Metal Ion Chelation and Oxidation Kinetics involving ... Swartz.pdfOxidation Mason Swartz, Philip...
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Model System UV-Vis All of the biological aspects of the system were eliminated, providing a bare-bones assessment of binding characteristics for specific metal ions to our synthetic peptides. Small, six amino acid peptides were synthesized to have a hairpin turn, representative of a larger protein structure, as well as a (L)- dopa residue, representative of an oxidatively damaged tyrosine residue. Theoretical binding would happen through chelation at the dopa residue and possibly through the coordination with other aromatic ring systems within the peptides. The Metal Ion Hypothesis Previous research has suggested the amyloid plaques implicated in the progression of Alzheimer’s Disease (AD) contain higher levels of metal ions such as iron (Fe 2+ ) and zinc (Zn 2+)1 . Other literature stresses the importance of genetics in maintaining metal ion homeostasis; these genes and pathways are typically disturbed in patients with AD 1 . The metal ions thought to play a role in AD are typically strong chelators. Metal chelation occurs through coordination with amino acid residues that have hydroxyl groups, most notably tyrosine and its oxidatively damaged form dopa. Though the actual role of the metal ions is not know, it is thought that they interact with and stabilize the oligomerized form of a peptide called amyloid- β 1 . Oligomerization reduces solubility of the peptides, thus forming the plaques indicative of Alzheimer’s Disease. Background Oxidation Mason Swartz, Philip Tubergen and Chad Tatko, Calvin College 3201 Burton Ave, Grand Rapids, Michigan Figure 3. A. Raw ultra-violet/ visible spectral data of Cu 2+ titrated into Dopa1Nal. The absorbance of the solution increases with the addition of Cu 2+ , specifically at 230nm and 280nm. There is a small, broad peak around 475nm, indicative of peptide : copper binding. B. The solution composition and concentrations of species. As copper concentration decreases, it complexes two coppers to one Dopa1Nal, followed by one copper to one Dopa1Nal, followed by one copper bound with two Dopa1Nal peptides. C. Binding scheme of peptides and metal ions. The aromatic ring with two hydroxyls is representative of a ( L)-dopa residue on the end ofthe syntheticpeptide. One peptide can bind to one metal ion, two metal ions, two peptides to one metal ion, and three peptides to one metal ion. Each of these complexes produces a different color, rendering it possible to differentiate the prominent species found in a specific solution. M n+ O O M n+ O O 2 M n+ O O O O O O M n+ O O O O A C NH 2 H N N HN N H H N NH 2 O O O O O O H H NH 3 OH OH 1Nal X dopa OH OH Figure 2. Synthetic generic peptide used for this research. The N- terminus is an (L)-dopa residue, followed by a valine, (D)-proline, glycine, ornithine, and a variable position; the C- terminus was amidated. The variable X position contained either a histidine, tryptophan, tyrosine, phenylalanine, 1-napthylalanine (1Nal), or 2-napthylalanine (2Nal). Both the dopa and the 1Nal are depicted. HO OH O NH 2 TYROSINE HO HO OH NH 2 O DOPA Figure 1. A modeled oligomer of amyloid-β. The hairpin turn creates two β sheets. The meal ion causes stabilization on both Phe20 and Val18 in this figure, while stabilizing other peptides above (not pictured). The amassing of multiple oligomer systems causes a plaque, likely due to the loss of hydrophilicity and thus solubility. Metal Ion Chelation and Oxidation Kinetics involving Synthetic Peptides NMR Spectroscopy 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 f1 (ppm) 1 2 3 4 5 0.25 eq. Cu 2+ 0 eq. Cu 2+ 0.5 eq. Cu 2+ 1 eq. Cu 2+ 2 eq. Cu 2+ Dopa 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4 8.5 f1 (ppm) 1 2 3 4 5 1Nal Dopa 0 eq. Cu 2+ 0.25 eq. Cu 2+ 0.5 eq. Cu 2+ 1 eq. Cu 2+ 2 eq. Cu 2+ Figure 4. A. NMR spectra of copper titrated into 3,4-dihydroxyphenylalanine methyl ester at 0.25, 0.5, 1, and 2 equivalents of copper to methyl ester. As cmore opper is titrated into the system, the hydroxyl peaks broaden and eventually flat line. B. NMR spectra of copper titrated into Dopa1Nal, at same equivalents. The dopa peaks on the right side of the spectra broaden and disappear by 0.5 equivalents of copper, whereas the 1Nal peaks, on the