for the kicks in the pants when I needed them!
I would like to give special thanks to Kathy Winant who was always there for me, even when she had to bear the brunt of my moodiness during this thesis writing- her support and encouragement has meant a lot.
Finally, I would like to thank my parents, Phillip and Janet Boswell, my brother Eric and his wife Diane, and my grandmother Eveline Pelletier for all their support. Their faith in me has allowed me to come this far.
Table of Contents
Short Abstract ii
Abstract iv
Ackowledgements vi
Table of Contents viii
List of Tables xiv
List of Figures xv
Abbreviations xxix
1. INTRODUCTION 1
1.1. General 1
1.2. Biological Aspects of Chromium 1
1.2.1. Exposure 1
1.2.2. Carcinogenicity and Toxicity 2
1.3. Chromium(VI), d
1.3.1. Reduction of Chromium(VI) 6
1.3.1.1. Cysteine and other thiols 8
1.3.1.2. Metals 9
1.3.1.2.1 Iron(II) 11
1.3.1.2.2 Vanadium(III) and Vanadium(IV) 12
1.3.1.2.3 Arsenic(III) 13
1.3.1.2.4 Molybdenum(IV) 13
1.4. Chromium(V), d
1 151.4.1. Electron Paramagnetic Resonance 16
1.4.2. Reduction of Chromium(V) 18
1.4.2.1. Metals 18
1.4.2.1.1 Polyvalent metal ions 18
1.4.2.1.2 Titanium(III) 21
1.4.2.1.3 Iron(II), Vanadium(IV) 22
1.4.2.1.4 Uranium(IV) 24
1.4.2.1.5 Summary 24
1.4.2.2. Thiols 25
1.5. Goals of this Research 26
2 MATERIALS AND METHODS 28
2.1 Materials 28
2.2 Methods 29
2.2.1 Electron Paramagnetic Resonance Spectroscopy 29
2.2.1.1 Chromium(V) concentration determination. 29
2.2.1.2 Frozen glass studies (77 K) 30
2.2.1.3 Solution studies (297 K) 30
2.2.2 Electron Paramagnetic Resonance Detection of Chromium(V) and Chromium(III) Species Formed During the Reduction of Chromium(VI) by Thiols in the Presence or Absence of DNA 31
2.2.2.1 Synthesis of Na[Cr(L-cys)
2.2.3 Electron Paramagnetic Resonance Detection of Chromium(V) and Radical Species Formed During the Reduction of Chromium(VI) by
b-mercaptoethanol in the presence of chelating (DES, DETAPAC) and spin-trapping (DMPO) agents. 322.2.3.1 Purification of DMPO 32
2.2.4 Electron Paramagnetic Resonance Study of the Reaction of Chromium(VI) with Reducing Agents in the Presence of a Spin-Trap 33
2.2.5 Electron Paramagnetic Resonance Study of the Reaction of Chromium(V) Species with Reducing Agents 33
2.2.5.1 Synthesis of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) 36
2.2.5.2 Synthesis of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) 37
3. Electron Paramagnetic Resonance Detection of Chromium(V) and Chromium(III) Species Formed During the Reduction of Chromium(VI) By Thiols 38
3.1. Results and Discussion 38
3.1.1. Reaction of Chromium(VI) with
3.1.1.1. Discussion 41
3.1.1.2. Reaction of Chromium(VI) with
3.1.1.2.1 Discussion 44
3.1.1.3. Effect of DETAPAC, DES, and DMPO on the Reaction of Chromium(VI) with
3.1.1.3.1 Discussion 52
3.1.1.4. Summary of the Reduction of Chromium(VI) by
3.1.2. Reaction of Chromium(VI) with Dithiothreitol 59
3.1.2.1. Discussion 60
3.1.2.2. The Reaction of Chromium(VI) with Dithiothreitol in the Presence of DNA 62
3.1.2.2.1 Discussion 63
3.1.2.3. Summary of the Reduction of Chromium(VI) by Dithiothreitol 65
3.1.3. Reaction of Chromium(VI) with Glutathione 65
3.1.3.1. Discussion 67
3.1.3.2. Reaction of Chromium(VI) with Glutathione in the Presence of DNA 69
3.1.3.2.1 Discussion 70
3.1.3.3. Summary of the Reduction of Chromium(VI) by Glutathione 71
3.1.4. Reaction of Chromium(VI) with Cysteine 71
3.1.4.1. Discussion 73
3.1.4.2. Reaction of Chromium(VI) and Cysteine in the Presence of DNA 75
3.1.4.2.1 Discussion 76
3.1.4.3. Summary of the Reduction of Chromium(VI) by Cysteine 76
