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Transcript of A XAS Study of the Sulphur Environment Location in Human Neuromelanin and Synthetic Analogues P.R....
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A XAS Study of the Sulphur Environment Location in
Human Neuromelanin and Synthetic Analogues
P.R. Crippa, M. Eisner, S. Morante, F. Stellato, F. Vicentin, L. Zecca
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OutlineOutline
• Parkinson’s Disease
• Neuromelanin
• X-Ray Absorption Spectroscopy Experiments
• Conclusions
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Parkinson’s DiseaseParkinson’s Disease
Parkinsons’s Disease (PD):
• Progressive and fatal neurodegenerative disease
• Described in 1817 by James Parkinson
• Affects 1-2% of over 50 population
• <10% of PD is familial majority of cases are sporadic
• 5 clearly defined genetic causes
B. Thomas et al.(2007) Hum Mol Gen 16,R183.
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Dopamine and acetylcholine are neurotrasmitters that control body movement
Dopamine is produced in a small area in the base of the brain, called substantia nigra
PD Pathogenesis
In Parkinson’s disease NM pigmented neurons die
synapse nerve terminal
synaptic vesicle
dopamine
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Neuromelanin (NM):
• Dark pigment present in neurons of different brain areas
• Mixture of similar polymers which are made up of different structural units
• Accumulates with aging
• Contributes to the protection of neurons from oxidative processes
• Pigmented neurons are lost in Parkinson’s disease
• Relation between neuronal vulnerability and presence of NM still unclear
NeuromelaninNeuromelanin
Neurons containing neuromelanin pigment
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Polymeric compound composed by indolebenzothiazine groups
XRD: multilayer (graphite-like) three dimensional structure with planar overlapped sheets consisting of cyclic molecules of indolebenzothiazine ring
NM structure
15% Covalent bound peptide component 20% lipidic
component
Binds Fe and Zn Contains S
NMNM
L. Zecca et al.(2000) J Neurochem 74, 1758.
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Sulphur Content
Indolebenzothiazine groups contain S
The peptidic part contains Cysteine(about 3% in weight)No Methionine detected
Indolebenzothiazine
SS
Cysteine
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XAS ExperimentsXAS Experiments
X-ray Absorption Spectroscopy (XAS) study at the S K-edge
Measurement of X-ray Absorption coefficient (E)
XAS features
Selective for the absorber
Local probe (~5 Å)
No crystallization needed
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Experimental Setup
Incident energy selection
X-ray mirror
Synchrotron
White beam
X-ray source
Monochromator
Monochromatic beam
Solid State Detector
Ionization chambers
Sample
IF measurement
I0 and I measurement
μ(E)d0eII(E) :LawBeer -Lambert
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EXAFS spectra are analyzed in terms of
k
kkk
0
0
2
0Em2k
EXAFS region can be analyzed in the single scattering approximation:
)k(kR2sinee),k(AkR
NS)k( ii
)k(R2
i
k2i2
i
i20
i2i
2
characteristic of atomic type
indistinguishable for light atoms (N, O, C)
introduce multiple scattering terms
XAS spectrum
.