left side of the spectra, remain into 1 equivalent of copper added. A B B 0 0.5 1 1.5 2 2.5 200 300 400 500 600 700 800 Raw Absorbance (au) Wavelength (nm) pH4.3 pH6.9 pH11.69 OH OH O O OH OH OH OH HO Figure 4. A. A catechol moiety. Catechols are exceptionally good oxidizers; the basic structure of a catechol is contained within a ( L )-dopa reside. B. The flavonoid quercetin, found in antioxidant rich foods. Flavonoids and flavonols, among other antioxidants, are responsible for scavenging free radicals within the human body, making them extremely important in maintaining normal bodily function. They often contain catechol moieties. C. 3,4-dihydroxyphenyl methyl ester (D1507), used as a surrogate for ( L )-dopa. This molecule was used in these oxidation experiments. A B C 0 0.005 0.01 0.015 0.02 0.025 0.03 0 2 4 6 8 10 12 14 16 Absorbance (at 725 nm) Time (min) 5:1 3:1 1:1 MALDI Mass Spectrometry Conclusions Acknowledgments and References We would like to thank Calvin College Chemistry and Biochemistry Department for funding and lab space. We also thank all previous student researchers for their contributions to our projects and Chad Tatko for his guidance. 1 Kepp, Kasper P. "Bioinorganic chemistry of Alzheimer’s disease." Chemical reviews 112.10 (2012): 5193-5239. http://news.rice.edu/files/2013/07/0722_ALZHEIMERS-3-web.jpg Figure 5. A. UV/Vis spectra of a pH titration of iron and D1507. At high pH (11.69), complexation is most evident in a 3:1 species of D1507 to iron, observed at 298 nm. At neutral pH (6.9), complexation is most evident in a 2:1 species, observed at 575 nm. At low pH (4.3), complexation happens most readily in a 1:1 species, observed at 725 nm. B. Kinetics spectra of complexation of D1507 and iron, assessed at pH 4.5 and a wavelength of 725 ±3 nm. Concentration of D1507 was increased in comparison to iron, starting at 1:1 equivalents of D1507 to iron, increasing to 3:1 equivalents and 5:1 equivalents. As the concentration of D1507 increased, so did the initial rate of product formation (shown in a darker shade). A B It can be reasonably concluded that metal ions, specifically copper, interact strongly with oxidatively damaged amino acid residues. Characterization of solutions using mass spectrometry support complexation of both a 1:1 metal to peptide complex and a 2:1 metal peptide complex. In the solution phase, not only can one peptide coordinate one metal ion, but species containing multiple of each can also be readily formed, which seems somewhat dependent on pH value. These peptides may also play a role in the oxidation of metal ions, as evidenced by the increase in rate of oxidation of iron in higher concentrations of D1507. These findings may lend further credence to the metal ion hypothesis of Alzheimer’s Disease. https://www.nsf.gov/news/mmg/mmg_disp.jsp?med_id=75061&from= A B C Figure 5. A. Matrix assisted laser desorption ionization (MALDI) time of flight mass spectrum of Dopa1Nal complexed with copper in a pH 2 solution. The peak at 761 amu represents Dopa1Nal, uncomplexed; the small group of peaks at 783 amu is the peptide complexed with sodium, a result of buffered solutions used to produce desires pH values; the final group of peaks at 823 amu represent the peptide complexed with copper. B. MALDI mass spectrum of Dopa1Nal complexed with copper in a pH 12 solution. The first three sets of peaks are the same as presented in the pH 2 spectrum: bare peptide, peptide with sodium, and peptide with one copper ion. The spectrum also indicated the presence of peptide bound to both sodium and copper at 845 amu. A two copper and one peptide complex is also evident, presented on the spectrum at 885 amu. C. Isotopic distribution of copper found to complex with peptide. This peak distribution is characteristic of all complexes with copper and can be seen in both the one peptide one ion complex as well as the one peptide to two ions complex, shown in theoretical form in lightgrey, superimposed on the image. OH OH H 3 CO O NH 2 O O H 3 CO O NH 2 Fe 2+ [ ] n Wavelengths Concentration (M) Solution Number O O H 3 CO O NH 2 Cu 2+ NH 2 H N N HN N H H N NH 2 O O O O O O H H NH 3 OH OH X O O Cu 2+ Pep::Cu 2+ Peptide Pep:Na + Pep::Cu 2+ Pep::2Cu 2+ Pep::Na + Cu 2+ Pep::Na + Peptide
Transcript of Metal Ion Chelation and Oxidation Kinetics involving ... Swartz.pdfOxidation Mason Swartz, Philip...