3.1.5. Summary of Reduction of Chromium(VI) by Thiols 77
3.1.6. Comparison of the Chromium(III) Products Formed During the Reaction of Chromium(VI) or Chromium(III) with Thiols 78
3.1.6.1. Chromium(III) vs. Chromium(V) EPR Spectroscopy 78
3.1.6.2. Chromium Reactions with Thiols 80
3.1.7. Correlation of Chromium(V) Formation with Chromium-DNA Binding During the Reaction of Chromium(VI) with Thiols 84
4. Electron Paramagnetic Resonance Study of the Reaction of Chromium(VI) with Reducing Agents in the Presence of a Spin-Trap 167
4.1. Spin Trapping 167
4.2. Results and Discussion 168
4.2.1. Cysteine 168
4.2.1.1. Discussion 169
4.2.2. Cysteamine 172
4.2.2.1. Discussion 173
4.2.3. Ethanethiol 174
4.2.3.1. Discussion 175
4.2.4. Propanethiol 176
4.2.4.1. Discussion 177
4.2.5. Dimercaptosuccinic acid 178
4.2.5.1. Discussion 179
4.2.6. Glutathione 183
4.2.6.1. Discussion 183
4.2.7. Ascorbic Acid 184
4.2.7.1. Discussion 185
4.2.8. Summary 187
5. Electron Paramagnetic Resonance Study of the Reaction of Chromium(V) Species with Reducing Agents 212
5.1. Results and Discussion 212
5.1.1.
5.1.1.1. Discussion 217
5.1.2. Dithiothreitol 218
5.1.2.1. Discussion 222
5.1.3. Glutathione 224
5.1.3.1. Discussion 226
5.1.4. Oxidized Glutathione 227
5.1.4.1. Discussion 227
5.1.5. Glycine 228
5.1.5.1. Discussion 228
5.1.6. Oxalic Acid 228
5.1.6.1. Discussion 230
5.1.7. Ascorbic Acid 231
5.1.7.1. Discussion 232
5.1.8. Thioglycolic Acid 233
5.1.8.1. Discussion 235
5.2. Summary 236
6. Summary of Results and Implications for Chromium(VI) Carcinogenesis 326
Appendix 334
References 344
List of Tables
Table 1-1. Reduction potentials, structures, and second order rate constants (pH 7.4 and 25 °C) for the reactions between chromate and a number of thiols, dithiols, and ascorbate 10
Table 1-2. Reduction potentials for various metal ions discussed in the text 15
Table 1-3. Reduction of chromium(V) chelate [Cr(V)(EHBA)2O]- by various metal ions in aqueous media, pH 2-4. Lig = 2-ethyl-2-hydroxybutyrate, (EHBA). 25
Table 3-1. EPR spectral parameters for the chromium(V) and radical species formed in the reaction of
b-mercaptoethanol (9.6 mM) with chromium(VI) (0.48 mM) in the presence of DNA (0.048 mM), DETAPAC (0.48 mM), or DES (0.48 mM) in 50 mM Tris-HCl, pH 7.0. 53Table 3-2. Chromium(V) complexes formed during the reduction of Chromium(VI) by thiols. 78
Table 3-3. EPR spectral parameters for chromium(III) complexes formed upon reaction of chromium(III)/chromium(VI) with thiols. 83
Table 4-1. EPR signals (297 K) observed during the reduction of chromium(VI) by various reducing agents in the presence or absence of DMPO and in Tris-HCl, cacodylic acid, or unbuffered solutions. 210
Table 5-1. EPR spectral parameters for the chromium(V) signals observed upon reaction of chromium(V) complexes with reducing agents in various solvents. 323
List of Figures
Figure 1-1. The "uptake-reduction" model for chromium(VI) carcinogenicity. 4
Figure 1-2. Structures of hydrogen chromate, HCrO
4-, chromate, CrO42-, and dichromate, Cr2O72- 5Figure 1-3. Diagram showing the predominant chromium(VI) species in aqueous solution at 25 °C and I = 1 M. 6
Figure 1-4. Structure of dimeric molybdenum(V) cation used by Ghosh and Gould for the reduction of chromium(VI) in aqueous solutions 15
Figure 1-5. Structure of chromium(V) chelate of tertiary hydroxy acids. Sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) ([Cr(V)(EHBA)
2O] 16Figure 1-6. Computer-generated (see program EPRsim, Appendix A) representative 77K EPR spectra for chromium(V) complexes with various structural differences. 19
Figure 1-7. Proposed structure of the final chromium(III) species produced by the reduction of [Cr(V)(EHBA)
