A XAS Study of the Sulphur Environment Location in Human Neuromelanin and Synthetic Analogues

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A XAS Study of the Sulphur Environment Location
in Human Neuromelanin and Synthetic Analogues
Acta Biophysica Romana 2008

• P.R. Crippa, M. Eisner, S. Morante, F.
  Stellato, F. Vicentin, L. Zecca

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Outline

• Parkinsons Disease
• Neuromelanin
• X-Ray Absorption Spectroscopy Experiments
• Conclusions

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Parkinsons Disease

• Parkinsonss Disease (PD)
• Progressive and fatal neurodegenerative disease
• Described in 1817 by James Parkinson
• Affects 1-2 of over 50 population
• lt10 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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PD Pathogenesis
Dopamine and acetylcholine are neurotrasmitters
that control body movement
synapse nerve terminal
synaptic vesicle
dopamine
Dopamine is produced in a small area in the base
of the brain, called substantia nigra
In Parkinsons disease NM pigmented neurons die
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Neuromelanin

• 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 Parkinsons
  disease
• Relation between neuronal vulnerability and
  presence of NM still unclear

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NM structure
Polymeric compound composed by
indolebenzothiazine groups XRD multilayer
(graphite-like) three dimensional structure with
planar overlapped sheets consisting of cyclic
molecules of indolebenzothiazine ring
15 Covalent bound peptide component
20 lipidic component
NM
Contains S
Binds Fe and Zn
L. Zecca et al.(2000) J Neurochem 74, 1758.
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Sulphur Content
Indolebenzothiazine groups contain S
S
The peptidic part contains Cysteine (about 3 in
weight) No Methionine detected
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XAS Experiments
X-ray Absorption Spectroscopy (XAS) study at the
S K-edge Measurement of X-ray Absorption
coefficient m(E)

• XAS features
• Selective for the absorber
• Local probe (5 Å)
• No crystallization needed

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Experimental Setup
X-ray source
IF measurement
Solid State Detector
Monochromatic beam
White beam
X-ray mirror
Sample
Monochromator
Ionization chambers
Synchrotron
I0 and I measurement
Incident energy selection
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XAS spectrum
c(k)
X-ray Absorption Near Edge Spectroscopy XANES
region EXAFS region Extended X-ray Absorption
Fine Structure
m(E)
k
E
XANES EXAFS
EXAFS spectra are analyzed in terms of
EXAFS region can be analyzed in the single
scattering approximation
characteristic of atomic type indistinguishable
for light atoms (N, O, C) introduce multiple
scattering terms
.
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Samples

• 6 powder samples
• Human Neuromelanin (HNM)
• extracted from cerebellum
• 3 Synthetic Melanins prepared with different
  procedures
• 2 Model compounds (Cysteine and Trichochrome)

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Model Compounds
Trichochrome S is present as heteroatom in
aromatic rings
Cysteine S is present in the amino acid side chain
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Synthetic Melanins
Synthetic compounds similar to natural melanins

• Enzymatic Oxidation
• (With Tyrosinase)
• Dopamine Cysteine
• DEC
• Dopa Cysteine
• Pheomelanin

• Auto-oxidation
• Dopamine Cysteine
• DAC

Dopamine
Dopa
Cysteine
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Data Collection

• 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
• mI/I0

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Total Electron Yield
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
Fluorescence yield is low for low-Z elements
(for S ?Flt0.1) Xs, XA emission probabilities of
fluorescence photon and Auger electron
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Results
HNM Model Compounds
Model Compounds Cysteine Trichochrome Significan
tly different spectral features Natural
Melanin HNM different from both
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HNM Synthetic Melanins
Synthetic Melanins DAC DEC - Pheomelanin Similar
spectral features Natural Melanin HNM Similar to
Pheomelanin and DEC
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Difference Spectra
Qualitative findings are consistent with
quantitative analysis of difference spectra
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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
P1
P2
E0
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Data Analysis
E0 and P1 are the same in all spectra P2 is the
same in all spectra but Cysteine
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Fit
Fits of HNM are obtained minimizing
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HNM Fit
HNM 64 Cysteine 36 Trichochrome
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Pheomelanin 55 Cysteine 45
Trichochrome
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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