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Model System
UV-Vis
All of the biological aspects of the system were eliminated,providing a bare-bones assessment of binding characteristicsfor specific metal ions to our synthetic peptides. Small, sixamino acid peptides were synthesized to have a hairpin turn,representative of a larger protein structure, as well as a (L)-dopa residue, representative of an oxidatively damagedtyrosine residue. Theoretical binding would happen throughchelation at the dopa residue and possibly through thecoordination with other aromatic ring systems within thepeptides.
The Metal Ion HypothesisPrevious research has suggested the amyloid plaques implicated inthe progression of Alzheimer’s Disease (AD) contain higher levelsof metal ions such as iron (Fe2+) and zinc (Zn2+)1. Other literaturestresses the importance of genetics in maintaining metal ionhomeostasis; these genes and pathways are typically disturbed inpatients with AD1. The metal ions thought to play a role in AD aretypically strong chelators. Metal chelation occurs throughcoordination with amino acid residues that have hydroxyl groups,most notably tyrosine and its oxidatively damaged form dopa.
Though the actual role of the metal ions is not know, it is thoughtthat they interact with and stabilize the oligomerized form of apeptide called amyloid-β1. Oligomerization reduces solubility ofthe peptides, thus forming the plaques indicative of Alzheimer’sDisease.
Background
Oxidation
Mason Swartz, Philip Tubergen and Chad Tatko, Calvin College3201 Burton Ave, Grand Rapids, Michigan
Figure 3. A. Raw ultra-violet/ visible spectral data of Cu2+ titrated into Dopa1Nal. The absorbance of the solution increases with the addition of Cu2+, specifically at 230nm and 280nm. There is asmall, broad peak around 475nm, indicative of peptide : copper binding. B. The solution composition and concentrations of species. As copper concentration decreases, it complexes twocoppers to one Dopa1Nal, followed by one copper to one Dopa1Nal, followed by one copper bound with two Dopa1Nal peptides. C. Binding scheme of peptides and metal ions. The aromaticring with two hydroxyls is representative of a (L)-dopa residue on the end of the synthetic peptide. One peptide can bind to one metal ion, two metal ions, two peptides to one metal ion, andthree peptides to one metal ion. Each of these complexes produces a different color, rendering it possible to differentiate the prominent species found in a specific solution.
OHOH Mn+ Mn+
OO
- 2H+
Mn+O
OMn+
OO
OO
OO
Mn+O
OO
O
OHOH Mn+ Mn+
OO
- 2H+
Mn+O
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OO
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OO
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OHOH Mn+ Mn+
OO
- 2H+
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DopaGly: (Dopa)VpGOG-NH2
DopaTyr:(Dopa)VpGOW-NH2
Dopa1Nal:
(Dopa)VpGOF-NH2DopaPhe:(Dopa)VpGOY-NH2
DopaTrp:(Dopa)VpGO(1Nal)-NH2
dopa
OHOH
Figure 2. Syntheticgeneric peptide used forthis research. The N-terminus is an (L)-doparesidue, followed by avaline, (D)-proline,glycine, ornithine, and avariable position; the C-terminus was amidated.The variable X positioncontained either ahistidine, tryptophan,tyrosine, phenylalanine,1-napthylalanine (1Nal),or 2-napthylalanine(2Nal). Both the dopa andthe 1Nal are depicted.
HO
OH
O
NH2
TYROSINEHO
HO
OHNH2
ODOPA
Figure 1. A modeled oligomer ofamyloid-β. The hairpin turn createstwo β sheets. The meal ion causesstabilization on both Phe20 andVal18 in this figure, while stabilizingother peptides above (not pictured).The amassing of multiple oligomersystems causes a plaque, likelydue to the loss of hydrophilicity andthus solubility.
Metal Ion Chelation and Oxidation Kinetics involving Synthetic Peptides
NMR Spectroscopy
5.55.65.75.85.96.06.16.26.36.46.56.66.76.86.97.07.17.27.37.47.57.67.77.87.9f1 (ppm)
1
2
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0.25 eq. Cu 2+
0 eq. Cu 2+
0.5 eq. Cu 2+
1 eq. Cu 2+
2 eq. Cu 2+
Dopa
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1Nal Dopa
0 eq. Cu 2+
0.25 eq. Cu 2+
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1 eq. Cu 2+
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Figure 4. A. NMR spectra of copper titrated into 3,4-dihydroxyphenylalanine methyl ester at 0.25, 0.5, 1, and 2equivalents of copper to methyl ester. As cmore opper is titrated into the system, the hydroxyl peaks broaden andeventually flat line. B. NMR spectra of copper titrated into Dopa1Nal, at same equivalents. The dopa peaks on theright side of the spectra broaden and disappear by 0.5 equivalents of copper, whereas the 1Nal peaks, on the leftside of the spectra, remain into 1 equivalent of copper added.