2O]- by Ti(III) (aqueous solution at low pH, m = 0.5 M, 25 °C) (Reaction [1-40]) 22Figure 3-1 Structures of four of the thiol compounds used in this study. Probable ligating groups are indicated by an asterisk. 40
Figure 3-2. EPR spectra of chromium(V) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with
b-mercaptoethanol (1.44, 2.4, 4.8 or 9.6 mM) 88Figure 3-3. Time courses for chromium(V) formation during the reaction of potassium dichromate (0.48 mM chromium(VI)) and
b-mercaptoethanol (1.44 - 9.6 mM) in 0.050 M Tris-HCl, pH 7.0 at 37 °C. 90Figure 3-4. EPR spectra of chromium(III) complexes formed upon reaction of either chromium(III) or chromium(VI) with
b-mercaptoethanol. Spectra were obtained upon reaction of chromium(III) nitrate (0.48 mM chromium(III)) with b-mercaptoethanol (9.6 mM) 92Figure 3-5. Comparison of the EPR spectra of chromium(V) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with
b-mercaptoethanol (1.44-9.6 mM) 94Figure 3-6. Time courses for chromium(V) formation during the reaction of potassium dichromate (0.48 mM chromium(VI)) and
b-mercaptoethanol (1.44 - 9.6 mM) in the presence or absence of double-stranded CT DNA. 96Figure 3-7. EPR spectra of chromium(V) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with
b-mercaptoethanol (9.6 mM) 98Figure 3-8. Time courses for formation of two separate chromium(V) species during the reaction of potassium dichromate (0.48 mM chromium(VI)) and
b-mercaptoethanol (9.6 mM) in the presence of double-stranded CT DNA. 100Figure 3-9. EPR spectra of chromium(V) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with
b-mercaptoethanol (9.6 mM) 102Figure 3-10. Time courses for chromium(V) formation during the reaction of potassium dichromate (0.48 mM chromium(VI)) and
b-mercaptoethanol (9.6 mM) in the presence of CT DNA (0.003-0.048 mM), poly(dG) (0.048 mM), 5'dGMP (0.048 mM), or absence of DNA. 104Figure 3-11. Time courses for chromium(V) formation during the reaction of potassium dichromate (0.48 mM chromium(VI)) and
b-mercaptoethanol (9.6 mM) in the presence or absence of double-stranded CT DNA or DETAPAC. 106Figure 3-12. EPR spectra of chromium(V) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with
b-mercaptoethanol (9.6 mM) 108Figure 3-13. EPR spectra of chromium(V) (A) or DMPO-radical adduct (B) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with
b-mercaptoethanol (9.6 mM) 110Figure 3-14. EPR spectra of chromium(V) (A) or DMPO-radical adduct (B) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with
b-mercaptoethanol (9.6 mM) 112Figure 3-15. Computer simulation study of the EPR spectra of DMPO-radical adduct complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with
b-mercaptoethanol (9.6 mM) 114Figure 3-16. Comparison of the RT EPR spectra of chromium(V) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with
b-mercaptoethanol (9.6 mM) 116Figure 3-17. EPR spectra of chromium(V) (A) or DMPO-radical adduct (B) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with
b-mercaptoethanol (9.6 mM) 118Figure 3-18. Comparison of the EPR signal intensities of (A) chromium(V) and (B) DMPO-radical adduct complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with
b-mercaptoethanol (9.6 mM) 120Figure 3-19. EPR spectra of chromium(V) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with dithiothreitol (1.44, 2.4, 4.8, or 9.6 mM) 122
Figure 3-20. Time courses for chromium(V) formation during the reaction of potassium dichromate (0.48 mM chromium(VI)) and dithiothreitol (1.44 - 9.6 mM) in 0.05 M Tris-HCl, pH 7.0 at 37 °C. 124