X-ray Absorption Near Edge Spectroscopy
XANES region
EXAFS region
Extended X-ray Absorption Fine Structure
(E)
(k)
E
k
XANES EXAFS
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6 powder samples
• Human Neuromelanin (HNM)extracted from cerebellum
• 3 Synthetic Melanins prepared with different procedures
• 2 Model compounds (Cysteine and Trichochrome)
Cerebellum
Samples
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Model Compounds
TrichochromeS is present as heteroatom in aromatic rings
CysteineS is present in the amino acid side chain
Cysteine
Trichochrome
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Synthetic Melanins
Synthetic compounds similar to natural melanins
Auto-oxidation
Dopamine + Cysteine
DAC
Enzymatic Oxidation
(With Tyrosinase)
Dopamine + Cysteine
DEC
Dopa + Cysteine
Pheomelanin
DopamineDopa
Cysteine
Model of Synthetic Melanin
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•Spectra collected at the D04B
bending magnet beam line of the
Brazilian Synchrotron Light Laboratory
•Total Electron Yield (TEY)
I0: incident current measured
with a 0.75 μm carbon foil
I: sample current collected
with an electrometer
=I/I0
Data Collection
Synchrotron
White beam
Monochromator
Monochromatic beam Carbon foil
Electrometer
Sample
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Auger effect (Non-radiative de-excitation)
-The photo-electron is emitted
-The core hole is filled by an electron of an upper level
-The energy is used up to eject an Auger electron
-TEY: detection of all electrons emitted by the sample
Total Electron Yield
Fluorescence yield
is low for low-Z elements (for S F<0.1)
Xs, XA: emission probabilities of fluorescence photon and Auger electron
AF
FF XX
Xη
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Results
0
-4 0 4 8
CysteineTrichochromeHNM
E-E0
(eV)
Model Compounds
Cysteine – Trichochrome
Significantly different spectral features
Natural Melanin
HNM
different from both
HNM & Model Compounds
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0
-4 0 4 8
DACDECPheomelaninHNM
E-E0 (eV)
Synthetic Melanins
DAC
DEC - Pheomelanin
Similar spectral features
Natural Melanin
HNM
Similar to Pheomelanin and DEC
HNM & Synthetic Melanins
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Difference Spectra
D (Emin> E0) D (Emin< E0)
DAC-DEC 0.10 0.17
DAC-Pheomelanin 0.09 0.17
DEC-Pheomelanin 0.03 0.10
HNM-DAC 0.08 0.15
HNM-DEC 0.04 0.10
HNM-Pheomelanin 0.02 0.06
MAX
min
E
E
mn dE(E)μ(E)μD
Qualitative findings are consistent with
quantitative analysis of difference spectra
0
-5 0 5 10
DAC-DECDAC-PheomelaninDEC-PheomelaninHNM-DACHNM-DECHNM-Pheomelanin
E (eV)
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-5 0 5
CysteineTrichochromeHNMDACDECPheomelanin
E-E0(eV)
Data Analysis
1- Determination of edge energy E0
2- Spectra shifted by E0
3- Identification of two peaks (white line and first peak) in all spectra
4- P1, P2: positions of the two peaks
5- A1, A2: amplitudes of the two peaks
E0
P1
P2
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Data Analysis
E0 and P1 are the same in all spectra
P2 is the same in all spectra but Cysteine
Sample E0 P1=E1-E0 P2=E2-E0
Model Compounds
Cysteine 2470.5 1.0 6.0
Trichochrome 2470.5 1.5 8.5
Synthetic Melanins
DAC 2470.5 1.5 9.0
DEC 2471.0 1.0 8.0
Pheomelanin 2471.5 1.0 8.0
Natural Melanin
HNM 2471.0 1.0 8.0
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Cysteine Trichochrome DAC DEC Pheo RHNM = 64% 36% 0.8HNM = 28% 72% 0.4HNM = 0% 100% 0.9HNM = 35% 65% 1.6HNM = 10% 90% 2.2HNM = 61% 39% 0.9HNM = 25% 75% 8.2Pheo = 55% 45% 2.0
(E)p)μ(1(E)pμp)(E,μ where
(E))(σ
(E)μp)(E,μ
2N
1R(p)
EXPm
EXPl
TH
E2EXP
2EXPTH
Fits of HNM are obtained minimizing
Fit
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—— HNM
—— Fit
HNM =
64% Cysteine + 36% Trichochrome
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Pheomelanin =
55% Cysteine + 45% Trichochrome
—— Pheomelanin
—— Fit
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Conclusions
We have performed a structural study on natural
Neuromelanin and Synthetic Analogues
• Identification of percentage of Trichochrome-like and a
Cysteine-like components in Human Neuromelanin
• S structure is similar in Human Neuromelanin and
Synthetic Melanins Pheomelanin and DEC