A
B
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0.5
1
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Raw
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orba
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pH4.3 pH6.9 pH11.69
OHOH O
OOH
OH
OH
OH
HO
Figure 4. A. A catechol moiety. Catechols are exceptionally good oxidizers; the basic structure of a catechol is contained within a (L)-dopa reside. B. The flavonoid quercetin, found inantioxidant rich foods. Flavonoids and flavonols, among other antioxidants, are responsible for scavenging free radicals within the human body, making them extremely important in maintainingnormal bodily function. They often contain catechol moieties. C. 3,4-dihydroxyphenyl methyl ester (D1507), used as a surrogate for (L)-dopa. This molecule was used in these oxidationexperiments.
A B C
0
0.005
0.01
0.015
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0 2 4 6 8 10 12 14 16
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rban
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t 725
nm
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Time (min)
5:1
3:1
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MALDI Mass Spectrometry
Conclusions
Acknowledgments and ReferencesWe would like to thank Calvin College Chemistry and BiochemistryDepartment for funding and lab space. We also thank all previousstudent researchers for their contributions to our projects and ChadTatko for his guidance.
1Kepp, Kasper P. "Bioinorganic chemistry of Alzheimer’s disease." Chemical reviews 112.10 (2012): 5193-5239.
http://news.rice.edu/files/2013/07/0722_ALZHEIMERS-3-web.jpg
Figure 5. A. UV/Vis spectra of a pH titration of iron and D1507. At high pH (11.69), complexation is most evident in a 3:1 species of D1507 to iron, observed at 298 nm. At neutral pH (6.9),complexation is most evident in a 2:1 species, observed at 575 nm. At low pH (4.3), complexation happens most readily in a 1:1 species, observed at 725 nm. B. Kinetics spectra ofcomplexation of D1507 and iron, assessed at pH 4.5 and a wavelength of 725 ±3 nm. Concentration of D1507 was increased in comparison to iron, starting at 1:1 equivalents of D1507 to iron,increasing to 3:1 equivalents and 5:1 equivalents. As the concentration of D1507 increased, so did the initial rate of product formation (shown in a darker shade).
A B
It can be reasonably concluded that metal ions, specificallycopper, interact strongly with oxidatively damaged aminoacid residues. Characterization of solutions using massspectrometry support complexation of both a 1:1 metal topeptide complex and a 2:1 metal peptide complex. In thesolution phase, not only can one peptide coordinate onemetal ion, but species containing multiple of each can alsobe readily formed, which seems somewhat dependent on pHvalue. These peptides may also play a role in the oxidationof metal ions, as evidenced by the increase in rate ofoxidation of iron in higher concentrations of D1507. Thesefindings may lend further credence to the metal ionhypothesis of Alzheimer’s Disease.
https://www.nsf.gov/news/mmg/mmg_disp.jsp?med_id=75061&from=
A B C
Figure 5. A. Matrix assisted laser desorption ionization (MALDI) time of flight mass spectrum of Dopa1Nal complexed with copper in a pH 2 solution. The peak at 761 amu represents Dopa1Nal,uncomplexed; the small group of peaks at 783 amu is the peptide complexed with sodium, a result of buffered solutions used to produce desires pH values; the final group of peaks at 823 amurepresent the peptide complexed with copper. B.MALDI mass spectrum of Dopa1Nal complexed with copper in a pH 12 solution. The first three sets of peaks are the same as presented in the pH2 spectrum: bare peptide, peptide with sodium, and peptide with one copper ion. The spectrum also indicated the presence of peptide bound to both sodium and copper at 845 amu. A two copperand one peptide complex is also evident, presented on the spectrum at 885 amu. C. Isotopic distribution of copper found to complex with peptide. This peak distribution is characteristic of allcomplexes with copper and can be seen in both the one peptide one ion complex as well as the one peptide to two ions complex, shown in theoretical form in light grey, superimposed on theimage.
OH
OH
H3CO
O
NH2
O
O
H3CO
O
NH2
Fe2+[ ]n
Wavelengths
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NH2Cu2+
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HNN
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O O
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DopaGly: (Dopa)VpGOG-NH2
DopaTyr:(Dopa)VpGOW-NH2
Dopa1Nal:
(Dopa)VpGOF-NH2DopaPhe:(Dopa)VpGOY-NH2
DopaTrp:(Dopa)VpGO(1Nal)-NH2
dopa
OHOH
O
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H3CO
O
NH2Cu2+
Pep::Cu2+
Peptide
Pep:Na+
Pep::Cu2+
Pep::2Cu2+Pep::Na+Cu2+
Pep::Na+
Peptide