Figure 3-21. EPR spectra of chromium(III) complexes formed upon reaction of either chromium(III) or chromium(VI) with dithiothreitol. 126
Figure 3-22. EPR spectra of chromium(V) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with dithiothreitol (1,44, 2.4, 4.8, or 9.6 mM) 128
Figure 3-23. Time courses for chromium(V) formation during the reaction of potassium dichromate (0.48 mM chromium(VI)) and dithiothreitol 130
Figure 3-24. EPR spectra of chromium(V) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with dithiothreitol (9.6 mM) 132
Figure 3-25. Time courses for chromium(V) formation during the reaction of potassium dichromate (0.48 mM chromium(VI)) and dithiothreitol (9.6 mM) in the presence of poly(dG) (0.048 mM) 134
Figure 3-26. EPR spectra of chromium(V) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with glutathione (1.44-9.6 mM) 136
Figure 3-27. Time courses for chromium(V) formation during the reaction of potassium dichromate (0.48 mM chromium(VI)) and glutathione(1.44 - 9.6 mM) in 0.05 M Tris-HCl, pH 7.0 at 37 °C. 138
Figure 3-28. EPR spectra of chromium(III) complexes formed upon reaction of either chromium(III) or chromium(VI) with glutathione. 140
Figure 3-29. EPR spectra of chromium(V) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with glutathione (9.6 mM) 142
Figure 3-30. Time courses for chromium(V) formation during the reaction of potassium dichromate (0.48 mM chromium(VI)) and glutathione (9.6 mM) in the presence of double-stranded calf thymus DNA (0.048 mM) 144
Figure 3-32. Time courses for chromium(V) formation during the reaction of potassium dichromate (0.48 mM chromium(VI)) and cysteine (1.44 - 9.6 mM) in 0.05 M Tris-HCl, pH 7.0 at 37 °C. 148
Figure 3-33. EPR spectra of chromium(III) complexes formed upon reaction of either chromium(III) or chromium(VI) with cysteine. 150
Figure 3-34. EPR spectra of potassium dichromate (0.48 mM chromium(VI)) incubated with cysteine (9.6 mM) 152
Figure 3-35. Electronic spectrum of potassium dichromate (0.48 mM chromium(VI)) incubated with cysteine (9.6 mM) 154
Figure 3-36. EPR spectra of chromium(V) complexes formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with cysteine (9.6 mM) 156
Figure 3-37. Time courses for chromium(V) formation during the reaction of potassium dichromate (0.48 mM chromium(VI)) and cysteine (9.6 mM) in the presence of double-stranded calf thymus DNA (0.048 mM) 158
Figure 3-38. EPR spectra of chromium(III) complex formed upon reaction of potassium dichromate (0.48 mM chromium(VI)) with cysteine (9.6 mM) 160
Figure 3-39. Energy level diagram showing the effect of zero field splittings on the fields at which the
DMs = 1 transitions occur in an S = 3/2 system 162Figure 3-40. EPR spectra observed upon reaction of Cr(NO
3)3•9H2O (0.475 mM) with b-mercaptoethanol. dithiothreitol, glutathione, or cysteine (9.6 mM) 163Figure 3-41. Proposed structures of the chromium(III)-thiol complexes formed upon incubation of hexaaquochromium(III) with (A) dithiothreitol, (B)
b-mercaptoethanol, and (C) glutathione. 165Figure 3-42. Crystal structure of the [Cr(L-cys)
2]- anion in Na[Cr(L-cys)2]•2H2O. 166Figure 4-1. Structures of the reducing agents used in this study. 189
Figure 4-2. The reaction of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (Diamagnetic), and with free radicals results in the "trapping" of the radical, forming a DMPO-R adduct (Paramagnetic), which can be observed in the EPR spectrum. EPR spectra were simulated using the computer program SpinSim (see Appendix).. 190
Figure 4-3. EPR spectrum of species formed upon reaction of potassium dichromate (5.0 mM chromium(VI)) with cysteine (5.0 mM) 192
