Transcript A XAS Study of the Sulphur environment Location in Human

A XAS Study of the
Sulphur Environment Location in
Human Neuromelanin and
Synthetic Analogues
P.R. Crippa, M. Eisner, S. Morante,
Stellato, F. Vicentin, L. Zecca
F.
Outline
• Parkinson’s Disease
• Neuromelanin
• X-Ray Absorption Spectroscopy
Experiments
• Conclusions
Parkinson’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.
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 Parkinson’s disease NM
pigmented neurons die
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 Parkinson’s disease
• Relation between neuronal vulnerability and
presence of NM still unclear
Neurons containing
neuromelanin pigment
NM structure
Polymeric compound
groups
composed by
indolebenzothiazine
XRD: multilayer (graphite-like) three dimensional structure
with planar overlapped sheets consisting of cyclic molecules of
indolebenzothiazine ring
20%
lipidic
component
Binds Fe and Zn
15% Covalent
bound peptide
component
NM
Contains S
L. Zecca et al.(2000) J Neurochem 74, 1758.
Sulphur Content
Indolebenzothiazine groups contain S
Indolebenzothiazine
S
The peptidic part contains Cysteine
(about 3% in weight)
No Methionine detected
Cysteine
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
Experimental Setup
X-ray source
IF measurement
Solid State Detector
Monochromatic beam
White beam
X-ray mirror
Monochromator
Sample
Ionization
chambers
Synchrotron
I0 and I measurement
Incident energy selection
Lambert- Beer Law :
I(E)  I0e
μ(E)d
XAS spectrum
(k)
X-ray Absorption Near
Edge Spectroscopy
XANES region
m(E)
k
EXAFS region
Extended X-ray
Absorption Fine
Structure
XANES
EXAFS spectra are analyzed in terms of k  
E
EXAFS
mk   m 0 k 
m 0 k 
k
2m  E 0 
2
EXAFS region can be analyzed in the single scattering approximation:
Ni
 2 k 2i2  2 R i
(k )  S 
A i (k, ) e
e
2
i kR i
2
0
(k )
sin2kRi   i (k)
characteristic of atomic type
indistinguishable for light atoms (N, O, C)
.
introduce multiple scattering terms
Samples
6 powder samples
• Human Neuromelanin (HNM)
extracted from cerebellum
Cerebellum
• 3 Synthetic Melanins prepared with different procedures
• 2 Model compounds (Cysteine and Trichochrome)
Model Compounds
Trichochrome
S is present as heteroatom
in aromatic rings
Trichochrome
Cysteine
S is present in the
amino acid side chain
Cysteine
Synthetic Melanins
Synthetic compounds similar to natural melanins
Auto-oxidation
Enzymatic Oxidation
Dopamine + Cysteine
(With Tyrosinase)
DAC
Dopamine + Cysteine
DEC
Dopa + Cysteine
Pheomelanin
Model of
Synthetic Melanin
Dopamine
Dopa
Cysteine
Data Collection
•Spectra collected at the D04B
bending magnet beam line of the
Brazilian Synchrotron Light Laboratory
Monochromatic beam
•Total Electron Yield (TEY)
I0: incident current measured
with a 0.75 μm carbon foil
m=I/I0
Sample
White beam
Monochromator
Electrometer
I: sample current collected
with an electrometer
Carbon foil
Synchrotron
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
XF
ηF 
XF  XA
is low for low-Z elements (for S F<0.1)
Xs, XA: emission probabilities of
fluorescence photon and Auger electron
Results
HNM & Model Compounds
Model Compounds
Cysteine – Trichochrome
Significantly different
spectral features
Natural Melanin
HNM
different from both
Cysteine
Trichochrome
HNM
0
-4
0
4
8 E-E (eV)
0
HNM & Synthetic Melanins
Synthetic Melanins
DAC
DEC - Pheomelanin
Similar spectral features
Natural Melanin
HNM
Similar to Pheomelanin
and DEC
DAC
DEC
Pheomelanin
HNM
0
-4
0
4
8 E-E0 (eV)
Difference Spectra
Qualitative findings are
consistent with
quantitative analysis of
difference spectra
D
E MAX
μ
n
(E)  μ m (E) dE
DAC-DEC
DAC-Pheomelanin
DEC-Pheomelanin
HNM-DAC
HNM-DEC
HNM-Pheomelanin
0
E min
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
-5
0
5
10 E (eV)
Data Analysis
1- Determination of edge
energy E0
P1
2- Spectra shifted by E0
P2
3- Identification of two
peaks (white line and first
peak) in all spectra
4- P1, P2: positions of the
two peaks
E0
5- A1, A2: amplitudes of
the two peaks
Data Analysis
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
1.0
8.0
Natural Melanin
HNM
2471.0
E0 and P1 are the same in all spectra
P2 is the same in all spectra but Cysteine
Fit
Fits of HNM are obtained minimizing
R(p) 
where
1

N2 E
μ
TH

(E,p)  μ EXP (E)
2
(σ EXP (E))2
μ TH (E,p)  pμ lEXP (E)  (1 p)μ EXP
m (E)
Cysteine Trichochrome DAC DEC Pheo
HNM =
64%
36%
HNM =
28%
72%
HNM =
0%
100%
HNM =
35%
65%
HNM =
10%
90%
HNM =
61%
39%
HNM =
25%
75%
Pheo =
55%
45%
R
0.8
0.4
0.9
1.6
2.2
0.9
8.2
2.0
—— HNM
—— Fit
HNM =
64% Cysteine + 36% Trichochrome
—— Pheomelanin
—— Fit
Pheomelanin =
55% Cysteine + 45% Trichochrome
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