Figure 4-4. EPR spectrum of species formed upon reaction of potassium dichromate (1.8 mM chromium(VI)) with cysteamine (5.45 mM) 194
Figure 4-5. EPR spectrum of species formed upon reaction of potassium dichromate (20.0 mM chromium(VI)) with cysteamine (20.0 mM) 196
Figure 4-6. EPR spectra of species formed upon reaction of potassium dichromate with ethanethiol in the presence or absence of DMPO (100 mM). 198
Figure 4-7. EPR spectra of species formed upon reaction of potassium dichromate with propanethiol in the presence or absence of DMPO (100.0 mM) 200
Figure 4-8. EPR spectra of species formed upon reaction of potassium dichromate with dimercaptosuccinic acid in the presence or absence of DMPO (100.0 mM). 202
Figure 4-9. EPR spectrum of species formed upon reaction of potassium dichromate (400.0 mM chromium(VI)) with glutathione (40.0-400.0 mM) 204
Figure 4-10. EPR spectrum of species formed upon reaction of potassium dichromate (20.0 mM chromium(VI)) with ascorbic acid (20.0 mM) 206
Figure 4-11. Computer simulation study of the radical species produced during the reaction of potassium dichromate (20.0 mM chromium(VI)) with ascorbic acid (20.0 mM) 208
Figure 5-1. EPR spectra of chromium(V) (A) and chromium(III) (B) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (50.0 mM chromium(V)) with
b-mercaptoethanol (50.0 mM) 239Figure 5-2. EPR spectra of chromium(V) (A) and chromium(III) (B) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (5.0 mM chromium(V)) with
b-mercaptoethanol (5.0 mM) 241Figure 5-3. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (5.0 mM chromium(V)) with
b-mercaptoethanol (5.0 mM) 243Figure 5-4. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (5.0 mM chromium(V)) with
b-mercaptoethanol (5.0 mM) 245Figure 5-5. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (5.0 mM chromium(V)) with
b-mercaptoethanol (5.0 mM) 247Figure 5-6. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (5.0 mM chromium(V)) with
b-mercaptoethanol (10.0 mM) 249Figure 5-7. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (5.0 mM chromium(V)) with
b-mercaptoethanol (10.0 mM) 251Figure 5-8. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (5.0 mM chromium(V)) with
b-mercaptoethanol (100.0 mM) 253Figure 5-9. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (5 mM chromium(V)) with
b-mercaptoethanol (500 mM) 255Figure 5-10. Solution EPR spectra of chromium(V) complexes formed upon reaction of potassium perchromate, K
3CrO8 (5.0 mM chromium(V)) with dithiothreitol (15.0 mM) 257Figure 5-11. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with dithiothreitol (10.0 mM) 259
Figure 5-12. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (5.0 mM) with dithiothreitol (100.0 mM) 261
Figure 5-13. (A) Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with dithiothreitol (100.0 mM) 263
Figure 5-14. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with dithiothreitol (25.0 mM) 265
Figure 5-15. (A) Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with dithiothreitol (100.0 mM) 267
Figure 5-16. (A) Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with dithiothreitol (25.0 mM) 269
Figure 5-17. (A) Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with dithiothreitol (7.5 mM) 271
Figure 5-18. Time course of the g = 1.980 EPR signal observed during the reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with dithiothreitol (20, 5, or 1.5:1 dithiol:Cr ratio) 273
Figure 5-19. Comparison of the solution EPR spectra of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) in various solvents (50% acetic acid, acetonitrile, N,N-dimethylformamide, or methanol) at 25 °C. 275
Figure 5-20. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium dichromate (5.0 mM chromium(VI)) with dithiothreitol (25.0 mM) 277
Figure 5-21. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (5.0 mM) with glutathione (5.0 mM) 279
Figure 5-22. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (0.5 mM) with glutathione (5.0 mM) 281
Figure 5-23. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (5.0 mM) with glutathione (15.0 mM) 283
Figure 5-24. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with glutathione (100.0 mM) 285
Figure 5-25. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with glutathione (100.0 mM) 287
Figure 5-26. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with oxidized glutathione (50.0 mM) 289
Figure 5-27. Frozen solution (77K) EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with oxidized glutathione (50.0 mM) 291
Figure 5-28. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (5.0 mM) with glycine (5.0, 50.0, or 500.0 mM) 293
Figure 5-29. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (4.0 mM) with oxalic acid (200.0 mM) 295
Figure 5-30. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (4.0 mM) with oxalic acid (200.0 mM) 297
Figure 5-31. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (4.0 mM) with oxalic acid (200.0 mM) 299
Figure 5-32. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (4.0 mM) with oxalic acid (200.0 mM) 301
Figure 5-33. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with oxalic acid (1.0 M) 303
Figure 5-34. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with ascorbic acid (2.5 mM) 305
Figure 5-35. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with ascorbic acid (100.0 mM) 307
Figure 5-36. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with thioglycolic acid (2.5 mM) 309
Figure 5-37. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with thioglycolic acid (5.0 mM) 311
Figure 5-38. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with thioglycolic acid (100.0 mM) 313
Figure 5-39. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with thioglycolic acid (100.0 mM) 315
Figure 5-40. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with thioglycolic acid (100.0 mM) 317
Figure 5-41. Solution EPR spectra of chromium(V) complexes formed upon reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with thioglycolic acid (100.0 mM) 319
Figure 5-42. Electronic spectra of the reaction of sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0 mM) with thioglycolic acid (100.0 mM) 321
Abbreviations
b
-ME, b-mercaptoethanol; CT-DNA, calf thymus DNA; CYS, cysteine; DETAPAC, diethylenetriaminepentaacetic acid; DES, deferoxamine mesylate; dGMP, deoxyguanosine monophosphate; dGTP, deoxyguanosine triphosphate; DMF, N,N'-dimethyl formamide; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; DMSA, dimercaptosuccinic acid; DNA-P, DNA nucleotide; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical; DTT, dithiothreitol; E°', reduction potential at pH 7.4 extrapolated from standard conditions using the Nernst equation; EDTA, ethylenediaminetetraacetic acid; ehba, 2-ethyl-2-hydroxybutyric acid; EPR, electron paramagnetic resonance; GSH, glutathione; GSSG, oxidized glutathione; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; hmba, 2-hydroxy-2-methylbutyric acid; salen, N,N'-ethylenebis(salicylideneiminato); poly(dA), poly deoxyadenosine; poly(dC), poly deoxycytosine; poly(dG), poly deoxyguanosine; Tris-HCl, Tris-(hydroxymethyl)aminomethane hydrochloride.